United States
Environmental Protection
Agency
Environmental Research
Laboratory
Athens, QA 30613
EPA/600/9-90/011
Mar. 1990
Research and Development
Fish Physiology, Fish
Toxicology, and Fisheries
Management:

Proceedings of an
International Symposium,
Guangzhou, PRC,
September 14-16, 1988

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                                EPA/600/9-90/011
                                March 1990
    FISH PHYSIOLOGY, FISH TOXICOLOGY,
        AND FISHERIES MANAGEMENT
Proceedings of an International Symposium,
  Guangzhou, PRC, September 14-16, 1988
                Edited by
             Robert C. Ryans
   ENVIRONMENTAL RESEARCH LABORATORY
   OFFICE OF RESEARCH AND DEVELOPMENT
 U.S. ENVIRONMENTAL PROTECTION AGENCY
         ATHENS, GEORGIA 30613

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                                 DISCLAIMER

      The information in this document has been funded in part by the United
States Environmental Protection Agency.  Papers describing EPA-sponsored re-
search have been subject to the Agency's peer and administrative review, and
have been approved for publication.  Mention of trade names or commercial
products does not constitute endorsement or recommendation for use by the
U.S. Environmental Protection Agency.
                                     ii

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                                   FOREWORD

     Joint ecological research involving scientists from every country in the
world is essential if global environmental problems are to be solved with
international expertise.  Recognition of this international aspect of environ-
mental pollution is reflected in the joint activities undertaken under Annex
3, Item 4 of the USA-PRC Protocol for Environmental Protection.  This, com-
ponent of the protocol provides for cooperative research on the environmental
processes and effects of pollution on freshwater organisms, soils, and
groundwater and on the application of transport and transformation models.

     Specific areas of cooperation in environmental research include inorganic
chemical characterization and measurement; inorganic chemical transport and
transformation process characterization; biological degradation process
characterization; oxidation/reduction process characterization; field evalua-
tion of selected transport, exposure and risk models; and application of
models for environmental decision-making concerning organic pollution in semi-
arid conditions, heavy metal pollution, and permissible loading of conven-
tional and toxic pollutants in Chinese rivers.   Activities include seminars,
workshops, joint symposia, training programs, joint research, and publications
exchange.

     The first symposium presented under this portion of the protocol was held
on the campus of Zhongshan University, Guangzhou, PRC, on September 14-16,
1988.  Sponsored jointly by the National Science Foundation of the PRC,
Zhongshan University, the U.S. Environmental Protection Agency, the Academy of
Sciences of China, the Canadian Society of Zoology, and the American Fisheries
Society, the symposium attracted presentations from eight countries who
recognized the need for knowledge exchange on a global scale.

     Symposia are an effective means of fostering cooperation among scientists
from different countries as environmental organizations seek to efficiently
gain the information necessary to predict the effects of pollutants on
ecosystems and apply the results on a global scale.  The symposia provide a
forum through1 which distinguished scientists from laboratories and institutes
from several countries can exchange scientific expertise on environmental
problems of concern to EPA and the international environmental community.

                                      Rosemarie C. Russo, Ph.D.
                                      Director
                                      Environmental Research Laboratory
                                      Athens, Georgia
                                     iii

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                                  ABSTRACT

      Scientists from eight countries presented papers at the first Interna-
tional Symposium on Fish Physiology, Fish Toxicology, and Fisheries Management,
which was held on the campus of Zhongshan University, Guangzhou, PRC.  This
proceedings includes 30 papers presented in sessions on the reproduction and
growth of fish; on the physiology, behavior and genetics of fish; and on tox-
icology and risk assessment in aquatic systems.. Papers address hormone func-
tions, sex manipulation in aquaculture, cloning and induced breeding in fish,
calcium and phosphorus requirements, fish diets and metabolism, and utilization
of inorganic nitrogen.  Descriptions are provided concerning research on gas
transfer, gill ventilation control, carbon dioxide effects, ammonia effects,
environmental acidification effects, phototaxis, sex pheromones effects, gonad
maturation, antifreeze protein genes, and bioenergetics modeling.  Presenta-
tions also covered fish conservation, water quality protection, predictions of
chronic effects levels, chemical toxicity, and toxicant modeling.
                                     IV

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                                 CONTENTS
FOREWORD	     ill
ABSTRACT	      iv
ACKNOWLEDGMENT,	  .     vii
SESSION 1.  REPRODUCTION AND GROWTH OF  FISH
            Dr. Lin Haoren, Dr. Edward  Donaldson, Dr. Yoshita
            Nagahama, and Dr.  Chen HongxiChairpersons

   Structure and Function of Gonadotropin-releasing Hormones in Fish       1
      Nancy M. Sherwood
   Manipulation of Sex in Aquaculture	      13
      Edward M. Donaldson,  Tillman J.  Benfey, and Francesc Piferrer
   Asexual Reproduction of Fish	      24
      Chen Hongxi
   Induced Breeding of Cultured Fish in China  .	      34
      H.R. Lin and R.E.  Peter
   Role of Steroid Hormones in Gonadal Growth and Maturation
      in Teleosts	      46
      Yoshita Nagahama
   Inducing Egg Cell Fusion with Laser	      62
      Ger Guo-chang,  Zhang Wen-di,  Zhau Pei,  and Wang Qu
   Testosterone and 11-Oxotestosterone Changes During an Annual
      Cycle and Induced Spawning in Blunt Snout Bream
      Megalobrama amblycephala   	      66
      Zhao Wei-xin
   Nutrient Requirements of Juvenile Allogynogenetic Crucian
      Carp, Carassius auratus gibelio  	  	      73
      He Xiqin, Jia Lizhu,  Li Zhongjie,  and Yang Yunxia
   Potential for Using Canola Meal and Oil  in Fish Diets   ......      88
      D.A.  Higgs,  J.R. McBride,  B.S. Dosanjh,  and U.H.M.  Fagerlund
   Mobilization of Body  Reserves During  Induced Gonadal Development
      in the Japanese  Eel, Anguilla japonica  (Temminck and Schlegel):
      The  Role of.the  Pituitary Gland  and Corpuscles of Stannius   .  .     108
      D.K.O.  Chan  and  E.Y.L. Lau
   Utilization of  Inorganic Nitrogen by  Tilapia nilotica   	     128
      Wang Ye  Qiang and  Shu Xue Bao

SESSION 2.  PHYSIOLOGY,  BEHAVIOR AND GENETICS OF FISH
            Dr. Robert Thurston, Dr.  Wu Shuisun,  Dr. Christopher
            Wood,  and Dr.  Edward TaylorChairpersons
   Oxygen, Carbon Dioxide and Ammonia Transfer Across Teolist
      Fish Gills     	,	
135
      David Randall
                                     v

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                                                                        Page

    Control of Ventilation in Fish	     146
       E.W. Taylor and D.J.  Randall
    Accumulation of Carbon Dioxide in Fish Farms  with Recirculating
       Water	     157
       J.F. Steffensen and J.P.  Lomholt
    Impact of Environmental Acidification on Gill Function in Fish  .     162
       Chris M.  Wood and D.  Gordon McDonald
    Ammonia Toxicity to Fishes  	     183
       Robert V. Thurston
    Environmental Tolerance of Some Marine Fish:   Applications in
       Mariculture Management	 .     197
       R.S.S. Wu
    Studies on Physiology of Fish and Marine Animals  in China ....     208
        Daren He
    Identified  Sex Pheromones in Fish:  Endocrine and Behavioral
       Effects	    216
       Norman E. Stacey and Peter W. Sorensen
    Effect of Female-specific Protein Compound Treatment of Hatching
       Rate of Fish Larval and Fish Gonad Maturation (Proteins
       Engineer I)  	    228
       Wang Hao and Liu Rongzhen
    Antifreeze Protein Genes:  Physiological Regulation and Potential
       Value to the Genetic Engineering of Freeze Resistant Fish   . .    230
       Garth L. Fletcher, Margaret A. Shears, Madonna J. King,
       Ming H. Kao, Peter L. Davies, and Choy L.  Hew
    Bioenergetics Modeling of Fish Growth   .... 	 ....    248
       Cui Yibo and R.J. Wootton

SESSION 3.  TOXICOLOGY AND RISK ASSESSMENT
            Dr. Ding Shurong, Dr. Rosemarie Russo, Dr. Jeffrey
            Black, and Dr. Zhang YongyuanChairpersons
    Role of Science and Technology in Conservation of the Sturgeon
       Resource   	
       Liu Jiankang and Yu Zhitang
    Protection of Water Quality in the United States  	
       Rosemarie C. Russo
    The Fish Embryo-larval Procedure:  Predicting Chronic Toxicity
       and Ecological Effects   	 .
       Jeffrey A. Black and Wesley J. Birge
    Toxicity of Fenvalerate to Six Species of Fish and Two Species
       of Fishfood Organisms	~	
       Ding Shurong, Zhou Fengfen, and Zhang Min
    Modeling the Effects of Toxicants on Fish Populations   .  .  . .
       Thomas G. Hallam, Ray R.  Lassiter, Jia Li, and William
       McKinney
    Toxicity of Isoprothiolane to Fishes  	
       Zhai Liangan, Zhao Xiaochun, Yao Alqin, and Li Jifang
    Fish Enzymatic Indicators in Aquatic Toxicology 	
       Xu Lihong, Zhang Yongyuan, and Wang Deming
    River Network Water Quality Modeling Using the Enhanced Stream
       Water Quality Model QUAL2E   	
258

262


270


284

299


321

327


337
       Qian Song
                                    vi

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                             ACKNOWLEDGMENTS

     Organizing and presenting a symposium and preparing a proceedings
is frequently a complex task, particularly when participants represent
organizations in several countries. Sponsoring organizations for this
symposium include the National Science Foundation of the Peoples' Re-
public of China; Zhongshan University; the U.S. Environmental Protec-
tion Agency; the Institute of Hydrobiology, Academy of Sciences of
China; the Canadian Society of Zoology; and the American Fisheries
Society.  The scientists, engineers, and environmental managers who
participated in the symposium, of course, are deserving of primary
recognition.  In particular, recognition is accorded to Dr. Zhang
Chunxiang, Vice President, Zhongshan University, and Dr. Liu Jiankang,
Honorary Director, Institute of Hydrobiology, Academy of Sciences of
China, and to the session chairpersons.  The latter group includes Dr.
David Randall, Dr. Lin Haoren, Dr. Edward Donaldson, Dr. Yoshitaka Na-
gahama, Dr. Chen Hongxi, Dr. Robert Thurston, Dr. Wu Shiusun, Dr. Chris-
topher Wood, Dr. Edward Taylor, Dr. Ding Surong, Dr. Rosemarie Russo, Dr.
Jeffrey Black, and Dr. Zhang Yongyuan.  Special appreciation is expressed
to Ms. Yvonne Hohe, Ms. Mimi Houston, Ms. Annie Smith, Ms. Martha Wilkes,
and Ms. Patricia Wyatt for applying their considerable word processing
skills to the preparation of the proceedings.

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viii

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                   STRUCTURE AND FUNCTION OF GONADOTROPIN-
                         RELEASING HORMONES  IN FISHES

                                      by

                              Nancy M.  Sherwood1
                         ENVIRONMENT, BRAIN, AND GnRH

     The brain is an important organ for the control of reproduction in all
vertebrates including the most primitive fishes.  In the brain, a number of
factors are produced that may ,affect reproduction, but the factor with the
most direct control is gonadotropin-releasing hormone (GnRH).   In fishes as in
other vertebrates, this hormone is a peptide of ten amino acids synthesized in
a few specialized nerve cells.  One function of GnRH after it is released from
the neurons is stimulation of the pituitary to synthesize and release gonado-
tropin hormones.  These pituitary hormones, in turn, activate the gonads.
Hence, GnRH is essential for internal control of reproduction.  This decapep-
tide also may be a critical node for some environmental effects on reproduc^-
tion in fishes.  A number of environmental factors that act on the nervous
system through the senses may inhibit the release of GnRH.  One supporting
argument for the relationship between the environment and GnRH inhibition is
that administration of exogenous GnRHs has been successful for the induction
of breeding in cultured fish that will not spawn in captivity.


                        SEVERAL FORMS OF GnRH IN FISHES

     GnRH has been detected in a variety of vertebrates from lamprey to
humans.  The evidence to date suggests the hormone is always ten amino acids
with the same end groups, but the individual amino acids within the molecule
vary.  The primary structure of GnRH has been determined for the five mole
cules shown below (Burgus et al. 1972; King and Millar 1982a,b; Matsuo et al.
1971; Miyamoto et al. 1982, 1983, 1984; Sherwood et al. 1983, 1986a).
Part of the structure of the molecule has been retained for millions of years.
The conserved portion is underlined  and includes the amino acids 1,  2, 4, 9,
10, and the G-terminal amide.
 Biology Department, University of Victoria, Victoria BC, Canada

                                       1

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                    123456789    10

        Lamprey  pGlu-His.-Tyr-Ser-Leu-Glu-Trp-Lys-Pro-Gly-NHg

         Salmon  pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2

     Chicken II  pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2

     Chicken I  pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-giro-Gl^-NH?

         Mammal  pGlu-Hig-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
     Other forms of GnRH  also  exist, but  their primary sequence  is not yet
known.  These other forms can  be detected, however, with cross-reacting
antisera that were made against one of the known forms.  Many of these
immunoreactive  (ir) GnRHs can  be separated from one another by chromatography,
such as high performance  liquid chromatography (HPLC).  The conclusion of
these studies is that fishes contain several forms of GnRH, some of which are
shared with higher vertebrates (Table 1).  Also, more than one form of GnRH
can exist in a  particular fish species and, indeed, this is the  common pattern
with only a few exceptions.
                        DISTRIBUTION OF GnRHs IN FISHES

     In addition to the salmon and lamprey forms of GnRH shown above, there
are several other forms, outlined in Table 1, for the fish studied to date.
The evidence varies, however, in strength and this is indicated.  For some
peptides, the primary structure has been determined (++++), but for others
only the HPLC elution position and cross-reactivity with a nonspecific GnRH
antiserum have been observed (+).

     A partial phylogenetic map for GnRH has begun to emerge from the dis-
tribution studies to date.  Lamprey GnRH has not been detected in other
fishes.  A chicken II-like form of GnRH is present in cartilaginous fish and
in small amounts in many bony fishes, but the phrase "chicken II-like" means
only that the molecules react with a nonspecific GnRH antiserum and have the
same HPLC mobility as chicken II GnRH.  It is not known whether the structures
are identical with chicken II GnRH or among the fishes.  The mammalian form of
GnRH has been detected in primitive bony fish with a GnRH antiserum specific
for the mammalian form.  It also has been observed with a nonspecific GnRH
antiserum (cross reacts with more than one form of GnRH) in two teleosts (eel
and winter flounder),  but otherwise does not appear to be present in teleosts
studied to date.  Rather,  the mammalian form is detected in tetrapods such as
amphibians and man.   Finally, the salmon form is present in the teleosts
studied to date except for the African catfish,  which appears to have its own
form.   The chicken I-like form has been reported for only three species of
teleosts.   Other forms also may be present in fish species not yet studied.

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TABLE 1.  GnRH IN FISHES
Species
GnRH form
                                 L-I   L-II
                 C-II  C-I   M  Novel
                                                                        Ref.
hagfish (Eptatretus stouti)
lamprey (Petromyzon marinus)      Mil  +++

ratfish (Hydrolagus colliei)
dogfish (Squalus aca-nthias)
dogfish (Poroderma africanum)                 +

reedfish  (Calamoichthys
  calabaricus)
sturgeon  (Acipenser
  transmontanus)
alligator gar  (Lepisosteus
  spatula)
bowfin  (Amia calva)

moray eel (Gymnothorax                        +
  fimbriatus
herring (Clupea harengus                      +
  pallasi)
milkfish  (Chanos chanos)                      +
goldfish  (Carassius auratus)                  +
catfish (Glarias gariepinus)
salmon  (Oneorhynchus  keta)                   ++-H
trout  (Salmo gairdneri)                       +
hake (Merluccius capensis)                    +
codfish (Gadus morhua morhua)                 +-H
molly  (Poecilia latipinna)                    +
snook  (Centropomus undecimalis)               +
sea bass  (Centropristis  striatus)             +
mullet  (Mugil  cephalus)                       +
tilapia (Tilapia sparrmanii)                  +
wrasse  (Coris  julis)                          +
winter  flounder  (Pseudo-                      +
  pleuronectes americanus)
                                       1
                                       2

                                       3
                                       1
                                       4

                                       5

                                       5

                                       5
                                       7

                                       8

                                       9
                                       10,11
                                       12
                                       13,14
                                       15
                                       4
                                       16
                                       17
                                       17
                                       17
                                       9
                                       4
                                       4
                                       18
 (1)  Sherwood & Sower,  1985;  (2) Sherwood et al.,  1986a;  (3) Lovejoy & Sher-
 wood,  1989;  (4) Millar & King,  1987;  (5) Sherwood et al.  1988a;  (6) Grim et
 al.,  1985;  (7) Shih et al.,  1988;  (8) Sherwood,  1986;  (9) Sherwood et al.,
 1984;  (10)  Sherwood & Harvey,  1986;  (11) Yu et al.,  1988; (12) Sherwood et
 al.,  1989;  (13) Sherwood et al., 1983;  (14) Sherwood,  1987b; (15) Sherwood et
 al.,  1986b;  (16) Wu et al.,  1986;  (17)  Sherwood & Grier,  unpubl.; (18) Idler &
 Everard,  1987.  Table 1 modified from Sherwood & Lovejoy, Fish Physiol.
 Biochem.  (in press).  Reprinted with permission.

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          STRUCTURAL VARIATION OF GnRH WITHIN A SINGLE SPECIES OF FISH

      Table 1 shows that most fish species have more than one form of GnRH.
 The amino acid composition of the minor form (II) of GnRH in lamprey is known
 and differs in three amino acids from the major form (I).  The other second
 forms of GnRH in fishes have been identified by immunological and chromato-
 graphic methods, but it is known that the second form is not a metabolic
 product that has lost its amide or been cleaved because such degraded forms
 are not recognized by some of the antisera.  The structures of the second
 forms need to be determined by sequencing of the amino acids or DNA nucleotide
 bases.  The quantity of GnRH in the brain is very small so that analysis is
 done on pools of brains.  Therefore, it also is not known whether multiple
 forms of GnRH are in individual fish or in the pools of fish.  The relative
 quantities of the various forms of GnRH in a species can be measured accurate-
 ly only by amino acid analysis; immunological measurements vary greatly
 depending on the percent cross-reactivity of an antiserum with a particular
 form of GnRH.   In goldfish,  for example,  the quantity of the two GnRH forms
 varied considerably in the radioimmunoassays (RIA)  depending on the form of
 GnRH that was used for antiserum production, RIA tracer,  and RIA standard (Yu
 et al. 1988).
             RELATIONSHIP OF GnRH STRUCTURE AND RELEASING FUNCTION

      Alterations in the GnRH molecule change  its  potency for  releasing
 gonadotropins  and its  binding  affinity to  the pituitary receptor.  Amino  acids
 1-3,  especially His2 and Trp3,  are essential for the release of gonadotropins
 (GtH), whereas amino acids  4-10  are  important for receptor binding in mammals
 (Burgus  et al.  1973, Schally and Coy 1977, Rivier et  al.  1981).  Analogs  with
 substitutions  in 2 and 3 positions bind competitively with the pituitary
 receptor,  but  lack releasing ability.

      The native forms  of the five GnRHs have  been compared for potency  in only
 a  few species  of fish.  Lamprey  GnRH was shown indirectly to  release  gonado-
 tropins  in lamprey; changes  in the gonadal steroid levels were measured (Sower
 et al. 1987) because the lamprey gonadotropins  have not been  characterized for
 use in RIA.

      The effect of lamprey GnRH  has  been observed in  the  goldfish where the
 peptide  was equipotent  with  the  salmon and mammalian  forms in vivo (Peter et
 al. 1985,  1987).   The other  forms of GnRH show  considerable overlap in potency
 in the various  fish tested.  Zohar et  al. (1989)  found  that mammalian and
 salmon GnRH were  equally effective both in vivo and in vitro  in the seabream,
 Sparus aurata.  Likewise, Van  Der Kraak et al.  (1987) found the same two
 peptides were equally effective  in vivo  in salmon.  Salmon GnRH was 10 times
 more potent than mGnRH, however, as  a  threshold dose for release of gonado-
 tropin from pituitary fragments  in goldfish (MacKenzie et al.  1984).

     In  the sterlet fish, which normally do not ovulate in captivity,  mam-
malian and chicken I GnRH did not advance ovulation, whereas salmon GnRH
 induced  30% to ovulate  (Horvath et al.  1986).   Salmon and mammalian GnRH (100
ugAg) .  but not chicken I or II GnRH, resulted in elevated gonadotropin levels

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in-testosterone-treated juvenile trout (L.W. Grim, pers. comm.)-  Further
testing of the native forms of GnRH including the lamprey form are needed in
fish species .that are quite separate in evolutionary terms.  But the mam-
malian, salmon and chicken forms appear to show more overlap of potency in
fish compared with"mammals.

     Part of the explanation for differing potencies of molecular forms of
GnRH in a particular species may be their affinities for the species receptor.
The native GnRHs differ primarily in positions 5-10 so that variation in
receptor binding is predicted.  Observations support the prediction, in that
binding affinity of GnRHs with rat anterior pituitary membranes is 100% for
mammalian GnRH, but only 13% for chicken II GnRH, 5% for salmon GnRH, and less
than 1% for chicken I and lamprey (Millar et al.  1986, Sherwood et al. 1983).
Receptor binding studies in the fish pituitary have been reported recently for
goldfish (Habibi et al. 1987), catfish (De Leeuw  et al. 1988), and winter
flounder (Grim et al. 1988).  Comparison of mammalian GnRH and salmon GnRH in
winter flounder showed they were equally effective in displacing a GnRH analog
bound  to the pituitary receptors.  Most deductions about nonmammalian GnRH
receptors, however, are based on comparison of releasing potency among native
GnRHs.  Information on the relative potency and binding affinity for each GnRH
is needed for different fish species to determine whether  structural changes
have evolved in the GnRH receptors in fish.


                           GnRH ANALOGS AND FUNCTION

     The relationships between the structure of native  forms of GnRH and
releasing function can be  altered considerably by making artificial forms
 (analogs) of GnRH.  Few analogs made with natural amino acids are more potent
than the native forms, although  [Phe7] chicken I  GnRH is equal  to salmon GnRH
for induced ovulation  in sterlets  (Horvath  et al. 1986) and there are five
agonists made with natural L-amino acids that have activity equivalent or
greater  than mGnRH  in  releasing LH and/or FSH in  rat assays  (Folkers et al.
1986).  A number  of GnRH analogs made with  unnatural or D  forms of  amino acids
are potent  in  fish  for release of GtH  or induction of ovulation/spermiation.
Two of the  most active and commonly used analogs  for fish  are shown below.


                       123456      789   10

    Mammalian  GnRH   pGlu-His-Trp-Ser-Tyr-Gly   -Leu-Arg-Pro-Gly-NH2

   agonistic analog   pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NH-CH2-CH3
        Salmon GnRH   pGlu-His-Trp-Ser-Tyr-Gly  -Trp-Leu-Pro-Gly-NH2

   agonistic analog   pdn-Hi Q-T-rr-Sp.r-Tyr-n-Ar-Trp-Leu-Pro-NH-CH2-CH3

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 Other analogs that are more active than the native GnRHs in fish (see Grim et
 al.  1987,  Grim.  (pers. conun.),  Horvath et al.  1986,  Van Der Kraak et al.  1987,
 Zohar et al.  1989) are shown in Table 2.
 TABLE 2.  ACTIVE GnRH ANALOGS IN FISHES.

Type of
GnRH analog
Mammalian
analogs
pGlu-His-Trp-Ser-Tyr-D-Trp
pGlu-His-Trp-Ser-Tvr-D-hArgfEto)
pGlu-His-Trp-Ser-Tyr-D-Leu
pGlu-His-Trp-Ser-Tyr-D-Naim
pGlu-His-Trp-Ser-Tvr-D-Trp
pGlu-His-Trp-Ser-Tyr-D-Ala
pGlu-His-Trp-Ser-Tyr-D-TLe
pGlu-His-Trp-Ser-Tvr-D-hArg(Eto')
pGlu-His-Trp-Ser-Tvr-D-SerYBu11)
pGlu-His-Trp-Ser-Tyr-D-His-fimBzl")
pGlu-His-Trp-Ser-Tvr-D-Naim
-Leu-Arg-Pro-Gly-NH2
-Leu-Arg-Pro-Gly-NH2
-Leu-Arg-Pro-Aza-Gly-NH2
-Leu-Arg-Pro-Aza-Gly-NH2
-Leu-Arg-Pro-NH-CH2-CH3
-Leu-Arg-Pro-NH-CH2-CH3
-Leu-Arg-Pro-NH-CH2-CH3
-Leu-ArE-Pro-NH-GH2-CH3
-Leu-Arg-Pro-NH-CH2-CH3
-Leu-ArE-Pro-NH-CH2-CH3
-Leu-Arg-Pro-NH-CH2-CH3
 Salmon
   analogs
pGlu-His-Trp-Ser-Tvr-D-hArg(Et2')
pGlu-His-Trp-Ser-Tyr-D-Phe
pGlu-His-Trp-Ser-Tyr-D-Ala
pGlu-His-Trp-Ser-Tyr-D-Arg
pGlu-His-Trp-Ser-Tvr-D-Trp
pGlu-His-Trp-Ser-Tvr-D-hArg(Et2')
-Trp-Leu-Pro-Gly-NH2'
-Trp-Leu-Pro-Gly-NH2
-Trp-Leu-Pro-NH-CH2-CH3
-Trp-Leu-Pro-NH-CH2-CH3
-Trp-Leu-Pro-NH-CH2-CH3
-Trp-Leu-Pro-NH-CH2-CH3
Chicken I
  analogs
pGlu-His-Trp-Ser-Tyr-D-Trp
pGlu-His-Trp-Ser-Tyr-D-hArgCEto")
pGlu-His-Trp-Ser-Tyr-D-Phe
pGlu-His-Trp-Ser-Tyr-D-Phe
pGlu-His-Trp-Ser-Tyr-Gly-
-Leu-Gln-Pro-Gly-NH2
-Leu-Gln-Pro-Gly-NH2
-Leu-Gln-Pro-Gly-NH2
-Leu-Gln-Pro-NH-CH2-CH3
-Phe-Gin-Pro-NH-CH2-CH3
Chicken II       pGlu-His-Trp-Ser-His-D^
  analogs        pGlu-His-Trp-Ser-His-D-hArgfEt2')
                                   -Trp-Tyr-Pro-Gly-NH2
                                   -Trp-Tyr-Pro-Gly-NH2
     The analogs appear to be more resistant to enzymatic breakdown  (Zohar et
al. 1989) and hence are effective in the body for a long time.  They  either
bind with higher affinity or are more resistant to degradation.  In  practical
terms, the analogues are long lasting and a number of them, regardless of

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whether fashioned on the backbone of the salmon, mammalian or chicken GnRHs,
are effective in fish.  Antagonistic GnRH analogs have been made for mammals,
but few for fish.  An example of an antagonist for lamprey is shown below:
       Lamprey GnRH
antagonistic analog
                                                          8
                                            10
pGlu-His  -Tyr  -Ser-Leu-Glu  -Trp-Lys-Pro-Gly-NH2
pGlu-D-Phe-D-Pro-Ser-Leu-D-Phe-Trp-Lvs-Pro-Gly-NH2
Prevention of reproduction can be a considerable advantage in fish such as
lamprey that destroy commercially available fish or in teleost fish that would
continue to grow if reproduction were blocked.  In both cases, however, large
numbers of fish would have to be treated.  Administration of the peptide in
water would be required, but a cost-efficient method has not been developed.
                   ASSESSING REPRODUCTIVE FUNCTIONS OF GnRH

     The use of GnRH for induced breeding has now been tested in a variety of
fish.  These fish span all the categories of cold freshwater fish, cold water
marine fish, warm freshwater fish, and warm water marine fish.  Use of GnRH in
fish from these categories is considered in detail in other papers in this
symposium.  To illustrate the use of GnRH analog forms, the warm water marine
sea bass will be considered.  The sea bass study shows the use of two dif-
ferent GnRH analogs and the importance of time, dose, and method of adminis-
tering GnRH.  Native GnRH is quickly metabolized; the half life in goldfish is
only 20 minutes (Sherwood and Harvey 1986).  Therefore, the method of ad-
ministering the peptide is important.

     The sea bass, Lates calcarifer. undergoes sex reversal from male to
female at about 4 or 5 years of age.  Spawning occurs in coastal waters and
males probably remain there, but females return to inland fresh water after
spawning.  Two GnRH analogs were compared in individual sea bass pairs to
determine their effectiveness in fish capable of multiple spawning (Almendras
et al. 1988).  The fish were reproductively mature.  The two analogs were  [D-
Ala6, Pro9 ethylamide]  mammalian GnRH and [D-Arg6, Pro9 ethylamide] salmon GnRH
as shown in the section above (GnRH analogs and function).  Prior to the
comparison, one of the analogs was used to test the best method of administra-
tion.  An injection of 60 to 100 ug of the D-Ala6 analog induced a single
spawning in fish, but multiple injections spaced 24 hours apart produced 1 to
4 spawnings in individual females.  The same analog also was placed in an
Alzet osmotic pump and implanted intraperitoneally in selected fish.  The pump
was  prepared so that only 9 ug of analog was released each day for 14 days.
The  result was that three fish spawned four times each on successive days and
one  fish spawned  five times.

     A similar effect was produced by embedding the analogs  (100 ug) in a
pellet made of cholesterol and cellulose.  Two pellets with analog in a matrix
of at least 95% cholesterol produced 1 to 4 spawnings on successive days in
individual  sea bass.  This slow release method of GnRH administration in
pellets appears to be an appropriate way to compare the structure-function
relationship of analogs because a number of effects can be observed:  number

-------
 of multiple spawnings/fish,  fecundity,  fertility rate,  hatching rate and
 rematuration of the broodstock.   The analogs were roughly equivalent in that
 both induced multiple spawnings  in individual females.   Each fish released 3
 to 7 million eggs with 40 to 70% fertility and 8 to 63% hatching rate.   In
 addition,  the same sea bass  that were induced to spawn  on several successive
 days in June could be induced to have multiple spawnings again in September
 with 1 to  3 million eggs, 36 to  86% fertile eggs and 32 to 81% hatched  larvae
 (Almendras et al.  1988).

      In conclusion,  the relationship between structure  and function of  GnRHs
 and their  analogs  depends on the species  of fish tested,  reproductive state,
 reproductive pattern of the  fish,  and the method by which the hormones  are
 administered.   Also a number of  factors need to be evaluated after induction.
 It appears from this and other studies mentioned above  that some analogs based
 on the structure of the mammalian,  chicken,  or salmon GnRHs are interchange-
 able in fish in terms of potency.
                        GnRH AND NON-RELEASING  FUNCTIONS

     The  functions  of  GnRHs have been  evaluated primarily by their releasing
function  for  the  gonadotropins.  Recently,  it  has become clear that GnRH also
has other functions.   GnRH nerve fibers  in  fish are widely distributed in the
brain  in  addition to terminating in the  pituitary.  GnRH receptors have been
detected  in nonhypothalamic regions of the  brain in rats, but have not been
reported  for  fish.  The distribution of  GnRH axons and receptors support the
idea that GnRH may  be  a neurotransmitter in the brain.  A separate collection
of GnRH neurons near the olfactory bulb  in  most fish have terminal axons in
the forebrain and in some species in the retina.  Three suggestions for their
function  are  (1)  transduction of olfactory  stimuli to reproduction behavior,
(2) induction of  pituitary gonadotrope maturation, and (3) modulation of
vision (for reviews see Sherwood 1987a,c)

     There is strong evidence that a nonmammalian form of GnRH is a neuro-
transmitter or modulator in the lumbar sympathetic ganglia of the bullfrog;
this has  not been reported for fish.   Similarly, the enhancement of sexual'
receptivity has been reported for other  vertebrates, but not yet studied in
fish.  The receptivity  evidence was obtained by application of GnRH directly
to the brain  to avoid effects secondary  to  gonadotropin and steroid release.
Finally,  GnRH neurons in the midbrain have  axons that travel in the spinal
cord to terminate on the caudal neurosecretory neurons (Miller and Kriebel
1986) .  It is hypothesized that the GnRH fibers modulate the release of a
secretion controlling contractility of oviducts and sperm ducts.

     The  story of GnRHs  and their functions will not be complete until these
novel functions have been elucidated for fish.   The relative potency and
binding to nonpituitary  receptors of the GnRHs may reveal that the functions
of GnRH are as diverse as the structures of this peptide family.

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Miyamoto,  K.,  Y. Hasegawa, M. Igarashi, N. Chino,  S.  Sakakibara,  K. Kangawa
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                                     10

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Miyamoto, K.,  Y. Hasegawa, M. Nomura, M. Igarashi, K. Kangawa, and H. Matsuo.
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Peter, R.E., C.S. Nahorniak, S. Shih, J.A. King, and R.P. Millar.  1987.
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Peter, R.E., C.S. Nahorniak, M. Sokolowska, J.P. Chang, J.E. Rivier, W.W.
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     ships of mammalian,  chicken, and salmon gonadotropin releasing hormones
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Rivier, J., C. Rivier, M. Perrin, J. Porter, and W.W. Vale.  1981.  In:  LHRH
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Schally, A.V., and D.H. Coy,  1977.  Stimulatory and inhibitory analogs of
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Sherwood,  N.M.  1986.  Evolution of  a neuropeptide family:  Gonadotropin-
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Sherwood,  N.  1987b.  Gonadotropin-releasing hormones in  fishes.  In:
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     pp.  31-60.

Sherwood,  N.M.   1987c.  Brain peptides  in the  control of  fish reproduction.
      In:   Reproductive  Physiology of Fish 1987.   Proceedings  of  the  Third
      International  Symposium on the  Reproductive  Physiology of Fish, St.
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Sherwood,  N.M.  and  B. Harvey.   1986. Topical  absorption of gonadotropin-
      releasing  hormone  (GnRH)  in goldfish.   Gen.  Comp. Endocrinol.   61:13-19.

Sherwood,  N.M.  and  D.A.  Lovejoy.  1989.   The origin  of the  mammalian form of
      GnRH in  primitive  fishes.   Fish Physiol.  Biochem. (in  press).

Sherwood,  N.M.  and  S.A.  Sower.   1985.   A  new family  member  for gonadotropin-
      releasing hormone.   Neuropeptides.   6:205-214.
                                      11

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 Sherwood, N.M., R. De Leeuw, and H.J.Th. Goos.  1989.  A new member of the
      gonadotropin-releasing hormone family in teleosts:  Catfish GnRH.
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 Sherwood, N.M., S.I. Doroshov, and V. Lance.  1988a.  Gonadotropin-releasing
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      (Lepisosteus spatula).  (Submitted).

 Sherwood, N.M.-, L. Eiden,  M.  Brownstein, J.  Spiess,  J.  Rivier,  and W.  Vale.
      1983.  Characterization of a teleost  gonadotropin-releasing hormone.
      Proc. Natl. Acad.  Sci. USA.   80:2794-2798.

 Sherwood, N.M., B. Harvey,  M.J.  Brownstein,  and L.E.  Eiden.   1984.   Gonado-
      tropin-releasing hormone  (Gn-RH)  in striped mullet (Mugil  cephalus),
      milkfish (Chanos Changs'),  and rainbow trout (Salmo eairdneri') :
      Comparison with salmon Gn-RH.   Gen. Comp.  Endocrinol.   55:174-181.

 Sherwood, N.M., S.A.  Sower, D.R.  Marshak,  B.A.  Fraser,  and M.J.  Brownstein.
      1986a.   Primary structure  of gonadotropin-releasing hormone from  lamprey
      brain.   J. Biol. Chem. 261:4812-4819.

 Sherwood, N.M. , R.T.  Zoeller, and F.L.  Moore.   1986b.   Multiple,  forms  of
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      crinol.   61:313-322.

 Shih, S.H., K.-L.  Yu, and R.E.  Peter.   1988.  Molecular forms of gonadotropin-
      releasing hormone  in the brain  areas  of  Gymnothorax fimbriatus  (Bennett).
      First International Symposium on  Fish Endocrinology,  Program and
      Abstracts,  June  12-17, 1988.  Univ. Alberta, Edmonton, AB,  Canada,  p.
      44.

 Sower, S.A. ,  J.A.  King, R.P. Millar, N.M.  Sherwood, and D.R. Marshak.  1987.
      Comparative biological properties  of  lamprey gonadotropin-releasing
      hormone  in vertebrates.  Endocrinology 120:773-779.

Van Der Kraak,  G., E.M. Donaldson, H.M. Dye, G.A. Hunter, J.E. Rivier, and
     W.W.  Vale.  1987.  Effects of mammalian and salmon gonadotropin-releasing
     hormones  and  analogues, on plasma gonadotropin levels and ovulation in
      coho salmon (Oncorhynchus kisutch).  Can. J. Fish. Aquat  Sci   44-1930-
      1935.

Wu, P.,  J.F. Ackland, N. Ling,  and I.M.D. Jackson.  1986.  Purification and
     characterization of luteinizing hormone-releasing hormone from codfish
     brain.  Regul. Pept.   15:311-321.

Yu, K.L., N.M.  Sherwood, and R.E. Peter.  1988.  Differential distribution of
     two molecular forms of gonadotropin-releasing hormone in discrete brain
     areas of  goldfish (Carassius auratus).  Peptides 9:625-630.

Zohar, Y. , A.  Goren, M.  Tosky,  G. Pagelson, D. Leibovitz, and Y.  Koch.   1989.
     The bioactivity of gonadotropin releasing hormones and its  regulation in
     the gilthead seabream,  Sparus aurata:   in vivo and in vitro studies.
     Fish Physiol. Biochem.   (in press).
                                      12

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                      MANIPULATION OF SEX IN AOUAGULTURE

                                      by

       Edward M. Donaldson1, Tillman J. Benfey2,  and Francesc Piferrer3


                                 INTRODUCTION

     The vigourous pursuit of techniques for the control of sex in cultured
teleosts over the last two decades saw its genesis in the publication of a
significant review  by Yamamoto (1969).  This review laid the foundation on
which our current, as yet incomplete, understanding of sex differentiation in
fish is based.  At the time of Yamamoto's review, there had been no report of
the application of hormonal treatment or chromosome set manipulation for sex
control in fish culture.  In fact, Bardach et al. (1972), in their text on
aquaculture, reported only on the application of interspecific hybridization
for the production of monosex stocks.  Since that time, much research activity
has occurred both in the development of methods for the hormonal regulation of
sex (for reviews, see Donaldson and Hunter 1982, Guerrero 1982, Hunter and
Donaldson 1983, Pandian and Varadaraj 1987, Piferrer and Donaldson 1988) and
for the genetic regulation of sex (for reviews, see Purdom 1983, 1986;
Thorgaard 1983, 1986; Benfey and Donaldson 1988).  Additional reviews discuss
both hormonal and genetic sex control and the integration of these techniques
(Stanley 1981, Yamazaki 1983, Donaldson 1986, Donaldson and Benfey 1987).

     This review discusses the current status of sex control techniques
focussed at specific end points, i.e., the production of all female, all male
and sterile groups of fish.  We will close with a summary of the current
status of the application of these techniques in fish culture.  The main
emphasis will be on Pacific salmonids, with reference to other  species where
appropriate.
 "West Vancouver Laboratory, Department of Fisheries and Oceans, West Vancouver
  BC,  Canada

 2MAFF, Directorate of Fisheries Research, Fisheries Laboraotry, LOWESTOFT
  Suffolk,  England

 3Consejo Superior de Investigaciones Cientificas,Institute de Acuicultura de
  Torre  de  La Sal, Ribera de Cabanes  Castellon,  Spain

                                      13

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                      PRODUCTION OF MONOSEX FEMALE STOCKS
 DIRECT FEMINIZATION
      This  procedure  involves  treatment of fish during early development with a
 natural  or synthetic estrogen (Figure 1).  Yamamoto (1969) postulated that
 treatment  must be  initiated prior to visible morphological sex differentia-
 tionand  continue until  sex differentiation is evident.  Treatment timing thus
 will  vary  between  species that differentiate early and those that differen-
 tiate later.  In a recent study in our laboratory in which coho salmon
 (Oncorhynchus kisutch)  eyed eggs, alevins and fry received single immersion
 treatments of 400  ug/L  17ft -estradiol at 10C, the most sensitive period to
 estradiol,  measured  by  the percentage of females in the resultant fry,
 occurred around the  time of hatching (Piferrer and Donaldson 1989).   By
 providing  two or more immersion treatments at intervals of approximately 1
 week  at  10C, it has been possible in experimental studies to obtain a high
 percentage of females in the  resultant offspring (Hunter et al. 1986);
 however, attempts  to utilize  the technique at a production scale at commercial
 hatcheries have provided variable results.  It should be noted that estrogens
 inhibit  growth at  moderate dosage levels and are toxic at higher levels.


 MONOSEX  FEMALE SPERMATOZOA

      In  many teleosts,  including for example the salmonids and the carps,  the
 female is homogametic and the male heterogametic.   Thus,  if monosex  female, X
 chromosome bearing, spermatozoa are used to fertilize  normal ova,  all the
 offspring  are normal females.   Currently, two methods  are used to  produce
 female spermatozoa, both of which involve sex reversal of genotypic  female
 embryos  into phenotypic males and subsequent grow-out  to  mature phenotypic
males that produce monosex female spermatozoa.

The two procedures are:

 1.  Masculinization of mixed sex larvae.  This  procedure,  which was  the
     first to be utilized on a practical  scale,  initially involves the
     masculinization (see below)  of larvae of mixed sex.   These mas-
     culinized larvae are then grown,to  sexual maturity and the sperma-
     tozoa from each phenotypic male are  used to fertilize separate
     batches of ova.   The eggs fertilized by each male  are then incubated
                             EYED EGG/ALEVIN


                          ESTROGEN 1  IMMERSION


                            PHENOTYPIC  FEMALE
                Figure 1.-  Direct feminization by estrogen
                  treatment.
                                    14

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separately and a portion of the  larvae from each batch  are mas-
culinized and grown on.  The remainder of the larvae from each batch
are grown to a size where they can be sexed visually or by histol-
ogy.   Fish from the groups of masculinized larvae are discarded if
their untreated counterparts are of mixed sex; masculinized fish
resulting from matings that gave 100% females in the progeny test
are retained.  The retained groups of masculinized fish are geno-
typic females that have a male phenotype.  At maturity, the sperma-
tozoa they produce are monosex female and can be utilized to
fertilize normal ova and produce genotypically and phenotypically
normal female offspring (Figure  2).

The procedure just described involves two generations of rearing
except in the special case of some studies in rainbow trout where
                       NORMAL  ALEVIN


                   ANDROGEN 1 IMMERSION


                      PHENOTYPIC MALE


              NORMAL  OVA X SPERM
     SEX RATIO                 ANDROGEN

  DETERMINATION              TREATMENT

 IN  SUBSAMPLE OF

   EACH MATING                 RETAIN

                            PHENOTYPIC MALE

                            GENOTYPIC FEMALES
                                            \
                           NORMAL  OVA X  SPERM
                          100% FEMALE OFFSPRING
  Figure 2.  Production of monosex female sperm, two gen
    eration technique (reproduced with permission from
    Donaldson and Benfey 1987).
                           15

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     adult masculinized trout of mixed genotype were sorted on the basis
     of whether the sperm  duct had developed or not.  In this  procedure,
     masculinized trout having no sperm duct were regarded as  being
     genotypic females  (Bye and Lincoln 1986).

2.   Masculinization of gynogenetic larvae.  This technique is being
     developed to obviate  the need for progeny testing after
     masculinization thus  reduce the process of producing monosex
     spermatozoa to one generation.  In this procedure,  haploid gynogene-
     sis is induced by  fertilization with UV-irradiated sperm.   Diploidy
     then is restored by application of a temperature,  pressure or
     chemical shock that prevents the separation of the second polar body
     (Thorgaard 1983, Donaldson and Benfey 1987).

     The gynogenetic embryos are reared to the stage at which  sex
     differentiation is initiated and then masculinized by androgen
     treatment.   The resultant fish are phenotypic male gynogens that
     produce monosex female spermatozoa at maturity (Figure 3).   These
     gynogens will be less heterozygous, but the use of their  spermatozoa
     to fertilize normal heterozygous ova should not cause a problem of
     inbreeding.
                        GYNOGENETIC ALEVIN
                                  \
ANDROGEN  I IMMERSION
                         PHENOTYPIC MALE
                              GYNOGENE
                                *
               NORMAL OVA X SPERM
                                 \
                      100% FEMALE OFFSPRING
         Figure 3.  Production of monosex female sperm,
           gynogenetic technique.
                                16

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                      PRODUCTION OF MONOSEX MALE STOCKS

DIRECT MASCULINIZATION

     This procedure involves treatment of fish during early development  with  a
natural or synthetic androgen (Figure 4).   This can be achieved by either
feeding, as is generally the case in tilapia culture (Guerrero  1982),  or by
immersion, as is used in coho and chinook salmon.   In our earlier studies  on
Pacific salmon, we used immersion treatments of eyed eggs and alevins  followed
by feeding of fry with androgen treated diet.   This treatment has since  been
reduced in chinook and coho salmon to a single 2-hour immersion treatment  that
is administered at or just after the time of hatching (Baker et al.  1988,
Piferrer and Donaldson 1989).  In the chinook study, monosex female larvae
were used and it was possible to detect some masculinization after immersion
in as little as 20ug/L 17a methyltestosterone (Baker et al. 1988).
                              EYED EGG/ALEVIN
                                      \
    ANDROGEN  I IMMERSION
            Figure  4.
             ment.
        PHENOTYPIC  MALE

Direct masculinization by androgen treat-
      A potential problem  in  the direct masculinization procedure is the effect
 referred to  as  "paradoxical  feminization" in which the proportion of females
 increases at high  dosages of androgen (Solar et al. 1986).  In recent studies
 using a non-aromatizable  androgen, we saw no evidence of paradoxical feminiza-
 tion (Piferrer  and Donaldson 1988).


 INDIRECT PRODUCTION OF MONOSEX MALES

      The generation of monosex male spermatozoa or monosex male ova for the
 purpose' of producing all-male offspring has not been yet achieved on a
 production scale.   For species in which the male is heterogametic, the
 generation of monosex male,  Y chromosome bearing, sperm is more complex than
 the generation  of  monosex female  sperm and involves the production of YY
 supermales.  This  can be  accomplished by inducing androgenesis and using the
 monosex male sperm from androgenetic males to  fertilize normal ova and produce
 normal XY all male offspring (Figure 5).  The  disadvantage of this technique
 is the technical difficulty of inducing androgenesis.  In androgenesis, the
 egg is subjected to gamma irradiation prior  to fertilization and diploidy  is
 restored by  preventing division  of  the first cell division (Parsons and
 Thorgaard 1985).
                                      17

-------
                        PRODUCTION OF STERILIZED STOCKS

     Fish can be sterilized by a variety of techniques (Figure  6)  that vary
both in practicality and in level of successful sterilization achieved.  The
sterilization techniques that are currently being  investigated  involve
androgen treatment with androgen, production of female triploids,  and exposure
to ionizing radiation.  In the future, it also may be possible  to  produce
transgenic fish in which gonadal development is blocked (N. Maclean, pers.
comm.)
HORMONAL STERILIZATION

     The technique of hormonal sterilization is  similar  to the technique for
direct masculinization, except that the treatment  continues for a longer
period and at a higher dosage (Figure 7) .   In coho salmon, the alevins are
treated with androgen by immersion and then by dietary administration from
first feeding up to a body weight of about  2 grams.  In  laboratory studies,
high level of treatment success has been achieved  (Goetz et al. 1979, Donald-
son and Hunter 1982); but, attempts to transfer  the technology to the produc-
tion level have had variable success.   Variations  in treatment protocol in
salmon can result in incomplete sterilization, a poor rate of multiplication,
or in the occurrence of morphological deformities.
                                                                           a
TRIPLOID FEMALES
     Studies in salmonids (Benfey 1989) and in other species have shown that
oocytes in triploid female fish do not normally undergo vitellogenic growth
and that the fish retain an immature appearance.  Triploid males, on the other
                          ANDROGENETIC ALEVINS
                                  I
      NORMAL  FEMALES                             SUPERMALES
                                                         \
                                        NORMAL  OVA X SPERM
                                           100% MALE  OFFSPRING
      Figure 5.  Production of monosex male sperm by androgenesis.

                                    18

-------
hand, do undergo testicular development, become mature  in appearance, and are
capable of producing  aneuploid spermatozoa (Benfey et al. 1986).

     Thus, to produce triploid fish that are sterile in appearance as well as
in reproductive capacity, it is necessary to produce female triploids.  This
can be accomplished in a variety of ways (Figure 8) (Donaldson and Benfey
1987).   Of the various techniques shown, the following  are the most straight-
forward:  (1) fertilization of ova with monosex female  spermatozoa followed by
induction of triploidy, (2) induction of triploidy in zygotes of mixed sex
followed by estrogen  treatment of larvae,  and (3)  fertilization of normal ova
with diploid sperm  followed by estrogen treatment of larvae.  The first
procedure is preferred as it will result in the production of 100% female
triploids.  As shown  in the diagram, it should be possible, in future, to
produce tetraploids that produce diploid monosex female sperm.

IONIZING RADIATION

     Studies in Japan on the effects of ionizing radiation on development in
fish indicated that irradiation during sex differentiation may cause selective
damage to the germ  cells and thus induce sterility (Egami and Ijiri 1979).
Recently, sterility was induced in Atlantic salmon (Salmo salar) by gamma
irradiation at the  eyed embryo stage (Thorpe et al. 1987).
      ANDROGEN TREATMENT

      DURING  ALEVIN AND

      EARLY FRY STAGES
INTERSPECIFIC

HYBRIDIZATION
FEMALE

TRIPLOID
                             STERILE SALMONID
     INDUCED GONADAL

     AUTOIMMUNITY
 SURGICAL

 REMOVAL

 OF  GONADS
X RAY OR  GAMMA

IRRADIATION OF

EMBRYOS OR FRY
    Figure  6.  Sterilization techniques  (reproduced with permission from
      Donaldson 1986).
                                    19

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                                   ALEVIN
                          ANDROGEN 1  IMMERSION
                                    FRY
                            DIETARY 1 ANDROGEN
                                  STERILE

               Figure 7.  Hormonal sterilization using androgen
                 treatment.
                      CURRENT STATUS AND FUTURE PROSPECTS

     Sex control techniques currently are  being applied in the  culture of
tilapia species, carps,  rainbow trout,  Atlantic salmon,  coho  salmon, and
chinook salmon'.  As understanding improves of the  methodologies for regulating
sex in fish, we can expect to see increased application of these techniques as
they promise to improve both the economics of the  culture  process--for
example, through the rearing of all female broodstock--and the  value of the
fish produced--for example, by improving quality at harvest.
                                  REFERENCES
Baker, I.J., I.I. Solar, and E.M. Donaldson.   1988.   Masculinization of
     chinook salmon (Oncorhynchus tshawytscha) by immersion treatments using
     17a-methyltestosterone around the time of hatching.   Aquaculture.   (in
     press)

Bardach, J.E., J.H. Ryther, and .0.  McLarney.  1972.   Aquaculture,  The
     Farming and Husbandry of Freshwater and Marine  Organisms.  Wiley Inter-
     science, New York.  868 pp.

Benfey, T.J., and E.M. Donaldson.  1988.  Triploidy  in the Culture of Pacific
     Salmon.  In:  Proceedings,  Aquaculture International  Congress,  September
     6-9, 1988, Vancouver, BC Canada.  549-554.

Benfey, T.J., H.M. Dye, I.I. Solar,  and E.M.  Donaldson.  1989.  The  growth and
     reproductive endocrinology of adult triploid Pacific  salmonids.  Fish
     Physiol. Biochem.  (in press)
                                     20

-------
Benfey, T.J., I.I. Solar,  G. de Jong,  and E.M. Donaldson.  1986.   Flow-
     cytometric confirmation of aneuploldy in sperm from triploid rainbow
     trout.  Trans. Am.  Fish. Soc.  115:838-840.

Bye, V.J.  and R.F. Lincoln.  1986.  Commercial methods  for the control of
     sexual  maturation in rainbow trout (Salmo gairdneri R.).  Aquaculture.
     57:299-309.

Donaldson, E.M.  1986.  The integrated application of controlled reproduction
     techniques in Pacific salmonid aquaculture.  Fish  Physiol. Biochem.  2:9-
     24.
SHOCK TREATMENT    TETRAPLOID
                           FEMALE SPERM X NORMAL EGG
AT  2ND MEIOTIC
                            SHOCK TREATMENT AT
DIVN. OF EGG
            DIPLOID
\       /
   ESTROGEN
  TREATMENT
1ST MITOTIC DIVN.  OF EMBRYO
                                      XXXX TETRAPLOID
                               FEMALE

                              TRIPLOID
 FEMALE SPERM X NORMAL EGG

 SHOCK TREATMENT AT

 2ND MEIOTIC DIVN.  OF EGG
                                          XX DIPLOID EGG

                                                  X

                                            FEMALE SPERM
                               XX DIPLOID SPERM

                                       X

                                  NORMAL EGG
                                                    ANDROGEN

                                                    TREATMENT
Figure  8.  Sterilization by production of female triploids (reproduced with
  permission from Donaldson and Benfey 1987.
                                   21

-------
 Donaldson,  E.M. and T.J. Benfey.  1987.  Current status of induced sex
      manipulation.  In:  Proc.  Third Int.  Symp.  Reprod. Physiol.  Fish.  D.R.
      Idler,  L.W.  Grim and J.M.  Walsh (eds.),  pp. 108-119.   Mem.  Univ.  NFLO
      St.  John's NFLD.  Aug.  2-7, 1987.

 Donaldson,  E.M. and G.A. Hunter.  1982.  Sex  control in fish with particular
      reference to salmonids.   Can.  J.  Fish. Aquat.  Sci.  39:99-110.

 Egami,  N.  and K-I. Ijiri.   1979.  Effects  of  irradiation on germ cells and
      embryonic development in teleosts.  Int.  Rev.  Cytol.   59:195-248.

 Goetz,  F.W.,  G.M.  Donaldson,  G.A.  Hunter,  and H.M.  Dye.  1979.   Effects of
      estradiol-17/3 and 17a-methyltestosterone on gohadal differentiation in
      the  coho salmon, (Oncorhvnchus kisutch) .  Aquaculture 17:267-278.

 Guerrero, R.D.   1982.  Control  of  tilapia  reproduction.  In:  The Biology and
      Culture  of Tilapias.  R.S.V.  Pullin and  R.H. Lowe-McConnell  (eds  )
      ICLARM.   pp.  309-316.

 Hunter, G.A.  and  E.M.  Donaldson.   1983.  Hormonal sex control and its  applica-
      tion to  fish culture.  In:  Fish Physiology, Vol.  9B.  U.S.  Hoar,  D.J.
      Randall,  and E.M.  Donaldson (eds.).   Academic  Press,  New York, NY USA
      pp. 223-303.

 Hunter, G.A.,  I.I.  Solar,  I.J.  Baker, and  E.M. Donaldson.   1986.   Feminization
      of coho  salmon (Oncorhvnchus kisutch) and chinook  salmon (Oncorhvnchus
      tshawvtscha)  by immersion  of alevins  in a solution of estradiol-17/3.
      Aquaculture.   53:295-302.
Pandian, T.J. and K. Varadaraj.  1987.
     breeding in tilapia.  Curr. Sci.
 Techniques to regulate sex ratio and
56:337-343.
Parsons, J.E. and G.H. Thorgaard.  1985.  Production of androgenetic diploid
     rainbow trout.  J. Hered.  76:177-181.

Piferrer, F. and E.M. Donaldson.  1989.  Gonadal differentiation in coho
     salmon, On'corhvnchus hisutch. after a single treatment with androgen or
     estrogen at different stages during ontogenesis.  Aquaculture (in press)

Piferrer, F., and E.M. Donaldson.  1988.  Progress in the development of sex
     control techniques for the culture of Pacific salmon.  In:  Proceedings,
     Aquaculture International Congress, September 6-9, 1988, Vancouver  BC
     Canada.  519-530.

Purdom, C.E.  1983.  Genetic engineering by the manipulation of chromosomes
     Aquaculture.  33:287-300.

Purdom, C.E.  1986.  Genetic techniques for control of sexuality in fish
     farming.  Fish Physiol. Biochem.  2:3-8.
                                     22

-------
Solar, I.I., E.M. Donaldson and G.A. Hunter.  1984.  Optimization of treatment
     regimes for controlled sex differentiation and sterilization in wild
     rainbow trout (Salmo gairdneri Richardson) by oral administration of 17a
     methyltestosterone.  Aquaculture 42:129-139.

Stanley, J.G.  1981.  Manipulation of developmental events to produce monosex
     and sterile fish.  Rapp. P.-v. Reun. Cons. Int. Explor. Mer 178:485-491.

Thorgaard, G.H.  1983.  Chromosome set manipulation and sex control in fish.
     In:  Fish Physiology, Vol. 9B.  Hoar, W.S., D.J. Randall, and E.M.
     Donaldson (eds.).  Academic Press, New York, NY USA.  pp. 405-434.
Thorgaard,  G.H.
     57:57-64.
1986.   Ploidy manipulation and performance.   Aquaculture
Thorpe, J.E., C. Talbot, C. andcM.S. Miles.  1987.  Irradiation of Atlantic
     salmon eggs to overcome early maturity when selecting for high growth
     rate.  In:  Selection, Hybridization, and Genetic Engineering in Aquacul-
     ture, Vol. II K. Tiews (ed.).  Heenemann Verlags.   mbH, Berlin,  pp. 361-
     374.

Yamamoto, T.  1969.  Sex differentiation.  In:  Fish Physiology, Vol. 3, W.S.
     'Hoar and D.J. Randall (eds.).  Academic Press, New York, NY USA.  pp.
     117-175.
Yamazaki, F.  1983.
     33:329-354.
    Sex control and manipulation in fish.  Aquaculture.
                                     23

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                         ASEXUAL REPRODUCTION OF FISH

                                      by

                                 Chen Hongxi1

      By means of microinjection or electric fusion, the asexual reproduction
of fish involves the transfer of somatic cell nuclei to the mature
unfertilized eggs or the enucleated eggs of fish of the same or different
species.  Just as with the zygote of the fertilized egg, the somatic cell
nucleus can promote the receiving egg to cleave and develop into a normal
individual.  There is no fertilization, however, of the male and female
pronuclear synapsis.  Thus the asexual reproduction of fish is quite different
not only from the normal sexual reproduction but also from the monosexual
reproduction in various types.  As the asexual reproduction of fish is a
special and artificial method of reproduction, it can be applied widely to
certain theoretical problems and to breeding practice.


            THE REPRODUCTIVE  MODES AND  ASEXUAL REPRODUCTION OF  FISH

      From hermaphroditic autogamy to dioecious amphimixis, fish have many
reproductive modes.  Of all these modes,  amphimixis is the most common
phenomena.  There also are a few genera,  however, whose fish belong to a so-
called- thelytoky, producing only female progeny.  Thelytoky can be divided
into two types:   gynogenesis and hydridogenesis.  Gynogenesis is a kind of
reproduction of apomixis or pseudogamy in which there is no male and female
pronuclear synapsis, and only female offspring are produced with a matromorphy
phenotype.  It is the result of diploid parthenogenesis.  On the other hand,
in hybridogenesis, the male and female pronucleus will be fused with each
other.  The progeny are real hybrids and their phenotype is matroclinous.   A
few examples of parthenogenesis have occurred in Osteichthyes due to
stimulations of some pathological or physicochemical factors.  The thelytoky
occurs depending on the sperm,  but parthenogenesis does not.

      Androgenesis is another kind of monosexual reproduction that
occasionally appears in artificial outcross.   Through artificially induced
androgenesis,  offspring could be produced that are both male and female.  The
asexual reproduction of fish described in this paper is an especially
artificial reproduction,  in which,  as mentioned above, the fish diploid
somatic nucleus  is transferred to the mature unfertilized egg or enucleated
egg by means of the nuclear transplantation or electric fusion.  Then the egg
     Institute of Hydrobiology, Academia Sinica, Wuhan, PRC.

                                      24

-------
will develop directly into an individual without the male and female
pronuclear synapsis as happens in amphimixis.  So it is neither diploid
parthenogenesis nor diploid androgenesis;  it is just asexual reproduction of
diploid somatic nuclei.  The offspring will be both male and female.  Their
phenotype resembles those of the nucleus donor fish.  In nuclear
transplantation of heterospecies,  however, some characteristics of offspring
may resemble those of the cytoplasm recipient fish.  A comparison of the modes
of reproduction in fish is provided in the Table 1.
           TABLE 1.   COMPARISON OF THE MODES OF REPRODUCTION IN FISH
Reproductive
   modes
Route of embryo
  development
Phenotype of
 offspring
                                                                  Occurrence
Amphimixis


Monosexual
   reproduction

    Gynogenesis


    Hydr i do gene s i s


    Androgenesis



    Parthenogenes is


Asexual reproduction
Dioecious or
autogamy
Pseudogamy
Gametogamy
Outcross or
female nucleus
inactive

Pathological or
induced

Diploid somatic
nucleus
                                                Intermediate
Matromorphy
Matroclinous
Patroclinous
Matromorphy
Similar to
donor fish
                  Most common
                  phenomena
Naturally or
induced

Naturally or
"synthesis"

Occas ionally
or induced
Occasionally
or induced

Artificially
induced
             PRESENT STATUS  OF STUDIES  ON  FISH ASEXUAL REPRODUCTION

       As  stated above,  fish asexual reproduction requires  transfer  of  a
 somatic cell nucleus to the cytoplasm  of  a recipient egg by microinjection or
 electric  fusion.   The recipient egg is then promoted by the donor nucleus  to
 cleave and develop into an individual.
                                       25

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 NUCLEAR TRANSFER MEDIATED BY MICROINJECTION     ;:

       The nuclear transplantation technique for :fish was  developed in China by
 the late professor Tong Dizhou and his colleagues.; ; By means  of nuclear
 transplantation,  Tong Dizhou and coworkers  (1980,  1984) carried out studies on
 the relationship of nucleus and cytoplasm,  in order to explore  the rule of
 phenotype variance and the new approachrof  fish.breeding.   They produced a
 hybrid fish from a carp (Cyprinus carpio) nucleus  and a crucian carp
 (Carassius auratus) cytoplasm,  and a hybrid fish from a crucian nucleus and
 carp cytoplasm.   Both male and female of  these, two nuclear-cytoplasmic hybrid
 fish developed normally and produced F2, F3 and: F4  ;filial  generations.
 Morphological characteristics of nuclear-cytoplasmic hybrid fish showed that
 some features are intermediates such as the number of lateral line scales in
 the combination  of carp nucleus and crucian cytoplasm.  Some  are inherited
 from the nucleus  donor fish,  such as the barbs, r^and some  seem to come  from the
 cytoplasm recipient fish,  such as the number of vertebrae in  hydrid fish.
 These results indicate that both nucleus and cytoplasm can  influence the
 expression of genetic information on the hybrid fish.   The  phenotype of hybrid
 fish result from the interaction of donor nucleus  and recipient 'cytoplasm.

       They also  transplanted blastula cell  nuclei  of grass  carp into cytoplasm
 of  blunt anout bream (Megalobrama amblvcephala) recipient eggs  and obtained
 nine subfamiliar  nuclear-cytoplasmic hybrid fish..   One male individual of
 these hybrid fish attained sexually maturity (1985).

       Taking three strains of domestic carassius goldfish (red  dragon  eye,  red
 carassius,  egg fish)  as  experimental materials, by means of nuclear
 transplantation technique,  Wu Shangqin et al.  (1980)  carried  out nuclear-
 cytoplasmic recombination  among strains, in order  to  explore  the effect  of
 nucleus  and cytoplasm to the  expression of  morphological characteristics  and
 interaction between nucleus and cytoplasm.   By means  of four  serial  nuclear
 transplantations--i.e.,  transplanting the blastula cell nuclei  of  double  tail
 red dragon eye goldfish  into  the cytoplasm  of single  tail red carassius
 recipient  egg, they found  increasing numbers of single tail goldfish in
 nuclear  transplanting fish.   If the  single  tail nuclear transplanting  fish
 were  crossed with red dragon  eye,  the number of single tail fish also
 increased.   So, cytoplasm  not only changes  the expression of  genetic
 characters but also  can  affect  the second generation.  This effect can be
 accumulated from  generation to  generation.  The -effect is still  not  fully
 understood.

       Chen Hong-xi et al.  (1986)  transplanted subcultured cell nuclei
 originating  from blastula  cells  of the crucian carp  (C. auratus) into the
 enucleated unfertilized  eggs  of  the  same species and obtained a nuclear
 transplanted fish  that survived  for more than 3 years.  They also transplanted
 the primary cultured  cell nuclei  originating from  the kidney of crucian carp
 or  goldfish  into  the  enucleated  eggs  of crucian carp or goldfish and obtained
mature adult fish.  This result  implies that the cultured cell nuclei
 originating from  the  specialized somatic cell of adult fish still can promote
 the recipient  eggs to develop to  an  adult fish and retain their  developmental
 totipotency.
                                     26

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      Lu Renhou et al. (1982) induced the diploid chromosomes of cultured
cells originating from grass carp tail fin doubling to autotetraploid cells by
colchicine.  Transplanting this tetraploid nuclei into loach (Misgurnus
anguillicaudatus) nucleated ,eggs, they also obtained interfamiliar nuclear-
cytoplasmic embryos, but'did .not obtain survival larva.
      According to the Porter's method, Liu Hanqin et al. (1987) inseminated
the eggs of Paramisgurnus dabryanus with the sperm of Misgurnus
anguillicaudatus.  In 7 to; 15 .minutes after artificial insemination, they
mechanically removed the;egg nucleus and obtained androgenetic haploid embryos
reconstituted from the sperm nuclei of M. anguillicaudatus and egg cytoplasm
of P. dabrvanus.  Then, they;transplanted androgenetic haploid embryonic cell
nuclei into the nucleated eggs of P. dabryanus and obtained seven androgenetic
diploid adult fish.  These androgenetic diploid fish were sexually mature and
the androgenetic haploid embryos or fries died of "haploid syndrome."  The
doubling mechanism of androgenetic haploid chromosomes in the egg cytoplasm of
P. dabryanus is not known yet.  According to the examination of morphological
characteristics, chromosomes, and isoenzymes of the androgenetic diploid fish,
however, they indeed came from the androgenetic haploid with nuclear
chromosomes doubling in recipient cytoplasm.  So the survival rate of nuclear
transplant fish obviously was enhanced.
NUCLEAR TRANSFER MEDIATED BY ELECTRIC FUSION

      There have not been many reports about fish cell fusion.  Tong Dizhou et
al.  (1973.) have qualitatively proven cell fusion of goldfish blastula cells
and  fusion between goldfish blastula cells and Ehrlich cells mediated by
Sendai virus.  Mizukami et al. (1979) studied the cell fusion process in which
the  isolated embryonic cells of niedaka (Oryzias latipes) could contact with
each other again in salt solution and then were led to be fused with each
other.  The factors that influenced the cell fusion also were studied.
Recently, many studies on fish cell fusion aiming at fish breeding by cell
engineering have been made by cytogenetic research group in the Institute of
Hydrobiology, Academia Sinica.  Yan Rang et al. (1984) used PEG to promote
cell fusion among CAB-80 cells and studied various experimental conditions
carefully.  They also used PEG to promote the cell fusion between blastula
cells and unfertilized eggs, but did not get normal fries.  Shu Xiaolin et al.
(1988) used PEG to promote minisegregant nuclei to fuse with fertilized eggs
in order to study the chromosome transfer mediated by minisegregant cells.
She  observed that the micronuclei in the cytoplasm of fertilized egg still
remained in the interphase nucleus stage and did not fuse with the nucleus of
the  recipient egg.

      Electric fusion is a new cell biological technique and is one of the
most effective method in cell fusion and gene transfer.  Its advantages--such
as high fusion rate, simple manipulation, nontoxicity, and visualization under
microscope--make it widely used in the study of cell fusion in animal, plant,
and  microbial cells and protoplasts.  Studies on cell fusion induced by
electric field in fish have not been reported yet, however.  Using Chinese-
made component and fittings, we have made an electric fusion device, and using
this device we have studied the cell fusion between cells or between cells and
eggs induced by electric field and the various factors and conditions that
affected the fusion rate.  The results are as follows.

                                      27

-------
      Liu Peilin et  al.  (1988) have gotten better results with P. dabryanus
blastula cell  fusion.  Under  the  conditions of 9 to 15C, 10 to 50
microseconds of time constant and 2.5 ~ 3xl04 cells/ml of cell density, the
biastula cells were  first  treated in an intermediate solution composed of 0.02
mol/L manitol  and  1  mmol/L CaCl2 with cells in tight contact for 2 to 5 min.
Cells then were treated  by electric pulse and finally were fused with  each
other (Table 2).
   TABLE 2.  EFFECTS OF FISH BLASTULA CELL FUSION INDUCED BY ELECTRIC FIELD*
            Dikaryocyte
                Multikaryocyte
                      FI,  %
            Viability(%)
XS
283174
506321
46.7110.8   88.614.4
"Calculated from eleven experimental results.
From Table 2, we can know that, under these conditions, the fusion rate of P^.
dabryanus blastula cells is 46.7% and the viability is 88.6%.  These results
indicate that the electric fusion device is basically fit for experimental
demands.

      Yi Yonglan et al. (1988) used P. dabryanus. M. anguillicaudatus and red
carassius as experimental materials to study the electric fusion of blastula
cells and unfertilized eggs.  In Table 3, it is shown that the hatching rates
of fries from blastula cells fused with recipient eggs of the same species,
from hybrid embryonic cells fused with P. dabryanus recipient eggs and from
common carp blastula cells fused with red carassius recipient eggs are about
18.2%, 16.7% and 3.5%, respectively.  In the control group, only 9 in 3778
unfertilized eggs could cleave irregularly.  Most eggs cannot normally develop
into the bastula stage.  Under the electric pulse, the donor nucleus may enter
the recipient egg and promote the egg to develop into a normal individual.
Judged from outer features of electrically fused fish, it is unquestionable
that the donor nucleus has participated in development of fused egg into fry.
The fusion and hatching rates in the experiment are not very high, however.

      Using P. dabryanus as experimental materials, Tian Jiunshi et al. (1988)
studied the main conditions of electric fusion between bastula cells and
unfertilized eggs and the various physicochemical and biological factors
influencing the electric fusion rate.  For certain electric field constants,
the variety and concentration of divalent cation, the concentration of
manitol, and the pretreatment condition of blastula cells have a significant
influence on the fusion rate.  Among effects of divalent cation, the effect of
Ca2+ on  electric fusion is the best.  When  the concentration  of  Ca2+ and
manitol is 10" M and  0.2M, respectively, the conductance and osmotic pressure
of intermediate solution are most beneficial to the fusion and hatching rates.
If the donor cells are pretreated with 100 ug/ml pronase E for 6 to 10 minutes
at 25C, the fusion rate can be enhanced markedly.  When the density of donor
                                      28

-------
TABLE 3.  COMPARISON OF EMBRYONIC DEVELOPMENT OF RECIPIENT EGGS AFTER ELECTRIC
FUSION                        '
                  Number of
Donor recipient   recipient eggs
cells, eggs
Number of
bastula stage
  Number
arrested in
gastrula stage
P. dabryanus  ( ) X
M. anguillicaudatus     1107
( ) -+ P. dabryanus

Cyprinus carpio          662
-> Red Carassius
      36
     113
      10
      18
 Fry
P . dabrvanus-*
P . dabryanus
296
33
12
6
(18.2%)
   6
(16.7%)

   4
(3.5%)
Blank control
Control
2230
1548
9
0
    donor cell nuclei  shocked  in  recipient eggs using electric pulses.
cells is below 105 cells/ml,  the direct proportion appears  between fusion rate
and donor cell density.  But when the density is above 10s  cells/ml,  the
fusion rate shows a declining trend.  The quality of recipient eggs also has
obvious influence on the fusion and hatching rates.  A properly matured
recipient egg is the basis of a successful experiment.  If fusion is
accomplished within 10, minutes after eggs are excited, a much higher fusion
rate can be obtained.  Under the optimum condition mentioned above, the fusion
and hatching rates can reach 80.0% and 20%, respectively.

      The cytological  studies on recipient eggs indicated that most recipient
eggs received 2 to 4 donor nuclei that were uncertainly located in the
cytoplasm.  Because every donor nucleus could develop to form a set of mitotic
apparatus, several blastomeres were formed after the first cleavage.  The
chromosome studies on  the electric fusion embryo are the same as that of donor
cell (P. dabryanus   X M. anguillicaudatus 2N - 74).  The outer features of
electric fusion fish are similar to that of donor fish.

      Liu Peilin et al.  (1988) improved experimental conditions further and
thus enhanced the fusion and hatching rates.  Using P. dabrvanus as
experimental material, they studied the electric fusion efficiency of blastula
cells and unfertilized eggs.  Normal artificial insemination eggs from the
same parents were used as the control group.  Here, the fertilized rate is the
percentage of fertilized eggs that can develop into middle gastrula stage in
all fertilized eggs.   The fusion rate is the percentage of fused eggs that
develop into middle gastrula stage in all recipient eggs.  Thus, the fusion
                                      29

-------
 efficiency is the ratio of fusion rate to the fertilized rate.   The hatching
 efficiency of fused eggs is also the ratio of two hatching rates of
 experimental group to control group.   The results in Table 4 are calculated
 from ten experimental results.
 TABLE  4.   ELECTRIC FUSION OF BASTULA CELLS  AND UNFERTILIZED EGGS  IN P.
 DABRYANUS                                       ,--,'
             Number of   Middle gastrula   Fries '
Group        recipient        stage
                eggs     No.         (%)     No.  (%.)
                    Fusion      Hatching
                    efficiency  efficiency
Experiment-     2273      1191
tal  group
5.2'. 30   191  16.04

Control
group
78.86
1420 930 65.49 354 38.06 2.8

42 . 14
2.41

      From Table 4,  it  is  seen that  the  electric  fusion efficiency  and  the
hatching efficiency  are 79.862.80%  and  42.142.41%,  respectively.  These are
average numbers from ten experimental  results and the average  standard
deviation is not above  5%, which  indicates  that the experimental conditions
are favorable and  the results  have reached  our demands.

      In addition, Liu  Peitin  et  al. (1988)  studied the conditions  of short-
term cultured embryonic cells  of  P.  dabryanus fused with the unfertilized eggs
of the same species.  Its  fusion  rate  was 30 to 32% and 2 larval fish survived
for 2 months.  It  seems that the  electric fusion  technique can cause the
cultured cell to fuse with the egg too.  When the nucleus enters the egg
cytoplasm, the recipient egg can  cleave  normally  and  develop into an
individual.  The condition of  electric fusion should  be improved, however, in
order to obtain satisfactory result  on the  fusion and hatching rates.


                                  CONCLUSION

      Microinjection and electric fusion are effective methods for  fish
asexual reproduction.   The nuclear transplantation by microirijection has been
studied for more than 20 years.   Its advantages have  been shown in  the studies
of nuclear-cytoplasmic  relationship, the formation of nuclear-cytoplasmic
hybrid, the influence of nucleus  and cytoplasm on the  phenotype and so on.
Microinjection, however, is a  complicated technique of micromanipulation.  It
is very difficult  to  avoid mechanical  damage in the operation.  So  the
survival rate of the nuclear transplantation with midblastula and cultured
somatic cell are only about 15% and 1%, respectively,   even for a well trained
technician.
                                      30

-------
      Compared with nuclear transplantation, electric fusion has many
advantages--such as high fusion and hatching rates, simple manipulation,
nontoxicity, simultaneous treatment of dozens of cells in electric field and
less mechanical damage.  On the other hand, in nuclear transfer mediated by
electric fusion, the recipient eggs whose nucleus is not picked out previously
always receive more than one donor cell nucleus, which increases complexity.
In spite of the complexity, the fusion and hatching rates are still high.
Reason for the high rates could be identified only after further investigation
of the mechanism of embryo development of fused eggs.  As to reasons for low
hatching and survival rates of cultured somatic nuclear transplantation by
microinjection or electric fusion, it also  seems that, except many technical
difficulties, there may be some unknown problems related to the developmental
potentiality of the somatic nucleus.  Only  after these problems about the
relation of somatic nucleus genotype and its totipotency become clearer and
the dedifferentiatiori condition of somatic  nucleus and the isolation of cell
clones capable of genetic totipotency is understood, we may greatly enhance
the hatching and survival rates.

      The compatibility between the donor  cell nucleus and the cytoplasm of
recipient egg in fish  is much greater than that  in amphibians.  According to
the present data, the  compatibility of intersubfamilies or intergenera  can
lead to producing normal individuals.  That provides  the favorable condition
on  the study about nuclear-cytoplasmic relationships  and the formation  of
nucleus-cytoplasmic hybrids.

      In  fish asexual  reproduction, compared with  amphimixis,  there are no
problems  such as reproductive isolation, crossing  incompatibility, F2
disjunction and hybrid sterility,  etc.   So, fish asexual reproduction can be
widely applied  in  as  a new approach fish breeding.

      The interaction of nucleus  to nucleus, nucleus  to cytoplasm, and  their
 influence on the phenotype are  still  not very  clear.   Great  efforts must be
made  in  order  to  solve these problems.

      Although  there  are many difficulties in  technique  and  theory at present,
 the prospects  of fish asexual reproduction will be very bright.   As  the
 techniques are  perfected,  fish asexual  reproduction will be  very useful not
 only  in theoretical research,  such as nuclear-cytoplasmic  relationship, the
 developmental potentiality of somatic nucleus  and somatic  cell genetics, but
 also  in the application of somatic cell breeding,  the formation of fish clones
 and nuclear-cytoplasmic hybrid fish and so on.   Especially when the  asexual
 reproduction technique is  combined with these  techniques  and methods  such  as
 modern cell engineering, development engineering,  gene engineering and
 classical breeding technique,  its applied values will be unforeseen.


                                   REFERENCES

 Chen H-x., Y-l. Yi.,  M-r Chen,  and X-q. Yang.   1986.  Studies on the
       developmental potentiality of cultured cell nuclei of fish.  Acta
       Hydrobiologica Sinica.  10(1):1-7.
                                       31

-------
 Liu P-l.,  Y-l.  Yi,  H-q.'Liu,  and H-z.  Chen.   1988.   Preliminary study on
       electric fusion of fish cells.   Acta Hydrobiologica Sinica.   12(1) -94-
       96.

 Liu P-l.,  H-q.  Liu,  and H-x.  chen.   1988.   A simple,  rapid,  high- efficiency
       technique for embryonic cells fused with the  unfertilized eggs of P_._
       dab ry anus .   (Unpublished data.)  .

 Liu P-l.,  H-q.  Liu,  and H-x.  Chen.   1988.   Viable larval fish produced from
       the  short-term cultured embryonic  cells fusing with the unfertilized
       eggs of P.  dabrvanus by the electric fusion technique.   (Unpublished
       data.)

 Liu H-q.,  Y-l.  Yi,  and H-x.  Chen.  1987.   Production of the androgenetic
       homozyous diploid loach (Misgurnus anguillicaudatusl .   Acta
       Hydrobiologica Sinica.   11(3) :241-246.

 LuR-h., Y-j . Li, Y-L.  Yi,  G-d.  Bai, and H-x.  Chen.   1982.  The tetraploidized
       cell strain GCC(4),  induced from the caudal fin cells  of grass carp,
       characteristics and nuclear transplantation experiment.   Acta Genetica
       Sinica.   9(5) :381-388.

 Laboratory of Cytology,  Peking Inst. of  Zool.  Academia Sinica.   1975.
       Technique for  nuclear transplantation  in fish.   Journal of Zoology
       (Beijing).  (2): 44-47.

 Mizukami,  S.  1979.   Cell  fusion of dissociation  embryonic cells of Medaka
       (Orvzias  latipes) .   Zool.  May.   88:17.

 Shu X-l. and Y-g. Jiang.   1989.   Study on  the  preparative  techniques of
       microcells  and minisegregant  cells in  fish.  Acta Hydrobiologica  Sinica
       (in  press) .

 Tung T. C., S-c. Wuy. , Y. Y-f Tung., S-y.  Yan. , M. Tu,  and T-y.  Lu.   1963.
       Nuclear transplantation in fish.   Scientia  Sinica.   (7):60-61.

 Tung T. C.  et al.  1980.  Nuclear transplantation in  teleosts.   I.  Hybrid
       fish from the  nucleus of carp and  the cytoplasm of crucian.   Scientia
       Sinica.   23(4) :517-523.

 Tung T. C., Y.  Y-f.  Tung., T-y.  Lu, F-m. Ton,  and M.  Tu.   1973.  Inter-
       subfamiliar nuclear transplantation  in fish.  Acta Zoologica  Sinica
Hybria
Scientia
Yan S-y. et al.  1984.  Nuclear transplantation in teleosts.  II.
      fish from the nucleus of crucian and the cytoplasm of carp.
      Sinica.  27(10) : 1029-1033.
Wu S-c.  1980.  Nuclear transplantation among interstrains of goldfish
      (Carossius auratus var.)  Acta biologie Experimentalis Sinica
      13(l):65-73.
                                      32

-------
Yi Y-l., P-l. Liu, H-q. Liu,, and H-x. Chen.  1988.  Electric fusion between
      blastula cells and unfertilized eggs in fish.  Acta Hydrobiologica
      Sinica.  12(2):189-192.

Yang K., M-r. Chen, and H-x. Chen.  1984.  Preliminary study on fusion of
      fish cells.  Hereditas (Beijing).  6(6):19-21.

Yan S-y. et al.  1985.  Nuclear transplantation in teleosts.  IVa. Inter-
      subfamiliar nuclear transplantation--hybrid fish from the nucleus of
      grass carp (Clenopharyngodon idellus) and the cytoplasm of bleam
      (Megalobrama terminalis).  Acta Biotechnologica Sinica.  l(4):27-33.
                                      33

-------
                   INDUCED BREEDING OF CULTURED FISH IN CHINA

                                       by       ..

                           H.R. Lin1, and R.E. Peter2
                                  INTRODUCTION
     "China has not only one of the largest freshwater areas in the world, but
 also a 3000-year history of fish culture.  The development of fish culture in
 China has made rapid progress since 1949.  Currently, not only ponds are used
 for fish culture, but also large water bodies, such as natural lakes,  reser-
 voirs ,  canals,  small rivers and their tributaries,  as well as many paddy
 fields.   Production from these different types of water bodies is rapidly
 increasing;  fish yields per unit in China is one of the highest in the world
 Recently, China produced more than 1 million tons of fish from freshwater fish
 culture,  which was about one-fifth of the total fisheries production,  and the
 largest  production of cultured freshwater fish in the world.

     Because of the development of fish culture,  more and more fry and
 fingerlings  are needed for stocking.   It is  obvious,  therefore,  that the
 artificial propagation of cultivated fishes  and the large scale  production of
 fry and  fingerlings are very important.   Although greater production of fry
 and fingerlings has occurred from year to year,  the demands can  still  not be
 satisfied.  ^Because the major Chinese carp do  not ovulate and spawn naturally
 in the confinement of ponds,  nature was  the  only  source  of fry.

     In  1958, under the efforts  of scientists  and fish farmers,  successful
 results were obtained by the  use of fish pituitary  and human  chorionic
 gonadotropin (HCG)  for the induction of  spawning  of silver carp  and bighead
 carp; the grass carp  and the  black carp,  on  the other hand, are  not sensitive
 with^HCG  treatments,  although both of them gave favourable response to  fish
 pituitary.   Since then,  this  technique has been spread country-wide.

     HCG, however,  is  effective  in only  a  few  species, and the carp  pituitary
 is very expensive.  For example,  it is often necessary to  collect pituitary
 from ten  or more  sexually  mature  carp to  induce spawning  in a single brooder
^Department of Biology,  Zhongshan University,  Guangzhou,  PRC
 Department of Zoology,  University of Alberta,  Edmonton AB,  Canada
                                    34

-------
Additionally, many brooder can not tolerate the injections and die after
ovulation or spawning, entailing a heavy loss to fish farmers every year.
Therefore, a highly effective, cheaper and safer ovulating agent was needed.

     In 1974, Chinese scientists attempted to use a synthetic analogue of
luteinizing hormone-releasing hormone, des-Gly10(D-Ala5)-LHRH ethylamide
(LSRH-A) as an ovulating agent (CTHAP 1977).   There are many problems with the
use of LHRH-A for induced ;oyulation and spawning of cultured fish, however,
and this polypeptide apparently has had relatively little practical impact on
fish farming in China.  Table 1 summarizes the so-called traditional methods
for induced ovulation and spawning of cultured carp in China.  It is evident
that LHRH-A currently is not being used alone for induced breeding of any
species of cultured carp in most fish farms (Peter et al. 1987)..

     Under the funding of the International Development Research Centre of
Canada  (IDRC) and National Science Foundation of China, the proposal titled
"The Basis for Application of GnRH Analogues and Dopamine Antagonists in Fish
Culture" has been carried out at both the Department of Biology, Zhongshan
University, Guangzhou, PRC, and Department of Zoology, University of Alberta,
Edmonton, Canada, since 1984.  The ultimate objective of our collaborative
research program is to determine the optimal conditions for application of
GnRH analogues and dopamine antagonists for induced ovulation and spawning of
cultured fish.


                     THE LINPE METHOD--COMBINATION OF GnRH
                       ANALOGUES AND DOPAMINE ANTAGONISTS

     Our basic studies have demonstrated that the neuroendocrine regulation of
GtH secretion in teleosts involves a dual control, with GtH release stimulated
by gonadotropin-releasing hormone  (GnRH) and inhibition of dopamine, which
acts as a gonadotropin release-inhibitory factor  (GRIF) on the action of GnRH
as well as a spontaneous GtH  release  (Peter et al. 1986).  The preovulatory
surge  of  gonadotropin, which  is responsible for ovulation, may be regulated by
a stimulation of GnRH and a release from inhibition by GRIF.

     For  example, in  common carp,  adding males and floating vegetation as a
spawning  substrate to the holding  pond  at 6 p.m.  resulted in spontaneous
ovulation the following morning.   A pronounced surge of GtH was  found in fish
that ovulated.  This  ovulatory  gonadotropin surge lasted at least 12 hours,
during which GtH levels Increased  about 10-fold above basal level.  Serum GtH
levels remained low during the  course of sampling in control fish and fish
that failed  to ovulate  (Lin et  al. 1986a).

     Therefore, the lack  of an  effect of LHRH-A alone  on ovulation  in carp
resulted  from a failure to stimulate  an increase  in  serum GtH levels of
similar magnitude to  that found in fish that ovulate spontaneously.  This  is a
result of the predominant influence  of  dopamine inhibition on gonadotropin
secretion.

     Figure  1 presents  a  general model  of  the neuroendocrine regulation  of  GtH
secretion in teleosts.  The levels of intervention and action of several
catecholaminergic drugs on GtH  release  are indicated.

                                      35

-------


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     Laboratory experiments on goldfish were the first to reveal the effect of
blockage of dopamine actions on GtH release and ovulation by determining the
response to LHRH-A, when given in combination with the dopamine receptor
antagonist pimozide(PIM).  In these studies, combined injections of PIM and
LHRH-A resulted in a rapid increase in the blood level of GtH and ovulation
within a predictable time following injection (Chang and Peter 1983; Peter et
al. 1986, 1987).

     Reserpine, a drug that causes general depletion of catecholamine, also
significantly potentiates the action of GnRH and its analogues.  The drug a-
methyl-para-tyrosine and carbidopa, which blocks catecholamine synthesis at
steps up to and including the production of dopamine, potentiates the actions
of LHRH-A on GtH release in goldfish (Peter et al. 1986) and Chinese loach
(Lin et al. 1986b); however; blocking conversion of dopamine to nofepinephrine
with the drug diethyldithioearbamate has no apparent effect on the actions of
LHRH-A.                   -:

     Results from experiments done on a number of species of cultured Chinese
carp (Lin et al. 1986a, 1987a,b; Peter et al. 1986, 1987, 1988) all confirm
that dopamine acts as GRIP and that dopamine receptor antagonists or drugs
that block dopamine synthesis potentiate the activity of GnRH analogues.  This
combined treatment is highly effective in,stimulating GtH secretion and
ovulation.

     To maximize the efficiency of this combined treatment for induced
ovulation, research was done to determine the most effective GnRH analog and
dopamine antagonist to use for induced ovulation of cultured fish.  Structure-
activity studies on gonadotropin-releasing hormone in the goldfish indicate
that an analogue of salmon gonadotropin-releasing hormone, (D-Arg6, Trp7,
Leu8, Pro9NEt)-LHRH(sGnRH-A),  is the most active peptide of all those tested
(Peter 1986; Peter et al. 1985).

     In the presence of PIM, sGnRH-A is 17  times more potent than LHRH-A in
stimulating GtH secretion in goldfish  (Peter et al. 1985).  In the presence of
a dopamine antagonist,  sGnRH-A also was more effective  than LHRH-A in stimu-
lating GtH secretion and ovulation  in  Chinese loach and common carp  (Lin et
al.  1988).  The activity of catecholaminergic drugs in  potentiating the
activity of LHRH-A was  investigated in goldfish  (Peter  et al. 1986) and
Chinese  loach  (Lin et al. 1986b).  Many of  these drugs, including a number of
dopamine receptor  antagonists that  are specific for D-2 type receptors,
potentiate the  actions  of GnRH analogues.   Domperidone  (DOM), a specific D-2
antagonist that does not cross the blood-brain barrier, is highly potent in
potentiating the actions of LHRH-A  and sGnRH-A  (Peter et al. 1986, Omeljaniuk
et al. 1987, Lin et al.  1988).

     For example,  common carp, when receiving the combination of sGnRH-A and
DOM, experience a  very  rapid increase  in  serum GtH level at 6 to 24 hours
after  injection, similar to a spontaneous ovulatory surge of gonadotropin.
This combination treatment  is highly effective  in inducing ovulation within 14
hours  following injection  (at 21-23C)  (Lin et al. 1987b).
                                      39

-------
      Experiments have been done on grass carp (Ctenopharvngodon idellus).
 silver carp (Hvpophthalmichthys molitrix),  bighead carp  (Aristichthys
 nobilis),  black carp (Mylopharyngodon piceus), mud carp  (Cirrhinus  molito
 rella), and bream (Parabramis  pekinensisl as  well  as  some  other cultured fish
 in China,  demonstrating that injection of the combination  of dopamine  antago-
 nist (PIM  or DOM)  plus GnRH analogues (LHRH-A or sGnRH-A)  is highly effective
 in inducing ovulation and spawning (Lin et  al. 1985,  1986a,b,  1987a,b;  Peter
 et al. 1987,  1988).   The effectiveness of this new technique,  called the Linpe
 method, for induced  ovulation  of cultured fish was judged  against several
 criteria.   The  criteria are a  high rate of  ovulation  that  occurs consistently
 from one group  of brooders to  another within  each  species, a series of
 ovulations that are  complete rather than partial,  a short  and .predictable
 time-to-ovulation following injection,  a consistent production of fertile and
 viable ovulated eggs,  and a no-effect consequence  for subsequent reproductive
 cycles by  the same brood fish  (Peter et al. 1988).  The  results of  the
 experiments on  each  species of cultured fish  were  highly successful in meeting
 these criteria  (Peter et al. 1987,  1988).   On the  basis  of these results, the
 optimal treatments in common carp,  silver carp,  mud carp,  grass carp,  bream,
 bighead carp, black  carp,  loach,  Thailand mud carp (Labeo  rohita).  Chinese
 catfish (Clarias  fuscus)  and African catfish  (Clarias gariepinus") are  sum-
 marized in Table  2.
                            RESULTS OF FIELD TRIALS

     In cooperation with several fish farms and fish hatcheries in Guangdong
Province, field trials were done on a number of species to test the Linpe
method.  The results of the field  trials demonstrate that the combination of
DOM plus LHRH-A or sGnRH-A is highly effective for induced ovulation or
spawning of silver carp, mud carp, grass carp, bighead carp, black carp and
Thailand carp  (Peter et al. 1988).  First and second spawning in the same
reproductive season have been induced with DOM and LHRH-A in silver carp,
grass carp, bighead carp, and mud  carp, and in successive reproductive seasons
with silver carp, grass carp and mud carp.  The results indicate that succes-
sive reproductive cycles are unaltered by induced ovulation and spawning with
the Linpe method.

     The results of field trials in 1986-1987 have provided an adequate basis
for commercialization of the Linpe method for induced ovulation and spawning
of the cultured freshwater fish species worked on, as well as on closely
related species.  With the help of IDRC, an adequate supply of 3 kg DOM has
been acquired for marketing and expanded field trials for approximately
600,000 to 800,000 kg of brood stock fish (based on our recommended dosages of
DOM).  A prototype spawning kit, using this DOM and LHRH-A made by Ningbo Fish
Hormone Factory, was marketed to the key fish hatcheries in 1988.   In the
Delta Area of Pearl River,  Guangdong Province,  more than 500 g DOM has been
utilized for induced spawning of about 125,000 kg of brood stock fish,
producing approximately 6,250 million fry.  For example,  the Nanhai Fish
Hatchery,  one of the largest in China,  completely adopted the Linpe method
instead of carp pituitary injection for inducing spawning of grass carp and
mud carp in 1988.   Hatchery personnel administered 80 g of DOM and suitable
amounts of LHRH-A, induced 1266 brooders of grass carp (average body weight,

                                      40

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                                              42

-------
10 kg) and more than 1500 brooders of mud carp (average body weight, 1 kg)
spawning, with a rate of spawning of 90%, a rate of fertilization of 80%, a
rate of hatching of 90%, and the production of about 750 millon fry.

     Thus, the Linpe method is now a consistently highly effective and
reliable technique that can be widely applicable to induce ovulation and
spawning of cultured fish.   The species tested include common carp (Cyprinus
carpio),  silver carp (Hypophthalmichthvs molitrix),  bighead carp (Aristichthys
nobilis), black carp (Mylopharyngodon piceus),  grass carp (Ctenopharvngodon
idellus), mud carp (Cirrhinus molitorella),  Thailand mud carp (Labeo rohita),
bream (Parabramis pekinensis),  loach (Paramis gurnus dabryanus),  Chinese
catfish (Clarias fuscus),  African catfish (Clarias gariepinus),  pacu (Clossoma
brachypomum),  mandarinfish (Siniperca chuatsi),  and silvery chub (Xenocypris
argentea).


                                    SUMMARY

     In summary, it is clear that the GRIF action of dopamine is found in a
wide phylogenetic range, and perhaps in all teleosts.  In many teleosts, the
endogenous GRIF activity of dopamine can effectively modulate the GtH-releas-
ing activity of exogenous GnRH to the extent that a high rate of ovulation
cannot be induced by injection of superactive GnRH agonist alone.  The use of
DOM, a highly potent dopamine receptor antagonist, in combination with GnRH
analogues, such as sGnRH-A, represents a highly effective means of inducing
ovulation and spawning in a number of cultured freshwater fish.

     The main advantages of the Linpe method compared to the traditional
methods include reduced cost of the synthetic drugs, long stability of the
drugs, high predictability of the time from injection to ovulation, decreased
stress on broodstock due to the necessity for only a single set of injections,
and lack of any side-effects on the reproductive cycle or immune system.  The
potential commercialization of the Linpe method for induced ovulation and/or
spawning of cultured freshwater fish will benefit the development of aquacul-
tural production.  Based on the results of our studies, a spawning kit
containing sGnRH-A and DOM will soon be marketed by Syndel Laboratories,
Canada, and Ningbo Fish Hormone Factory, China.
                                  REFERENCES
 Chang, J.P.  and R.G.  Peter.  1983.  Effects of pimozide and des Gly10,  [D-
     Ala6] luteinizing hormone-releasing hormone ethylamide on serum gonado-
     tropin  concentrations, germinal veside migration, and ovulation in female
     goldfish, Carassius auratus.  Gen. Comp. Endocrinol., 52:30-37.

 HCTHAP.   1977.  A new highly effective ovulating agent for fish reproduction.
     Team for Hormonal Application in Pisciculture.  Scientia Sinica, 20:469-
     474.
                                      43

-------
Lin, H.R.,  C. Peng, L.Z. Lu, S.J. Zhou, G. Van Der Kraak, arid R.E. Peter.
     1985.   Induction of ovulation in the loach (Paramisgurnus dabryanua)
     using pimozide and (D-Ala6, Pro9-N-ethylamide) -LHRH.  Aquaculture,
     46:333-340.

Lin, H.R.,  G.V.D. Kraak, J.Y. Liang, C. Peng, G.Y. Li, L.Z.  Lu, X.J. Zhou,
     M.L. Chang, and R.E. Peter.  1986a.  The effects of LHRH analogue and
     drugs which block the effects of dopamine on.gonadotropin secretion and
     ovulation in fish cultured in China.  In:  Aquaculture of Cyprinids.  (R.
     Billard and J. Marcel,  (eds.)  INRA, Paris, France,  pp. 139-150.

Lin, H.R. ,  C. Peng, G. Van Der Kraak, R.E. Peter, and B. Breton.'  1986b.
     Effects of (D-Ala6,  Pro9-NEt)-LHRH and  catecholaminergic  drugs  on
     gonadotropin secretion  and ovulation in the Chinese loach (Paramisgurnus
     dabryanus).  Gen. Comp. Endocrinol., 64:389-395.

Lin, H.R.,  X.J. Zhou, G. Van Der Kraak, and R.E. Peter.  1987a.  Comparison of
     (D-Arg6,  Trp7,  Leu8, Pro9NEt)-luteinizing hormone-releasing hormone
     (sGnRH-A), and (D-Ala6,  Pro9NEt)-luteinizing hormone-releasing  hormone
     (LHRH-A), in combination with pimozide (PIM) or domperidone (DOM), in
     stimulating gonadotropin release and ovulation in the Chinese loach
     (Par amis gurnus dabryanus) .  In:  Proceedings of the Third International
     Symposium on Reproductive Physiology of Fish.  L.W. Grim, D.R.  Idler and
     J. Walsh (eds.).  pp. 33.

Lin, H.R.,  J.Y. Liang, G. Van Der Kraak, and R.E. Peter.  1987b.  Stimulation
     of gonadotropin secretion and ovulation in common carp by an analogue of
     salmon GnRH and domperidone.  In:  Proceedings of the First Congress of
     the Asia and Oceanic Society for Comparative Endocrinology.  E. Ohnishi,
     Y. Nagahama and H. Ishizaki, (eds.).  pp. 155-156.

Lin, H.R.,  G. Van Der Kraak, X.J. Zhou, J.Y. Liang, R.E. Peter, J.E. Rivier,
     and W.W. Vale.  1988.   Effects of  [D-Arg6,  Trp7,  Leu8, Pro9NEt)-LHRH
     (sGnRH-A), and [D-Ala6,  Pro9NEt]-LHRH (LHRH-A),  in combination  with
     pimozide or domperidone, on gonadotropin release and ovulation in the
     Chinese loach and common carp.  Gen. Comp.  Endocrinol., 69(1):31-40.

Omeljaniuk, R.J., S.H. Shih, and R.G. Peter.  1987.  In vivo evaluation of
     dopamine receptor-mediated inhibition of gonadotropin secretion from the
     pituitary of the goldfish, Carassius auratus.  J. Endocrinol.  114:449-
     458.

Peter, R.E., C.S. Nahorniak, M., Sokolowska, J.P. Chang, J.E. Rivier, W.W.
     Vale,  J.A. King, andR.P. Millar,  1985.  Structure-activity relationships
     of mammalian, chicken,  and salmon  gonadotropin releasing hormone in vivo
     in goldfish.  Gen Comp. Endocrinol., 58:231-242.

Peter, R.E.  1986.  Structure-activity  studies on gonadotropin-releasing
     hormone in teleosts, amphibians, reptiles and mammals.   In:  Comparative
     Endocrinology:  Development and Direction.   C.L. Ralph (ed.).  Alan R.
     Liss,  New York, NY.  USA.  pp. 75-93.
                                     44

-------
Peter, R.E.,  J.P. Chang, C.S. Nahorniak,  R.J.  Omeljaniuk,  M.  Sokolowska,  S.H.
     Shih, and R. Billard.  1986.   Interactions of catecholamines and GnRH in
     regulation of gonadotropin secretion in teleost fish.  Recent Prog.  Horm.
     Res., 42:513-548.

Peter, R.E.,  H.R. Lin, and G. Van Der Kraak.  1987.  Drug/hormone induced
     breeding of Chinese teleosts.   In:  Proceedings of the Third Internation-
     al Symposium on the Reproductive Physiology of Fish,  St. John's, New-
     foundland, Canada.  D.R. Idler, L.W. Grim, and J.M. Walsh (eds.).  pp.
     120-123.

Peter, R.E.,  H.R. Lin, and G. Van Der Kraak.  1988.  Induced ovulation and
     spawning of cultured freshwater fish in China:  Advances in application
     of GnRH analogues and dopamine antagonists.  Aquaculture (accepted).
                                     45

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                      ROLE OF STEROID HORMONES  IN GONADAL
                       GROWTH AND MATURATION IN TELEOSTS
                                      by

                              Yoshitaka Nagahama
                                  INTRODUCTION

      It is well established that gonadotrophic hormones play important roles
 in the physiological regulation of gonadal function in teleosts.   In most
 cases,  however,  gonadotropin action on gonadal development is not direct,  but
 rather,  occurs through the biosynthesis of gonadal steroid hormones  that,  in
 turn,  mediate various stages of gametogenesis including oocyte growth,  oocyte
 maturation,  spermatogenesis,  and spermiation.   This article reviews  some  of
 our findings,  mainly based on the amago salmon,  Oncorhvnchus rhodurus.  on the
 physiological regulation of gonadal function by steroid hormones  and the  sites
 and mechanisms of gonadal steroid biosynthesis.

              STEROIDAL MEDIATORS OF OOCYTE GROWTH AND MATURATION

     Vertebrate  oocytes  grow while arrested in the first meiotic  prophase.   In
 teleosts,  as  in  other nonmammalian vertebrates,  the principal  events
 responsible  for  the  enormous  growth of the  oocyte  involve  the  sequestration
 and packaging  of a hepatically derived plasma  precursor, vitellogenin,  into
 yolk protein  (Wallace 1985).   It  seems  most likely that  vitellogenesis  is
 promoted by a  two-step mechanisms  in which  gonadotropins increase ovarian
 secretion  of estradiol-17j9, which  in turn stimulates the hepatic  synthesis and
 secretion  of vitellogenin (Figure  1).   Vitellogenin then is  selectively taken
 up  from the blood stream by developing  oocytes.  In all  of  the teleost species
 studied so far,  elevated levels of estradiol-17 have been  reported in females
 during active vitellogenesis  (Figure 2).

     Besides stimulating  estradiol-17y3 production,  gonadotropin is thought to
 stimulate the ovary to incorporate vitellogenin into the oocytes; however, the
 details of this  action of gonadotropins are unknown.  There  is now biochemical
 evidence that teleosts, similar to other vertebrates, probably possess two
 gonadotropins  (Idler and Ng 1983, Suzuki et al. 1988).   In my review, the term
gonadotropin is  taken  to refer to the glycoprotein-rich "maturational"
gonadotropin (Idler and Ng 1983) or GTH II  (Suzuki et al. 1988).
Laboratory of Reproductive Biology, National Institute of Basic Biology,
Okazaki, Japan
                                     46

-------
                 OOCYTE GROWTH
            Pituitary    OOCYTE MATURATION
          "VC-^L
       ^\^^
Gonadotropin            Gonadotropin
                                            bS<_17a,20p-Dihydroxy-4-pregnen-3-one

                                         Oocyte
                                             Maturation-promoting factor
        Granulosa cell
         Figure 1.   Hormonal control of oocyte growth and maturation
           in salmonids.              
     A period of oocyte growth  is  followed by a process called oocyte
maturation  (resumption of meiosis),  which is  accompanied by several
maturational processes in the nucleus  and cytoplasm of the oocyte.  These
changes occur prior  to ovulation and are prerequisite for successful
fertilization; they  consist  of  the breakdown  of the germinal vesicle (GVBD),
chromosome  condensation and  extrusion  of the  first polar body  (Masui and
Clarke 1979, Goetz 1983, Nagahama  1987c).

     In vitro systems have proven  useful for  investigating the mechanisms of
oocyte maturation  in teleosts.   It has been demonstrated that gonadotrophic
hormones  stimulate meiotic maturation  in follicle-enclosed oocytes, but not in
defolliculated oocytes  (Goetz  1983,  Nagahama 1987c).  Cyanoketone, a specific
inhibitor of 3/3-hydroxy-A5-steroid dehydrogenase,  completely abolished the
maturational effects of gonadotropin (Young et al. 1982).  These  findings
suggested that gonadotropins may trigger maturation by stimulating the
follicle  cells to  synthesize a A4  steroid that acts directly on the oocyte to
cause maturation (Figure  1).

     A variety of  C21-steroids have  been shown to induce oocyte maturation in
vitro  (Jalabert  1976,  Iwamatsu 1978, Fostier et al. 1983, Goetz 1983, Nagahama
et  al. 1983, Greeley et al.  1986,  Canario and Scott 1988, Trant and Thomas
1988).  These include  progesterone,  17a,200-dihydroxy-4-pregnen-3-one
(17a,20^-diOHprog),  17a,20/3,21-trihydroxy-4-pregnen-3-one, cortisol, and
deoxycorticosterone.  Testosterone as  well as other C19-steroids  have been
shown  to  induce  oocyte maturation only at high concentrations.  Estradiol-17/3

                                      47

-------
and other  CIS-steroids generally are not effective in including oocyte
maturation in  teleost oocytes.

     Recently  we purified and characterized,  for the first time in any
vertebrate,  the  maturation-including hormone  of amago salmon from media in
which  immature but  fully grown folliculated oocytes of amago salmon had been
incubated  with partially purified chum salmon,  Oncorhynchus keta.  gonadotropin
(SGA,  Syndel Lab.,  Canada)  (Nagahama and Adachi 1985).   Twenty separate
fractions  were obtained by the  use of reversed-phase high performance liquid
chromatography.   Maturation-inducing activity was found only in one fraction
that had a retention  time coinciding exactly  with l7a,20-diOHprog.   The
purity and final characterization of this active fraction were confirmed
further by thin  layer chromatography and mass spectrometry with authentic
17a,20j8-diOHprog.

     A specific  radioimmunoassay for 17,20y3-diOHprog developed in our
laboratory has been applied to  measure blood  concentrations of this  steroid
during the  sexual maturation  of female amago  salmon.   17a,20/3-DiOhprog levels
were low in vitellogenic females.   A dramatic increase  occurred in mature and
ovulated females  (Young et al.  1983b).   We also found that in masu salmon,
          15
        o
        1
        to
        UJ
                 June
July
                               Aug.   Sept.
                                                             75
                                       Ol
                                       c

                                       03
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                                       I
                                       CO
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                                                             50
                                                            oc
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                                       o
                                       O
                                       i
                                       O
                                       04
                          OOCYTE GROWTH
              -i-Q
Oct.   Oct.

    OOCYTE MATURATION
        Figure 2.  Changes in plasma estradiol-173 and 17a,20g-
          dihydroxy-4-pregnen-3-one levels during sexual maturation
          of female amago salmon.
                                   48

-------
Oncorhvnchus masou. plasma 17a,20y3-diOHprog levels peaked 2 to 4 days prior to
ovulation, coincident with the occurrence of GVBD of oocytes (Yamauchi et al.
1984).   The relative effectiveness of a range of pregnene derivatives in
inducing GVBD was investigated in vitro using amago salmon oocytes.  Of the
steroids tested, 17cc, 20/3-diOHprog was found to be the most effective inducer
of GVBD (Nagahama et al. 1983).  Taken together, these results indicate that
17a,20/3-diOHprog is the major naturally occurring maturation-inducing hormone
in amago salmon (Nagahama 1987b).

     Further studies from our laboratory and others suggest that 17a,20/3-
diOHprog functions as the maturation-inducing hormone common to several
salmonids (Fostier et al 1973, Jalabert 1976, Goetz 1983, Scott et al. 1983,
Canario and Scott 1988).  It also is possible that 17a,20/3-diOHprog acts as an
important steroidal mediator of oocyte maturation in some nonsalmonid teleosts
(Goetz 1983, Stacey et al. 1983, Scott et al. 1984, Greeley et al. 1986,
Upadhyaya and Haider 1986).   Recently, 17a,20/3,21-trihydroxy-4-pregnen-3-one
has  been  identified as  a naturally  occurring maturation-indueing  steroid of
Atlantic  croaker,  Micropogonias undulatus  (Trant et  al.  1986, Trant  and Thomas
1988).  17a,20y8,21-Trihydroxy-4-pregnen-3-one does not  appear to  be  involved
in the  induction of oocyte maturation in salmonids,  however, because there is
no evidence for the presence  of large amounts of this steroid in  the blood of
female  salmonids undergoing maturation or  ovulation  (Canario  and  Scott  1988).

     The  site of 17a,20/3-diOHprog action in inducing oocyte maturation  in
teleosts  is considered to be  at or  near  the oocyte surface (Nagahama 1987c)
 (Figure 1).   17a,20-DiOHprog was found  to be  ineffective in inducing oocyte
maturation  when microinjected into  fully grown  immature oocytes of the
goldfish, Carassius auratus.  but  was effective  when  applied externally.   These
results suggest that 17a,20-diOHprog acts indirectly on germinal vesicles to
induce  resumption of meiosis.  Previous  studies of meiotic maturation in
amphibian oocytes have revealed that cytoplasm  from  maturing oocytes induces
GVBD when injected into immature  oocytes (Masui and  Clarke 1979).  The
activity  responsible for causing  GVBD is called "maturation-promoting factor"
or MPF.

      It,  therefore, was anticipated that a cytoplasmic  factor similar to
 amphibian MPF is newly produced in fish oocytes under the influence of
 17a,20/3-diOHprog.  The hyaline layer obtained from goldfish oocytes treated
with 17a,20/3-diOHprog was able to induce meiotic maturation when injected into
 immature  recipient oocytes (Nagahama and Yamashita 1988).  Thus,  MPF activity
 was present in the cytoplasm of 17cc,20-diOHprog-treated goldfish oocytes
 undergoing GVBD.  Furthermore, MPF from mature oocytes  of goldfish-induced
 GVBD when injected into the South African clawed toad,  Xenopus  laevis,  oocytes
 and vice versa.  It also was shown previously that goldfish MPF induced
 maturation of immature oocytes of the starfish, Asterina pectinifera
 (Kishimoto 1988).  These results suggest that MPF is similar among vertebrates
 and invertebrates.  Moreover, MPF has been detected during metaphase in
 mitotic cells of many species, ranging from yeast to man.  Thus,  MPF may be  a
 widespread initiator of metaphase in both meiotic and mitotic cells  (Kishimoto
 1988).
                                      49

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          STEROIDOGENESIS IN THE OVARIAN FOLLICLE: TWO-CELL TYPE MODEL

      Ovarian follicles of teleosts, like those of other vertebrates, are
 composed of two major cell layers, an outer thecal cell layer and an inner
 granulosa cell layer (Nagahama 1983) (Figure 1).  Development of a simple
 dissection technique to separate the ovarian follicles of salmonids into two
 cell layers, the thecal and granulosa layers, has made it possible to
 elucidate the relative contributions of each layer and gonadotropin in the
 overall process of estradiol-17 and l7a,20/3-diOEprog.  Our in vitro studies
 indicate that the thecal and granulosa cell layers cooperate in the production
 of these two steroidal mediators of oocyte growth and maturation.

                  ESTRADIOL-170  PRODUCTION DURING  OOCYTE GROWTH

      In amago salmon, estradiol-17^ levels in the plasma increase  during
 oocyte growth and rapidly decline prior to oocyte maturation (Kagawa et al.
 1983).   The capacity of intact follicles to produce estradiol-17/3  in response
 to gonadotropin stimulation increases during oocyte growth,  but rapidly
 decreases in association with the ability of the oocyte  to mature  in response
 to gonadotropin (Kagawa et al.  1983).

      Using various follicular preparations  obtained from vitellogenic  amago
 salmon,  we examined the effects of purified salmon gonadotropin on estradiol-
 17 production.   Gonadotropin stimulates estradiol-17/3 production  by intact
 follicles and thecal and granulosa cell layer co-culture  preparations,  but not
 by the  isolated thecal  and granulosa cell  layers  (Kagawa  et  al.  1982).   These
 results  indicate that both cell layers  are  necessary for  gonadotropin-
 stimulated estradiol-17 production.   In contrast,  gonadotropins greatly
 stimulated testosterone production by  thecal  layers  but only slightly
 stimulated testosterone production by  the other  follicular preparations.
 Incubation of granulosa layers  with exogenous  testosterone resulted  in
 elevated estradiol  levels, whereas  isolated thecal  layers  incubated with
 testosterone produced relatively  small  amounts of estradiol-17, which  should
 be attributed  to  contamination  of  thecal layer preparations with granulosa
 cells.   Experiments  examining the  effects of conditioned media from incubates
 of one follicle  cell  layer on steroidogenesis by  the  other cell layer revealed
 that  the thecal  cell  layer produced a steroid precursor that was metabolized
 by estradiol-17^  in  the granulosa cell  layer.  We further  identified
 testosterone as  the major aromatizable  androgen produced by thecal cell layers
 in response to gonadotropin (Adachi and Nagahama, unpublished).

      In  consideration of all of these data, a two-cell type model in the
 production of follicular estradiol-17/S has been proposed (Figure 3).  In this
 model, the thecal cell layer, under the influence of  gonadotropin,  secretes
 the androgen substrate  (testosterone), which diffuses into the granulosa cell
 layer where the aromatase is localized exclusively (Kagawa et al. 1985).
 Estradiol-17^ then would have to be transported back  to the thecal layer for
 secretion into the blood stream, because the granulosa layer is devoid of
 capillaries.  In a recent study, we found that the thecal cell layer from
 amago salmon and the granulosa cell layer from the rainbow trout, Salmo
gairdneri, could produce the same effect as has been reported using
combination of thecal and granulosa cell layers form the  same species.   The
                                      50

-------
reciprocal use of amago salmon granulosa and rainbow trout thecal cell layers
also is effective.  This finding implies that there may be little species
specificity of each of these cell layers among salmonids (Nagahama 1987b).

     In Vitro production of testosterone by the isolated thecal layer
preparations obtained each month during oocyte growth and maturation has
revealed that the capacity of the thecal layer to produce testosterone in
response to gonadotropin gradually increased during the course of oocyte
growth and peaked during the post-vitellogenic period; this capacity of thecal
cell layers was maintained by the period oocyte maturation and ovulation
(Kanamori et al. 1988).  As identical seasonal pattern of stimulation of
testosterone production was observed when thecal cell layers were incubated
with forskolin and agents known to raise intracellular cAMP (Kanamori and
Nagahama 1988b).  Furthermore, specific gonadotropin receptors were
demonstrated in both thecal and granulosa cell layers from vitellogenic
follicles  (Kanamori and Nagahama 1988a).  Therefore, these results are
consistent with the general hypothesis  that gonadotropin acts on thecal cell
layers to  stimulate testosterone production through a receptor-mediated
adenylate  cyclase-cAMP system.
1 	 THECAL CELL 	
Cholesterol
CSCC enzyme j% 1
T
Pregnenolone
1
3P-HSD [^ 1
Progesterone
,. J, CH3
17a-Hydroxylase [y f ,1=0
_xxiX>!;"OH
j^^L^^f
I "7 * LJ^ i fA^f*i*tf\ /i*'M*^^"iQotoBpf^nf* ^^
vvs
^
i
1
1
1
1
1

1
r GRANULOSA CELL -



CH3
HO-C-H
^xjx'srOH
rrr
20P-HSD 0XtfixJ
_ 17a,20p-Dihydroxy-
i/a nyaroxyproges leroi it;  -q- pregnen o one
C17.20lyase ^ 1
Androstenedione
1
17&-HSD |^> iLx
rrr
0
-------
      Aromatase activity  is granulosa cell layers increased during
 vitellogenesis and decreased rapidly in association with the ability of the
 oocyte to mature in response to gonadotropin  (Young et al. 1983a, Kanamori et
 al. 1988).  This decrease in aromatase activity appears to be coincident with
 the decreased ability of intact follicles to produce estra.diol-17/3 in response
 to gonadotropin.  Because testosterone production in thecal cell layers did
 not decline during this  time, the reduced production of estradiol-17/3 by
 postvitellogenic follicles is due, in part, to decreased aromatase activity in
 granulosa cell layers.  Although the mechanism of the induction or activation
 of the salmonid granulosa cell aromatase system is unknown at this point, we
 recently have found that in the medaka, Oryzias latipes.  which has a
 reproductive cycle much shorter than that of salmonids, gonadotropin
 stimulates the de novo synthesis of aromatase via an adenylate cyclase-cAMP
 dependent step (Matsuhisa et al.,  unpublished).  It is possible that prolonged
 treatment of salmonid granulosa cell layers with gonadotropin may stimulate
 aromatase activity.

 17o!,20-DiOHprog PRODUCTION DURING OOCYTE MATURATION

      17a,20-DiOHprog production by intact follicles at different stages of
 development showed that the capacity of the follicles to  respond to
 gonadotropin by synthesizing this  steroid was acquired immediately prior to
 the natural maturation period (Young et al.  1983b,  Kanamori et al.  1988).
 Using incubation techniques similar to  those used for the studies on
 follicular estradiol-17 production,  a  two-cell type model  has been proposed,
 for the  first time  in any vertebrate, for the follicular  production of
 maturation-inducing hormone (Figure 3).   In this  model, the thecal  cell  layer
 produces  17/3-hydroxyprogesterone that traverses the  basal lamina and is
 converted to 17tt,20y3-diOHprog by the  granulosa cell  layer where gonadotropin
 acts  to enhance  the activity of  200-hydroxysteroid dehydrogenase (20/3-HSD),
 the key enzyme  involved in  the conversion of 170-hydroxyprogesterone  to
 17a,20-diOHprog  (Young et  al. 1986,  Nagahama 1987a,b).

      The  first  step of the  stimulating  effect of  gonadotropin  in thecal cell
 layers is receptor-mediated activation  of adenylate  cyclase and formation of
 cAMP, the major site of action probably occurring at  the  steroidogenic step
 between cholesterol and pregnenolone  (cholesterol side-chain cleavage enzyme
 systems  (Kanamori and Nagahama 1988a,b).   Gonadotropin appears  to promote
 formation of one or more  labile  proteins  required for the delivery of
 cholesterol  to the mitochondrial cytochrome  P-450 system  in thecal cell
 layers.   In  contrast, granulosa  cells lack the  side-chain cleavage enzyme
 systems.   The action of gonadotropin on 200-HSD enhancement: in granulosa cells
 was mimicked by forskolin, by dbcAMP, but not dbcGMP, and by two
 phosphodiesterase inhibitors (Nagahama et al. 1985a, Kanamori and Nagahama
 1988b).   Furthermore, gonadotropin and forskolin caused a rapid accumulation
 of cAMP with maximum levels at 30 to 60 min.  These findings are consistent
with the view that cAMP is the second messenger of gonadotropin action.

     In addition to cAMP, calcium appears to play an important role in the
 gonadotropin regulation of steroidogenesis in amago salmon granulosa cells.
Galmodulin inhibitors such as trifluoroperazine (TFP), N-(6-aminohexyl)-l-
naphthalenesulfonand.de hydrochloride (W5) and N-(6-aminohexyl)-5-chloro-l-
                                    52

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naphthalenesulfonamide (W7) strongly prevented gonadotropin, cAMP, and
forskolin-stimulated 20^-HSD activation in granulosa cells in a do'Se-dependent
manner (Nagahama, unpublished).

     In vitro experiments utilizing both protein synthesis inhibitors
(cycloheximide and puromycin) and RNA synthesis inhibitors (actinomycin D,
cordycepin and a-amanitin) have revealed that the gonadotropin and cAMP
induction of 20/3-HSD activity in graulosa cell layers is dependent on the
activation of new RNA and protein synthesis.  Our time course studies further
suggest that de nove synthesis of 20j8-HSD in vitro in response to gonadotropin
and dbcAMP occurs, and consists of gene transcriptional events within the
first 6 hours of exposure to gonadotropin and dbcAMP and transcriptional
events 6 to 9 hours after the exposure to gonadotropin and cAMP (Nagahama et
al. 1985b).  Thus, these results suggest that gonadotropin causes the de novo
synthesis of 20/3-HSD in the amago salmon granulosa cell through a mechanism
dependent on RNA synthesis.  The induction of 20/3-HSD activity by gonadotropin
in amago salmon granulosa cells is a good example of differentiated  functions
expressed by the target cells in response to peptide hormone stimulation.

     Immediately prior to oocyte maturation, intact follicles acquire an
increased ability to produce ^a.^O^S-diOHprog in response to gonadotropin.
Although granulosa cell layers first acquired the ability to convert exogenous
17a-hydroxyprogesterone to 17a, 20/3-diOHprog in response to gonadotropin about
2 months prior to oocyte maturation, thecal cell layers did not develop the
ability to produce I7a,20p-diOHprog in response to gonadotropin until
immediately prior to oocyte maturation.  Thus, thecal cell function  seems to
be especially critical for the acquisition of intact follicles to produce
170,20)8-diOHprog in response to gonadotropin (Kanamori et al. 1988).

            STEROIDAL MEDIATORS OF SPERMATOGENESIS AND SPERMIATION

     Spermatogenesis and  spermiation in teleosts have been considered to be
under gonadotropin control  (Clemens and Grant 1965, Billard et al. 1982).   It
generally is assumed, however, that gonadotropin does not act directly to
induce spermatogenesis and spermiation in teleosts but works in concert with
testicular somatic elements to stimulate the production of  steroidal
mediators.  In male amago  salmon, plasma levels of testosterone and  11-
ketotestosterone are high  during the later stages of spermatogenesis and
rapidly decline  after the  onset of spermiation  (Ueda et al. 1983a, Sakai,
unpublished)  (Figure 4).   In contrast, the levels of 17a,20/3-diOHprog are low
during spermatogenesis and increase sharply at  the time of  spermiation  (Ueda
et al. 1983b)  (Figure 4).  These results suggest that, in amago salmon,
androgens are  involved in the  later stages of spermatogenesis, whereas
17a,20)8-diOHprog is involved in spermiation.

     The  involvement of  17a, 20/3-diOHprog in the process of  spermiation was
strongly  supported by our studies demonstrating that a single  injection of
gonadotropin  to  non-spermiating amago  salmon induced precocious spermiation 1
to 2 months prior to the  normal spermiation period, concomitant with a marked
increase  in plasma  levels  of 17a, 20/3-diOHprog.  Similarly,  two  successive
injections of 17a, 20/3-diOHprog caused  precocious spermiation, but the  response
to 17a, 20/3-diOHprog was  of lesser magnitude than to gonadotropin.  Neither
                                      53

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                                                      SPERMIATION

                                        SPERM A TOGENESIS
                                                                  12
                                                                  10 g>

                                                                  6!
                                                                     X

               Feb.  Mar.  Apr.   May  June  July  Aug. Sept.  Oct.  Nov.
         Figure 4.  Changes in plasma 11-ketotestosterone and 17a,20g
           dihydroxy-4-pregnen-3-one levels during sexual maturation
           of male amago salmon.
 testosterone nor 11-ketotestosterone were effective (Ueda et al.  1985).
 Considered together,  these results provide evidence to suggest that 17a  200-
 diOHprog is a testicular steroidal mediator of gonadotropin-induced
 spermiation in salmonids.   Similar suggestion has  been made for goldfish
 (Kobayashi et al.  1986).

      It  seems most likely that  the development of  sperm motility in mammals is
 dependent on hormones,  especially androgens (Hoskins  et al.  1978).   Nothing is
 known, however,  about the  role  of hormones in the  acquisition of sperm
 motility in teleosts.   We  have  begun to explore this  question by acquiring
 basic information  about the effects  of steroid hormones on  the acquisition of
 sperm motility in  salmonids.  In  masu salmon,  sperm in the  testis and  sperm
 duct are  not motile.  If sperm  duct  sperm  are  diluted with  fresh water,  then
 for the  first time, they gain motility.  On the contrary, if testis  sperm are
 diluted with fresh water,  they  will  not become  motile.  Thus,  two separate
 processes  are involved  in  the induction of sperm motility in salmonids   One
 is the acquisition of motile ability when  shifting  from the  testis to  sperm
 duct, and  the  other is  the initiation of motility when  diluted with  fresh
water.

     Early in  the breeding season, however, sperm from  the sperm duct do not
gain motility even when they are diluted with fresh water.  This
                                      54

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characteristic,  namely the period during which sperm become motile, makes the
masu salmon a good system for studying the endocrinological mechanisms
involved in the acquisition of sperm motility.  Effects of two successive
daily intramuscular injections of three steroid hormones (17a,20/3-diOHprog,
testosterone, 11-ketotestosterone) on the acquisition of sperm motility were
examined in masu salmon during the early breeding season.  Injections of
17a,200-diOHprog significantly raised the percentage of motile sperm and the
duration of sperm motility.  In contrast, the other two steroids showed no
significant effect on both values.  Similar stimulatory effects of 17a,20/3-
diOHprog on sperm motility were observed in the Japanese eel, Anguilla
japonica.  These results suggest that 17a,20y8-diOHprog is involved in the
acquisition of sperm motility in masu salmon and eel (Yamauchi et al. 1987).
Further studies are currently under way to define the mechanisms by which
17a,20-diOHprog injections stimulate sperm motility.

                          TESTICULAR STEROIDOGENESIS

     In vitro incubation studies on 11-ketotestosterone production by
testicular fragments at different stages of development have revealed that  the
capacity of the fragments to produce this steroid in response to gonadotropin
is high during the later stages of spermatogenesis and rapidly declines after
the onset of spermiation.  ,In contrast, the capacity of testicular fragments
to produce 17a,20/3-diOHprog is low during spermatogenesis and increases
sharply at the time of spermiation.  Thus, a distinct shift in the
steroidogenic pathway from 11-ketotestosterone to 17a,20/J-diOHprog appears  to
occur in the testis of amago salmon around the onset of spermiation (Sakai  et
al., unpublished).

     The preceding findings led to the conclusion that the testis is the
principal source of 11-ketotestosterone and 17a,20;9-diOHprog.  Using three
different testicular preparations (intact testicular fragments, sperm-free
testicular fragments and sperm preparations), we examined the effects of
gonadotropin, 17a-hydroxyprogesterone, and testosterone on 11-ketotestosterone
and 17a,20/3-diOHprog production.  Incubation of intact or sperm-free
testicular fragments with gonadotropin, 17a-hydroxyprogesterone or
testosterone resulted in a highly significant increase in 11-ketotestosterone
levels in the incubation medium.  Sperm preparations did not produce 11-
ketotestosterone at any incubation condition.  Intact testicular fragments'
produced a large amount of 17a,20/3-diOHprog in response to gonadotropin or
17o;-hydroxyprogesterone.

     Neither sperm-free testicular fragments nor sperm preparations alone were
capable of producing 17a,20/3-diOHprog in response to gonadotropin.  17a-
Hydroxyprogesterone markedly stimulated 17a,20/?-diOHprog production by sperm
preparations, but not by sperm-free testicular preparations (Ueda et al.
1984).  After incubation of intact testicular fragments with  [1AC]17a-
hydroxyprogesterone, 17a,20/3-diOHprog and 11-ketotestosterone were identified
as major metabolites (Figure 5).  In contrast, only 17a,20/3-diOHprog was
identified when sperm preparations were incubated with [14C]17o;-
hydroxyprogesterone.  No further metabolite was obtained when sperm
preparations were incubated with testosterone.  These results indicate that
different testicular elements are the sites of synthesis of 11-
                                     55

-------
ketotestosterone and 17a,20/3-diOHprog.  Furthermore, it is suggested the
involvement of sperm in  the production of 17a,20/3-diOHprog in the testis of
spermiating salmonids.

                                 CONCLUSION

     Inasmuch as 17a,20/3-diOHprog was identified as the maturation-inducing
hormone,  we now have two known biologically  important mediators of oocyte
growth and maturation in salmonids,  estradiol-17a and 17a,20/3-diOHprog.   It is
now established that the granulosa cells  are the site of production of  these
two mediators but production by the ovarian  follicle depends on the  provision
of precursor steroids by the thecal cell  layer  (two-cell type model).   A
dramatic shift in the steroidogenic pathway  from estradiol-17a to 17a,20/3-
diOHprog occurs only in ovarian follicle  cells  immediately prior to  oocyte
maturation.   These results suggest that differential changes in the
steroidogenic capacity of thecal granulosa cell layers are prerequisite for
growing oocytes to enter the final stages of maturation.   Resolution of the
                               Progesterone

                             Androstenedione
                         17a-Hydroxyprogesterone
                               Testosterone

                        17a,20p-Dihydroxy-4-
                                   pregnen-3-one
                           11-Ketotestosterone

                         11P-Hydroxytestosterone
            Testis
                                  ORIGIN
Sperm
       Figure  5.  Metabolites of [14C]17a-hydroxyprogesterone  (*)
         by  incubation of intact testicular fragments (Testis)  and
         sperm preperations (Sperm)  obtained from spermiating  amago
         salmon.  Authentic steroid  standards are indicated.
                                   56

-------
molecular events regulating these changes may provide new insight into the
hormonal events regulating oocyte growth and maturation.

     17a,20-DiOHprog also has been shown to be a major mediator of
gonadotropin-induced spermiation in amago salmon.  This steroid also was shown
to be involved in the acquisition of sperm motility in masu salmon and eel.  A
shift in steroidogenic pattern from 11-ketotestosterone to 17a,20/3-diOHprog
seems to occur in the testis immediately prior to or during the spermiation
period.  Although the question of which cell type is the principal source of
testicular production of 11-ketotestosterone and 17a,20-diOHprog is still
unresolved, it is likely that different testicular elements are the sites of
synthesis of 11-ketotestosterone and 17cc,200-diOHprog.  These findings may be
one of the best documented examples of the relationship between testicular
steroidogenesis and male germ cell maturation in nonmammalian vertebrates, and
will certainly provide the basis for a study at the molecular levels of
hormonal regulation of male germ cell development.

                                ACKNOWLEDGMENTS

     The research of Y. Nagahama was supported by Grant-in-Aid for Special
Project Research (Project No. 63640001) and Scientific Research from the
Ministry of Education, Sciences, and Culture of Japan.

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Nagahama, Y., K. Hirose, G. Young, S. Adachi, K. Suzuki,  and B. Tamaoki.
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Nagahama, Y., H. Kagawa, and G. Young.  1985a.  Stimulation of 17a,20/3-
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Oncorhynchus rhodurus. by cyclic nucleiotides.  J. Exp. Zool.  236:371-375.

Nagahama, Y., G. Young, and S. Adachi.  1985b.  Effect of actinomycin D and
cycloheximide on gonadotropin-induced 17a,20/3-diOHprog-4-pregnen-3-one
production by intact follicles and granulosa cells of the amago salmon,
Oncorhynchus rhodurus.  Develop.  Growth and Differ.  27:213-221.

Scott, A.P., D.S. MacKenzie, and N.E. Stacey.  1984.  Endocrine changes during
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Hormones.  Gen. Comp. Endocrinol.  56:349-359.

Scott, A.P. J.P. Sumpter, P.A. Hardiman.  1983.  Hormone changes during
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Stacey, N.E., R.E. Peter, A.F. Cook, B. Truscott, J.M. Walsh, and D.R. Idler.
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 Suzuki, K.,  H. Kawauchi, and Y. Nagahama.  1988.   Isolation and
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 glands.  Gen. Comp. Endocrinol.  71:292-301.

 Trant,  J.M.  and P. Thomas.  1988.  Structure-activity relationships of
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 Trant,  J.M. , P.  Thomas, and C.H.L.  Shackleton.  1986.   Identification of
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 Ueda, H.,  A. Kambegawa, and Y.  Nagahama.   1984.   In vitro 11-ketotestosterone
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 isolated sperm of rainbow trout,  Salmo gairdneri.   J.  Exp.  Zool.  231:435-439.

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 Ueda, H.,  Y. Nagahama,  F.  Tashiro,  and L.W. Grim.   1983a.   Some endocrine
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 Ueda, H.,  G.  Young,  L.W.  Grim,  A. Kambegawa,  and  Y.  Nagahama.   1983b.
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 Upadhyaya, N. and S.  Haider.  1986.  Germinal vesicle  breakdown in oocytes  of
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 Yamauchi,  K.,  H.  Kagawa, M.  Ban,  N. Kasahara,  and Y. Nagahama.  1984.   Changes
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 final oocyte maturation of the  masu salmon, Oncorhynchus masou.  Bull.  Jap.
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 Yamauchi, K., T. Miura, and Y.  Nagahama.   1987.  Involvement of 17a,20/3-male
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Young, G., S. Adachi, and Y. Nagahama.  1986.  Role of ovarian thecal and
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Young, G., H. Kagawa, and Y. Nagahama.  1982.  Oocyte maturation in the amago
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Young, G., H. Kagawa, and Y. Nagahama.  1983a.  Evidence for a decrease in
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Reprod. 29:310-315.

Young, G. L.W. Grim, H. Kagawa, A. Kambegawa, and Y. Nagahama.  1983b.  Plasma
17a,20)8-dihydroxy-4-pregnen-3-one levels during sexual maturation  of  amago
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vitro production by ovarian follicles.  Gen. Comp. Endocrinol.  51:96-106.
                                      61

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                       INDUCING EGG CELL FUSION WITH LASER

                                       by

             Ger Guo-chang1, Zhang Wen-di2,  Zhau Pel?, and Wang Qu3
                                  INTRODUCTION

       Cell fusion was first demonstrated with, inactivated viruses by Okada
 (1960).   A chemical method was  established with polyethylene  glycal  (PEG)  by
 Kao  (1970),  and an electrofusion method was developed by  Zimmermann  (1980).
 Such methods have several disadvantages,  including  a  low  viability rate  for
 fused  animal cells.   The  recent development of a laser microbeam technique,
 which^has been especially successful  in micro-surgery at  the  subcellar level
 for  single living cells,  offers a new cell, fusion tool that could produce  less
 damage than other methods.   In  our work,  inducing,egg cell fusion in the loach
 with the  laser was successful.   Fused eggs  can cleave and develop.   Some of
 them developed to gastrul stage;  a few even,to hatch  stage and lived on.

      A Nd:  YAG laser of  nano-second  pulse  and high peak  power density is  the
 basic source.   A 0.53-um  laser  generated by second-harmonic crystal  KDP and a
 0.59-um laser generated by a dye  laser,  which  is  pumped by a  0.53-um laser,
 are  employed as a working laser for microbeam  irradiation of  cells.  An
 auxiliary He-Ne laser beam isfor aiming.   Both laser beams are collinearally
 focused through a microscope on the specimen.   The radius  of  the spot affected
 by the laser can be  adjusted by objective magnification of the microscope and
 the  laser energy.

      Breeding  was induced with HCG out  of  the  loach breeding season.  After
 fertilization,  the eggs were put  into  a medium  containing  0.002 gram of
pronase per  milliter.  Three minutes  later, the membrane was peeled.   The eggs
were washed  three  times and placed in  a 10% Holtfreter solution.   The eggs
 then were  loaded  onto  a special plastic plate that placed  two adjacent eggs
close together.  The plate with the eggs was put on the stage of.microscope.
The He-Ne  laser first was aimed at the contact region of two adjacent eggs and
then the working laser beam was irradiated onto the eggs.   The magnification
of microscope objective and energy was just within the range in which the pore
of membrane made by laser beam could reseal automatically.  If the  density of
     ^quaculture Department,  Ocean University of Qingdao,  PRC.

     2Physics  Department,  Ocean University of  Qingdao,  PRC.

     3Biology  Department,  Ocean University of  Qingdao,  PRC.

                                      62

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energy was too high, the membrane would be unable to reseal itself and the
pore would be permanent so that the intercellular organelles would flow out of
cells and affect the viability of the egg.  The threshold should be measured
by experiment.  For fertilized loach egg, it was 10 uJ/um2 by our practice.
After the irradiation, the eggs are returned to the cultured medium and
examined, and the development behavior is recorded.


                                    RESULTS

      After irradiation, the paired eggs at first appeared as a dumbbell.
About 2 hours later (at 24C), the fusing eggs were rounding into a spheroid,
the lipid at the contact region between the two cells disappeared, and the
whole volume was doubled compared with a normal egg.  The two eggs were fused.
The experiment has been repeated on 120 pairs of eggs; of these, 37 pairs were
fused.  Compared with the normal fertilization of eggs, the cleavage rate was
90% and the fusion rate was about 40%.

      Most of the fused eggs cleaved.  In two cases, the eggs did not cleave,
and it was found that some of fused eggs cleaved rather slower than the normal
egg cell.

      The development patterns of embryo were divided into two types,
depending on the relative position of the two blastodiscs in the fused eggs.

      1. The two blastodiscs were so far apart that they could not join
together.  Most of  the fused eggs belonged to this type.  For most of them,
development was arrested prior to the blastula stage.  Some of the further
developing embryos  apparently maintained separative cleavage; a few of them
developed to mophopoiesis.  For the others, one of the two blastodiscs ceased
cleavage at certain stage of development; another continued to develop
further.

      2. The two blastodiscs were on the same side and close to each other.
They bean to cleave separately.  But as the number of blastomeres gradually
increased, they joined together and continued to develop to single blastula,
gastrula, embryonic coelm, even newly hatched larvae.

      The number of embryos developing to various stage are listed in Table  1.
            TABLE  1.  NUMBER OF  EMBRYOS DEVELOPING TO VARIOUS STAGES
                              Blastula
 Development
 type         Cleavage
 Larva
               Single
                Double
                                             Gastrula
    Single
Double
     1

     2
27

 8
9

5
5
1
3
        1
        1
        2
                                      63

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      In on-going work  in  1988, three pairs of fused eggs developed--one into
a normal larvae, with one  head and one tail, normal in external structure.  It
developed faster than the  unfused control egg, however, and lived for 10 days.
The other two also hatched--one with two heads and one tail, the other with
the tail fused together, but with two separate bodies.  They died in a few
days.
                                  DISCUSSION

      The results showed that the larvae, fused from two eggs by laser
developed well and  that there was no margin in the middle of the body.  The
unfused fish could  join two bodies into one, but without laser inducing, there
was a very clear margin within the body that separated the fish into two.  It
also was found that the size of the fused egg was bigger than a normal one.
The diameter of a normal egg was 0.925 mm and the volume of it was 0.31 mm3.
The fused one was 1.175 mm in diameter and 0.63 mm3 in volume.   It was about
twice the size of a normal egg.

      The laser inducing fusing could occur only when two closed egg cells
were arranged closely, and the two blastodiscs were on the same side and
close.  If the two  bla:stodiscs were on contrary sides, or if one was up and
the other was down, the fused egg cell could only develop into an abnormal
fish with two heads or other deformities.

      The wavelength of the working laser used in our experiment was 530 nm
and 590 nm.  In another experiment, a laser beam with a wavelength of 266 nm
was used to treat the fertilized eggs of the loach.  Results showed that it
was harmful to the  cell and caused many embryo variations.   So in this
experiment, the wavelength of the working laser used was 530 nm, 590 nm to
avoid any variations, and it was found it was not harmful for the cell--the
embryo developed normally.

      The technique did not work on the walking catfish.  The inner pressure"
of the catfish egg  was higher than loach.  After laser irradiation, the eggs
were broken.  The method and dosage of laser for fish egg cell fusion needs
more research.
                                    SUMMARY

      The results showed that the laser-induced cell fusion is available in
fertilized fish eggs, and a high survival rate was achieved.  It was
considered that the method is new and valuable for egg cell fusion.  The work
is still going on with more analysis and research on embryo development and
egg fusion needed.
                                  REFERENCES
Ger, G. C. and Y. L. Zan.  Unpublished.
      normal breeding season.
Inducing loach breeding out of the
                                     64

-------
Kao, K. N.  1974.  A method for high frequency intergeneric fusion of plant
      protoplasts.  115(4):355-367.

Okada, Y. et al.  1961.  Isolation of a new variant of HVJ showing low cell
      fusion activity.  4(3):217-220.

Teissie, J. et al.  1984.  Electrofusion a new highly efficient technique
      for generating somatic cell hybrids.  150:477-482.

Zimmermann, U. and P. Scheurich.  1981.  High frequency fusion of plant
      protoplasts by electric fields.  151(1):26-37.
                                      65

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          TESTOSTERONE AND 11-OXOTESTOSTERONE CHANGES  DURING AN ANNUAL
                           CYCLE AND INDUCED SPAWNING
                IN BLUNT SNOUT BREAM (MEGALOBRAMA AMBLYCEPHALM

                                 Zhao Wei-xin1

                                  INTRODUCTION

      Higher vertebrate  androgen comes  from two parts,  adrenal cortex and
 gonads  (testis  and  ovary),  but  the  testis  is the major  place where  androgen  is
 synthesized and excreted.   Among the excreted androgens,  the biological
 activity  of testosterone is the  highest.   The testis  of fish can  synthesize
 not only  testosterone and androstenedione  but also  another  androgen,  11-
 oxotestosterone (11-OT or 11-ketotestosterone, 11-KT),  which has  very high
 activity.   The  11-OT  level  in blood of  Atlantic salmon  (Salmo  salar)  is much
 higher  than the testosterone level  (Hunt and Wright 1981).   In Atlantic salmon
 (Hunt and Wright  1981),  rainbow  trout (Salmo gairdneri")  (Schulz 1984,  Scott  et
 al. 1980, Simpson and Wright 1977)  and  goldfish (Carassius  auratus)  (Kobayashi
 et al.  1986), testosterone  levels in blood of female  fish are  higher  than
 those of  male fish  of the same species.  This also  is observed in female
 amphibians  and  reptiles.  The 11-OT level  in blood  of female fish is very low,
 however (Simpson  and  Wright 1977, Wright 1976).  In this experiment, we
 investigate the changes  of  androgen excreution and  attempt  to  discuss  the
 physiological function of androgen  during  the annual  reproductive cycle based
 on the  studies  of oestradiol by  the author  in 1986.


                             MATERIALS AND  METHODS

      Sampled fish were  the  first sexual maturation of blunt snout bream
 (Megalobrama  amblvcenhala')  collected from a  pond at the Wuj iang County
 Fisheries Institute in Jiangsu province.  Artificially  induced  spawning had
not been used and the  fish had not  spawned spontaneously.   Study fish  (7-13
male and 9-14 female  fish weighing  250  to 700g) were taken  randomly.  Blood
 samples were  removed  from the caudal vein of  each fish at the end of each
month from January to  December 1984.  The serum was collected and stored at
 -40C until assay.  At same  time, body  and gonad weights of sampled fish were
recorded,  and the gonadsomatic index (GSI-gonad wt.  x 100/body wt.)  was
determined.
     1Shanghai Fisheries University, Shanghai, PRC.

                                     66

-------
       Spawning was  induced with human  chorionic  gonadotrophin  (HCG)  (1500  I.
U./kg  body wt. for  female,  a half  dose for male) or HCG plus dried pituitary
gland  of  carp  (500  I. U. HCG + Img CPG/kg body wt. for female,  a half  dose for
male)  in  May 1985.   Blood  samples  were taken before and after  injection, and
the serum was collected and stored at  -40C until assay.

       Radioimmunoassay (RIA) was used  to measure serum T and 11-OT levels
according to the method of Zhao Wei-xin (1987).  3H-testosterone (3H-T) and
3H-ll-oxotestosterone (3H-11-OT)  are the products of British Amersham Company.
T standard and T antiserum were obtained from Shanghai Institute of
Endocrinology, 11-OT standard came from Sigma Company, 11-OT antiserum was
provided  by Dr. R.  S. Wright of the Marine Laboratory, Aberdeen UK.  The
ranges of T and 11-OT standards are 0  to 400 pg  and 0 to 600 pg, respectively.
A method  of comparison between heating and extraction was made  during  T and
11-OT  measurements;  no significant difference was found (P > 0.05).


                                    RESULTS

       The peak level of T  (2.45   0.35 ng/ml, X  SE) was shown in March,  a
month  before GSI'peak (see Figure  1).   The T level then declined to  1  to 2.5
ng/ml  from April to  July with a minimum closing  to base line level in  August.
The second peak (1.17  0.06 ng/ml) was found in November.  At  that  time,  GSI
rose slightly.  The  T level declined again in December.  The annual  change of
serum  T levels was not correlated  with the annual change of GSI, r-0 2892  (P >
0.05).

       The peak level of 11-OT (32.84 5.30 ng/ml) was shown in April, in
accordance with the  peak level of  GSI.  The 11-OT level was 14.30   1.15 ng/ml
in May, about a half-peak  level.   11-OT level decreased continuously to a
minimum (0.31  0.05 ng/ml) in September.  The annual change of 11-OT  levels
was highly correlated with the annual  change of GSI, r=0.8355  (P < 0.01).

       Five mature male and female  fish were injected with HCG and then put
into a concrete-lined pool  (water  temperature 25C).  After 12  hours,  two
females had ovulated, one had spawned, and two had neither ovulated nor
spawned.  At this time, serum T level  of males rose from basic  line level
before injection to  1.53   0.62 ng/ml  (Figure 2).  A high-significance
difference (P < 0.01) was  found during induced spawning.   The serum 11-OT
level  of  males was 4.49   0.35 ng/ml (n-4) 12 hours after injection  (Figure
3).  No significant  difference (P  > 0.05) was found during induced spawning
although  the T level showed a tendency to decrease.

       For the year (Figure  4),  the T level was minimum (0.49   0.08 ng/ml)  in
January and then rose gradually.   After March,  the T level increased rapidly.
The peak  level (5.03  0.45 ng/ml) was shown in April.   The T level declined
slightly  in May and  decreased sharply  in June.   A small peak was shown in July
(2.53  0.26 ng/ml).  There was no obvious change of T level from August to
December  (0.7 to 1.2 ng/ml).  The  annual changes of T level was correlated
with the  annual change of GSI,  r=0.5151 (P < 0.05).
                                     67

-------
                      FKAMJ  JASON
       Figure 1.  Annual changes in serum T, 11-OT and GSI in male
         blunt snout bream  (	T, 	11-OT,	GSI).
j 
E 2-
0)
c
H
1-
rt
T









                        B
Figure 2.  Changes in serum T
  level during induced spawning
  in male blunt snout bream
  (Abefore injection, B24
  hours after injection).



I
8)
C
1-
0
"


7,
6-
5'
4'



31
2.
1-
r>








r












 '

















T


                      B
Figure 3.  Changes in serum 11-OT
  level during induced spawning
  in male blunt snout bream
  (Abefore injection, B12
  hours after injection).
                                      68

-------
      Changes for the first group (in Qinpu Freshwater Fish Farm in May 1985
are shown in Figures 5 and 6.  The mature male and female fish were injected
with HCG + CPG and then put into a concrete-lined pool (water temperature
22C)   After 14 hours, males had produced milt; females had not spawned or
ovulated.  Measurements of serum T and 11-OT levels during induced spawning
indicated that the T level was 1.95  0.53 ng/ml (n-10) in the female before
injection and that the T level increased rapidly to 17.96  4.32 ng/ml (n-10)
14 hours after injection.  A highly significant difference (P < 0.01) was
found during induced spawning.  Serum 11-OT level in females was very low
(0 98  0.08 ng/ml) (n=7) before injection and increased to 1.53  0.18 ng/ml
(n=7) 14 hours after injection.  A significant difference (P < 0.05) was found
during induced spawning.

      From  the second  group  (in Wujiang County Fisheries Institute, June
1985), five mature male  and  female fish were  injected with HCG before being
placed in a concrete-lined pool  (water temperature  25C).  After 12 hours,
three females had spawned or ovulated.  At this  time,  the average T level  of
three spawners was 8 ng/ml and the T  levels of  two  non-spawners were all over
10 ng/ml.


                                  DISCUSSION

      It is generally  thought that T  has  an effect  on  spermatogenesis.  The
metabolic substances of  T, released out of the body with urine, can attract
the  opposite  sex and affect  sexual behavior.  In addition, T  is a precursor of
estrogen biosynthesis.   T may promote gonadotrophin (GTH) to  stimulate  sperm
release  and ovulation.   T has synergism with  GTH and strengthens GTH,  inducing
                                                              ?o
                                                              16
               E
                                                              i?
                                                                CO
                                                                o
                                   M   J   J

                                    Month
           Figure 4.  Annual changes in serum T level and GSI in female
             blunt snout bream (	T,	GSI).
                                      69

-------
        20.
     I  10
1
en
o
1
0

-


T


               . A         B

   Figure 5.  Changes in serum T
     level during induced spawning
     in female blunt snout bream
     (Abefore injection, B24
     hours after  injection).
                                           B
                  Figure  6.  Changes in serum 11-OT
                    level during induced spawning
                    in female blunt snout bream
                    (Abefore injection, B14
                    hours after injection).
 17a-hydroxy-20fi-dihydroprogesterone (17a-20P)  production.   l7a-20P  is
 considered to be  an effective maturation-inducing hormone released by testis
 and ovary.

       Our  results showed that the  GSI  of male fish  reached  its maximum in
 April  (Figure 3).   The  serum T level increased  obviously after January; the
 peak level of T was about a  month  earlier  than  that of GSI.  This indicated
 that T stimulated testis development and spermatogenesis.   After September, T
 level  increased again and formed a small peak in  Autumn.  It may be associated
 with the testis development  of the next sexual  cycle and stimulate the
 spermatogonia proliferation  and spermatocyte formation.  In point of  sexual
 cycle  of female fish (Figure 6), the peak  level of  T appeared in April, also
 about  a month earlier than maximum GSI of  female  fish.  A higher level of T
 was  maintained in May (reproductive season).

       During  a female sexual cycle, the T  level alternated between 1  and 5
 ng/ml  and was  above 2 ng/ml  for 4  months in a year.  But during a male sexual
 cycle,  the T  level  was  lower,  only between 0.2  and  2.5 ng/ml.  This also was
 found  in salmonid--the  T level  was  higher  in female than that in male.  This
 may  closely associate T as a precursor of  estrogen  synthesis.  In blunt snout
bream,  the annual change of  E2 was  similar to that of T (Wright  1976).  The
peak level of  E2 appeared in April, followed by a lower peak in  June,  and a
 still  smaller  peak  maintained from  October to December.  These peaks  indicated
 that the changes of E2  synthesis and release are closely related to  the
changes of T level  in the annual cycle.

      The T level increased  in both sexes after hormone injections.   In the
female, the T  level greatly  increased after injection,  and decreased during
                                      70

-------
ovulating or spawning.  This change also was found in salmonid (Zhab et al.
1987).  At this time, T is not viewed as a precursor of E2 synthesis.   It
seems to be a synergist that acts with greatly released GTH to induce steroid
production and -lead to germinal vesical movement, polarization, and
disappearance.  The metabolite of T, a kind of 5-reduced androgen, was
released as a sex pheromone to attract male fish.  In the male, the T level
also increased.  The T level decreased after injection in male silver carp
(Hypophthalmichthys molitrix) found by author (unpublished).   The changes of T
level during induced spawning requires further study.

      It generally is thought that 11-OT markedly stimulates the development
of secondary sex characteristics in males.  In females, the 11-OT level was
very low and usually not able to be determined.   Therefore, 11-OT has been
used to identify the sexes before the appearance of secondary sex
characteristics (Wright 1976).  In our experiment, the annual changes of male
GSI, T and 11-OT (Figure 6), showed that the releasing content of 11-OT
increased as GSI increased gradually.  The serum 11-OT level rapidly increased
1 or 2 months before reproductive season (May) and the secondary sex
characteristics of male were becoming apparent.   The peak level of 11-OT
occurred in April, simultaneous with the peak of GSI.  The 11-OT peak level
was about 13 times greater than the peak T level.  T synthesized and released
in the Spring may be used to stimulate spermatogenesis and to serve as a
precursor converting into 11-OT.  In the late period of spermatogenesis
(April), however, T mainly converts into 11-OT.

      There was a tendency for 11-OT to decrease in the male with hormone
injection, but no significant difference was found in comparison with pre-
injected.  This was similar to the author's results (unpublished), which were
obtained by culturing mature testis in vitro.  This indicates that 11-OT has
no direct effect on sperm release (Fostier et al. 1984).  The 11-OT level
increased in females after hormone injection.  This may be caused by the
increase of T level and the increase of converting into 11-OT at this time.
11-OT may have no direct effect on oocyte final maturation.


                                  REFERENCES

Fostier, A., R. Billard, and B. Breton.  1984.  Plasma 11-oxotestosterone
      and gonadotrophin in relation to the arrest of spermiation in rainbow
      trout (Salmo gairdneri).  Gen. Comp. Endocrinol.  54:378-381.

Hunt, S. M. V. and R. S. Wright.  1981.  Seasonal change in the levels of
      11-oxotestosterone and testosterone in the serum of male salmon, Salmo
      salar L., and their relationship to growth and maturation cycle.  J.
      Fish Biol.  20:105-119.

Kobayashi, M., K. Aida, and I. Hanyu.  1986.  Annual changes in plasma levels
      of gonadotrophin and steroid hormone in goldfish.  Bull. Japan.  Soc.
      Sci. Fish.  52(7):1153-1158.
                                     7.1

-------
 Schulz,  R   1984.  Serum levels of 11-oxotestosterone in male 17-estradiol
       in female rainbow trout (Salmo gairdneri)  during the first reproductive
       cycle.   Gen.  Comp.  Endocrinol.  56:111-120.

 Scott, A.  P.,  V.  J.  Bye,  S.  M.  Baynes,  and J.  R.  C.  Springate.   1980
       Seasonal variations in plasma concentration of 11-ketotestosterone and
       testosterone  in male rainbow trout,  Salmo  gairdneri Richardson   J  Fish
       Biol.   17:459-505.

 Simpson, T. H.  and R.  S.  Wright.   1977.  A radioimmunoassay for  11-oxotesto-
       sterone:  its  application in the measurement of levels  in  blood  serum of
       rainbow  trout  (S. gairdneri).   Steroids.   29)3):383-398.
Wright, R. s.  1976.  Sexing  live  fish.
      Exploration of the Sea.
International Council for the
Zhao, W-xo Weixin, R-l. Jiang Renliaang, and S-j. Huang Shijiao.  1987.  An
      improved method for the radioimmunoassay of oestradiol and a study of
      annual cycle of serum oestradiol in female blunt snout bream
      (Megalobrama amblvcephalal .   Acta Hydrobiologica Sinica.  11(2) :97-104.

Zhao, W-xo. Weixin.  1987.  Changes of serum steroid levels during ovulation
      in rainbow trout (Salmo gairdneri") .   J. Fish. China.  11(3):205-213.
                                     72

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              NUTRIENT REQUIREMENTS OF JUVENILE ALLOGYNOGENETIC
                   CRUCIAN CARP, CARASSIUS AURATPS GIBELIO

                                     by

              He Xiqin1, Jia Lizhu, Li Zhongjie, and Yang Yunxia
                                 INTRODUCTION
      A nutritionally well-balanced and low-cost formulated diet is essential
to fish farming, especially to the intensive culture of high quality food
fish.  A reasonable feed formulation and high efficiency diet can only be
based on an understanding of the nutrient requirements of cultivated fish.  On
the other hand, because the feed represents up to 50% of aquaculture expense,
one cannot afford the economic loss caused by improper feed and feeding
regimens.  Therefore, the study of nutrient requirements of fish is important
both in basic fish nutrition and in practical fish production.

      Since DeLong et al. studied the optimal protein level in food for
chinock salmon, Oncorhynchus tshawytscha. in 1958, the literature on nutrient
requirements in fish has grown rapidly (Millikin 1982a, NRC 1983).  So far,
the results show that the protein requirements range from 23 to 55% in
different fishes.  Because of different diet compositions and experimental
conditions, there are marked differences in protein requirements even within
the same fish species.  For example, requirements for the grass carp,
Ctenopharyngodon idella. have been reported as 27.66% (Lin Ding et al. 1980),
36.7% (Mao Yongqing et al. 1985a), and 41 to 43% (Dabrowski 1977).
Consequently, the determination of nutrient requirements of fish is not a
matter of simple measurement but a complicated problem involving many factors.

      Crucian carp is a commercially important food fish in China.  Although
crucian carp successfully maintain large populations in ponds and lakes, they
take 3 year to grow to market size (250 g).  Allogynogenetic crucian carp is
an improved breed obtained by using heterologous sperm to initiate the nucleus
development of female crucian carp.  The new breed exhibited good growth
performance in the ponds, cages, and lakes.  Little is known, however, about
the nutrient requirements of this species.  Our experiment was aimed at
investigating the optimum levels of dietary protein, fat, carbohydrate, and
cellulose that would promote the maximum growth of juvenile allogynogenetic
crucian carp.
"Institute of Hydrobiology,  Academia Sinica,  Wuhan,  PRC.

                                      73

-------
                             MATERIALS AND METHODS

      An orthogonal design method was employed in two experiments for a period
of 30 days each.  Nine experimental diets with different levels of nutrients
were prepared according to the orthogonal table Lg(3A) .  A nonprotein diet was
arranged so that NPU could be calculated by the method of Miller and Bender
(1955).  The factors and levels being examined and the composition of
experimental diets are shown in Tables 1 and 2, respectively.  Experimental
diets were formulated by using purified food.  Casein, corn oil and fish liver
oil, potato starch and dextrin, and cellulose were used as protein, fat,
carbohydrate, and cellulose sources, respectively.   Mineral and vitamin premix
were prepared according to NRG methods (1977, 1983).

      The test fish were provided by an aquaculture experiment station and
reared in the fish pond before use.  Before the start of the experiment, the
fish were transferred to indoor, 57 x 33 x 32 cm plastic aquaria of 40-liter
capacity.  Fifteen fish were stocked in each aquarium and feel with commercial
feed containing 30% protein.  Tap water was filtered with activated charcoal,
heated, aerated, and run into each aquarium at the rate of 0.6 1/min.  Test
diets were fed according to the quantity designed by the orthogonal table in
experiment I and fed at 3 to 4% of fish wet weight daily in Experiment II.
The fish were fed three times a day at 0800, 1200 and 1600 hours.  All fish
were weighed every 10 days in order to regulate the feeding quantity or rate.
In Experiment I, the average body length was 4.25 + 0.17 cm, with an average
body weight of 2.53 + 0.31 g.  In Experiment II, the average body length was
5.0  0.4 cm, and the body weight was 3.58 + 0.20 g.
temperature of 23.1  0.8C and 28.8  0.6C,
               The water had a
       a dissolved oxygen content of
5.43  0.83 mg/1 and 5.29  0.71 mg/1,
and II, respectively.
and a pH of 6.9 to 7.1 in Experiments I
 TABLE 1.  NUTRIENTS AND THEIR LEVELS IN EXPERIMENT I AND II

           (g/lOOg body weight/day)
                    Experiment I
                   Experiment II
 Level      Protein   Fat   Carbohy-   Cellu-   Protein   Fat   Carbohydrate
                            drate      lose
1
2
3
0.8
1.2
1.6
0.1
0.2
0.3
0.7
0.9
1.1
0.3
0.4
0.5
1.1
1.3
1.5
0.07
0.17
0.26
0.93
1.11
1.29
                                     74

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TABLE 2.  COMPOSITION OF THE EXPERIMENTAL DIETS (g/100 g body weight/day21)
Experiment I
Diet
number
1
2
3
4
5
6
7
8
9
Protein
0.8
1.2
1.6
0.8
1.2
1.6
0.8
1.2
1.6
Fat
0.3
0.1
0.2
0.2
0.3
0.1
0.1
0.2
0.3
Carbohy-
drate
0.7
0.9
1.1
0.7
0.9
1.1
0.7
0.9
1.1
Cellu-
lose
0.4
0.3
0.5
0.3
0.5
0.4
0.5
0.4
0.3
Protein
1.1
1.3
1.5
1.1
1.3
1.5
1.1
1.3
1.5
Experiment II
Fat
0.26
0.07
0.17
0.17
0.26
0.07
0.07
0.17
0.26
Carbohydrate
0.93
0.93
0.93
1.11
1.11
1.11
1.29
1.29
1.29
a6% mineral premix, 3% vitamin premix and 1% binder (taro powder)

 were  added to  each diet.
      Twenty fish at the beginning and at the end of each treatment were taken
for carcass analysis.  Crude protein was measured by Kjedahl's method, crude
fat by Soxhlet ether extract, ash by burning at 600C for 12 hours, crude
fiber by acid-base washing method, dry weight by drying at 105C for 24 hours,
and carbohydrate by the indirect method of difference.  The obtained data were
compared directly with the range values of orthogonal design.
                            RESULTS AND DISCUSSION
NUTRIENT REQUIREMENTS
      The results of Experiment I are shown in Table 3 and Figure 1.  Among
the nutrients, the change of dietary protein level played the most important
role in the growth of juvenile allogynogenetic crucian carp.  The range value
was 0.45%.  Carbohydrates and cellulose were less important for growth and the
range values were 0.21 and 0.15% respectively.  The dietary fat level seemed

                                      75

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                                    (g/100gBW/day)
      Figure  1.   Relationships between dietary nutrient levels and
        SGR,  FCR,  PDR,  PER,  and NPU of juvenile allogynogenetic
        crucian  carp  for experiment I.
to have no definite effects  on the growth.   The range value was only 0.02%.
As shown in Figure 1,  daily  SGR increased proportionally with increasing
dietary protein level.   The  rate of growth increased rapidly while protein
level increased from  0.8 g to  1.2 g and the increase of growth rate slowed
with further increase in protein intake.   The lowest FCR and the highest PDR,
PER and NPU fell on the  second level of dietary protein, i.e., 1.2 g.  These
results indicated that the optimum protein level lay between 1.2 and 1.6 g.
Although the fat level seemed  to have no definite effect on SGR, the FCR
increased and  the PDR and NPU  decreased with the increasing dietary fat
levels.  The optimum  dietary fat level was 0.1 g.  The dietary carbohydrate
level played a less important  role on the growth of fish.  An increase in SGR,
PDR, PER, and  NPU and a  decrease in FCR were observed as the dietary
carbohydrate level increased.   The optimum dietary carbohydrate level was 1.1
g.  Because the optimum  fat  and carbohydrate level lay on the margin of the
test range, it is necessary  to extend the test level range.  The optimum
dietary cellulose level  was  noted at the middle level, i.e., 0.4 g.  The
growth rate and the utilization of feed and protein decreased on either side
of cellulose level.   So  there  is no need to examine the cellulose level
further.  The  results from Experiment I indicated that the nutrient
requirements were:  protein, 1.2 to 1.6 g;  fat, 0.1 g; carbohydrate, 1.1 g;
and cellulose, 0.4 g/lOOg body weight/day.
                                       77

-------
       The results of Experiment II are shown in Table 4 and Figure 2.  The
 value for dietary protein level was highest in all the parameters examined.
 Dietary protein also played the main role in the diet of fish; carbohydrate
 and fat ranged next in importance.  As shown in Figure 2, the highest SGR,
 PDR, PER, and NPU and the lowest FCR occurred at the middle level of dietary
 protein, i.e., 1.3 g.  No differences in growth rate and little difference, if
 any, in FCR value between fat levels of 0.17 g and 0.26 g were observed.  How-
 ever, the PDR, PER, and NPU were better in 0.17 g than in 0.26 g.  So it could
 be considered that the optimum fat level was 0.17 g.   There was not much
 increase in SGR (only 0.02%),  as the carbohydrate increased from 1.11 to 1.29
 g.  On the other hand, FCR increased and PER and NPU decreased as the carbo-
 hydrate level increased.   The optimum dietary carbohydrate level was 1.11 g.

       On the basis of this study,  it can be concluded that,  under the
 experimental conditions,  the nutrient requirements of juvenile allogynogenetic
 crucian carp were:  protein,  1.3 g;  fat,  0.17 g;  carbohydrate, 1.1 g; and
 cellulose,  0.4 g.   Transferring to percent content of nutrients,  the diet
 should contain 39.3% protein,  5.1% fat,  36% carbohydrate (including 2.5% of
 potato starch in the vitamin premix)  and 12.1% cellulose with an additional 6%
 mineral premix,  0.5% vitamin,  and 1%  binder.   The fish obtained maximum growth
 when a diet formulated according to  this  composition  was fed to the fish at
 the daily rate of 3.3% body weight.


 EFFECTS ON FISH BODY COMPOSITION

       The carcass  composition  between the  fish at the end and at  the  start of
 the experiment are shown  in Figure 3.  Dry matter and ash content in  the  fish
 at the end were  2.2% and  11.4% lower;  protein and fat content in  the  fish at
 the end were 1.4%  and 7.8%  higher  than the fish at the start.   In fish  fed
 with the nonprotein diet, protein  and ash  content decreased  dramatically,
 being 17.4%  and  17%  lower,  and fat and dry matter content  increased markedly,
 being 34.9%  and  12%  higher  than that  of the initial specimens.

       The effects  of nutrients and their level  on the  body composition  are
 shown in Table 5.  There was no  significant difference in body composition  due
 to  the  different diets.  The range values  of protein  for gross body
 composition  were 0.6 to 0.8%,  thus there was no definite correlation between
 the protein  content  of the  fish  and the diets.  This result agrees with the
 results  for  channel  catfish, Ictalurus punctatus.  (Page and Andrew 1973);
 striped bass, Morone saxatilis.  (Millikin  1982b); and  red drum, Scaiaenops
 ocellatus, (Daniels  and Robison  1986) but  differs from the results for plaice,
 Pleuronectes platessa. (Cowey  et al.  1972); and rainbow trout, Salmo
gairdneri. (Satia 1974, Austreng and Refatie 1979).

      The lack of correlation between body protein and diet protein is
probably either due to the fact that duration of the experiment was not long
enough  (Millikin 1982b) or that the selected test range is too close to the
optimum level (Papaparaskeva-Papoutsoglou and Alexis 1986).  The dietary fat
level, however, seemed to have some effect on the fish body composition.
Dietary fat was negatively correlated with body protein content and positively
correlated with body fat content (Figure 4).  The regression of body protein
                                     78

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  Figure  2.   Relationships between dietary nutrient levels and
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           60%

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                                   80

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         0.1
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               (g/100gBW/day>
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            0.3
             Figure 4.  Relatinships between dietary fat level and body
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   content on dietary fat level and body fat content on dietary fat level were
   calculated and the equations obtained are Y = -18.5x + 60 77 (r = 0 999)  and
   L".6
-------
      To check whether the two differently designed method produced similar
test results,  the determination of dietary protein requirement by using the
graded design method has been conducted in parallel under the same test
condition as the orthogonal design method.  Results are shown in Figure 6
SGR increased proportionally with increasing dietary protein level up to 40%.
Beyond this level there was a slight retardation of the growth.  This growth
pattern in response to the dietary protein level agrees with results for eel,
Anguilla japonica. (Nose and Arai 1972); plaice (Cowey et al. 1972); grass
carp (Dabrowski 1977); tllapla, Tila^ia ziUii, (Mazid et al. 1979); and black
carp  Mvlonharvngodon piceus. (Yang Guohua et al. 1985), but different from
the results for chinock salmon, (DeLong et al. 1958); carp, Cyprinus cjipio
(Oeino and Saito 1970) gilthead bream, Chrvsoohrvs aurata, (Sabaut and Laquet
1973)- and rainbow trout (Satia 1974).  The relationship between dietary
protein content and the growth rate of juvenile allogynogenetic crucian carp
can be expressed by a parabolic curve and the polynomial regression formula
was Y - 0.0975 + 0.0223x - 0.000173x  (r - 0.874).  Obviously,  the results
obtained by both experimental design methods were almost the same. . Used
properly, both methods could produce reasonably comparable results.


RELATIVE AND  ABSOLUTE NUTRIENT REQUIREMENTS

       The  nutrient requirements of fish are  usually expressed as  the percent
protein content of the diet.   The protein requirement of juvenile      _
allogynogenetic crucian carp was found to be 39.3%. by our study,  which 
higher than that of grass carp at 22.77 to 27.66% or 36.7%,  that of Tilapia
aurea at 36%  (Davis and Stickney 1978),  but  comparable with that of black carp
at 41% (Yang Guohua'et al. 1985); mud carp,  Cirrhinus molitorella,  at  38.86%
 (Mao Yongqing et al.   1985b);  rainbow trout  at 40% (Satia 1974),  and lower
 than that of snakehead,  Channa microoeltes.  at 52% (Wee and TAcon 1982);  and
plaice at 50% (Cowey et al.  1972).

       The percentage of protein, however, is a ratio numerator whose value-
 depends directly on the quantities of other components in the diet that make
 up the denominator (Bowen 1987).  Moreover,  the common expression of nutrient
 requirements solely in terms of a "dietary percentage" has itself limited
 value unless it is related to the food intake and subsequent growth of the
 animal (Tacon and Cowey 1985).  For example, Ogino (1980) reported a decrease
 in the dietary protein requirement of juvenile carp and rainbow trout from 60
 - 65% to 30  - 32% when the feeding level was increased from 2 to 4% body
 weight per day in both species.

       Thus,  the relative  requirement expressed as dietary percentage is by no
 means a good measure of nutrient requirements except that it  is convenient for
 formulating  diet.  The absolute requirement expressed as protein (gram ) per
 (100 g) body weight per day not only accurately expresses the daily protein
 requirement  by the fish but also allows valid comparison of data obtained by
 different laboratories.   The relationship between relative protein requirement
 and absolute protein requirement could be expressed  as:
       Absolute protein  requirement  (g protein/lOOg body weight/day) 
  protein requirement  (%) x  feeding rate  (g feed/lOOg body weight/day.
relative
                                       83

-------
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0.4-
    0.3-
            14 -
                     0.7          0.9          1.1
                          Dietary carbohydrate level
                             (g/100gBW/day)

            Figure 5.  Relationships between  dietary
               carbohydrate level and body ash content.
            10    20    30   40    50    60
                       Dietary protein level (%)
                                           i
                                           70
80
    Figure 6.  Effect of dietary protein level on the
      specific growth rate of juvenile gynogenetic
      crucian carp.
                                 84

-------
      By contrast, if the relative protein requirement of fish is converted to
the absolute protein requirement, the protein requirement of juvenile
allogynogenetic crucian carp is found to be 1.3 g/100 g body weight/day by
this study, which is higher than that of mud carp at 0.8 g/100 g body
weight/day, that of snakehead at 1.04 g/lOOg body weight/day, that of plaice
at 0.75g/100g body weight/day, comparable with black carp at 1.23 g/100 g body
weight/day, and lower than that of grass carp at 1.65 to 1.76 g/100 g/100 g
body weight/day, that of rainbow trout at 1.8 g/100 g body weight/day, that of
Tilapia aureus at 3.18g/100g body weight/day.  Obviously the comparison of
protein requirement in different fishes in terms of absolutes requirement is
quite different from that of the comparison of relative requirement between
fishes.  More data should be accumulated and further research is needed in
this respect.

                                ACKNOWLEDGEMENT

      The authors wish to thank Prof. Jiankang K. Liu for his continued
support and advice throughout the study and critical review of the manuscript.
                                   REFERENCES

 Austreng,  E.  and T.  Refatie.   1979.   Effect of varying dietary protein level
       in different families of rainbow trout.   Aquaculture 18:145-156.

 Bowen,  S.H.   1987.  Dietary protein requirements of fishes -  a reassessment.
       Can. J. Fish.  Aquat.  Sci. 44:1995-2001.

 Cowey,  C.B.,  J.A. Pope,  J.W.  Adron,  and A.  Blair.   1972.   Studies on the
       nutrition of marine flatfish - the protein requirement  of plaice
       (Pleuronectes platessa).   Br.  J. Nutr. 28:447-456.

 Dabrowski, K.  1977.  Protein requirements  of grass carp  fry  (Ctenopharvngodon
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 Daniels, W.H.  and E.H.  Robison.  1986.  Protein and energy requirements of
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 Davis,  A.T.   and R.R. Stickney.  1978.  Growth responses  of Tilapia aurea to
       dietary protein quality and quantity.  Trans. Am. Fish. Soc. 107:479-
       483.

 DeLong, D.C., J.E. Halver,  and E.T.  Mertz.   1958.   Nutrition  of salmonid
       fishes VI.  Protein requirements of chinock salmon at two water
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 Lin Ding, Mao Yongqing,  and Cai Fasheng.  1980.  Experiments  on the protein
       requirements of grass carp (Ctenopharyngodon idellus (C. & V.))
       juvenile.  Acta Hydrobiologica Sinica 7:207-212.
                                       85

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 Mao Yongqing,  Cai Fasheng,  and Lin Ding.   1985a.   Studies  on the  daily
       requirements of protein,  carbohydrate,  fat  minerals  and fiber  of
       juvenile grass carp (Ctenopharvngodon idellus  C.& V.).   Transactions  of
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 Mao Yongqing,  Cai Fasheng,  and Lin Ding.   1985b.   Studies  on the  nutritional
       requirements for optimum growth of  mud carp, Cirrhinus  molitorella  (C. &
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 Mazid, M.A. , Y.  Tanaka,  T.  Katayama,  M. A.  Rahman, K.L.  Simpson,  and
       C.O.  Chichester 1979.   Growth response of Tilipai  zillii fingerlings  fed
       isocaloric diets with variable protein levels.  Aquaculture 18:115-122.

 Miller, D.,  and A.  Bender.  1955.   The determination of  the net utilization
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 Millikin, M.R.   1982a.   Qualitative and quantitative nutrient requirements  of
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 Millikin, M.R.   1982b.  Effects of dietary protein concentration  on  growth,
       feed efficiency, and  body composition of striped  bass,   Trans. Am.  Fish
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 Nose,  T.,  and  S. Arai.  1972.   Optimum level of protein in purified  diet  for
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 NRG.   1977.  Nutrient requirements of domestic animals.  Nutrient requirements
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 NRG.   1983.  Nutrient requirements of warmwater fishes and shellfishes.
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 Ogino, C.   1980.   Protein requirements of carp and rainbow trout.  Bull  Jap
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 Page, J.W.  and  J.W. Andrews.  1973.   Interaction of  dietary levels of protein
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 Papaparaskeva-papoutsoglou,  E. and M.  N. Alexis.   1986.   Protein requirements
      of young  grey mullet,  Mugil  capito.   Aquaculture 52:105-115.

 Sabaut, J.J. and P. Luquet.   1973.  Nutritional requirements of the gilthead
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 Satia, B.P.  1974..  Quantitative protein requirements of rainbow trout.
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Tacon, A.G.J. and C.B. Cowey.   1985. Protein and amino  acid requirements.
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                                     86

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Watanabe, T.  1982.  Lipid nutrition in fish.   Comp.  Biochem.  Physiol
watan ^.^   ^ R  _  &nd A G j  Tacon   1982.  A preliminary study on
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      Sci. Fish. 48:1463-1468.

Yang Guohua, Li Jum  , Guo Luji, and Gu Daoliang.  1985.  Optimum level of diet
      protein for black carp fingerlings.  Scientific Reports of the Shanghai
      Fisheries Research Institute No. 1, 1-7.
                                      87

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              POTENTIAL FOR USING CANOLA MEAL AND OIL IN FISH DIRTS

                                        by

                   D. A. Higgs1,  J.R.  McBride1, B.S.  Dosanjh1,

                               U.H.M.  Fagerlund1



                           DEVELOPMENT OF CANOLA SEED
       Canola seed and rapeseed share a common history as the former has been
 derived from the latter by genetical selection.   The English word rape in
 rapeseed originates from the Latin word rapum, which means turnip.   Both rape
 and turnip belong to the Brassica genus of crops,  which also include mustard,
 cabbage,  rutabagas, broccoli,  and kale (Sauer and Kramer 1983).   The oilseed
 forms of rape (rapeseed) have a long history;  Indian Sanskrit writings dating
 from 3500 to 4000 years ago refer to rapeseed as  a source of cooking and
 illumination oil (Boulter 1983).   In Europe,  rapeseed was employed  for
 illumination and soap making before the thirteenth century (Bell  1982).

       The impetus for Canadian rapeseed production stemmed from a critical
 need for  rapeseed oil as a lubricant in naval and  merchant ship steam engines
 during the Second World War (Miller 1988).  Following the war, rapeseed  oil
 was  used  increasingly as a food product until studies on rats  given rapeseed
 oil  caused human health concern because of  the oil's  high content of eico-
 senoic and erucic acids.   This  concern proved to be fallacious because there
 is no  known hazard to humans from ingesting high erucic  acid rapeseed oil
 but  it nevertheless  stimulated  Canadian plant breeders to  develop cultivars of
 ^a?oNSed  that haVS 1<>W erucic acid content  (Boulter 1983,  Sauer and Kramer
 1983).  This initiative  was enhanced further by additional observations of
 myocardial  fat infiltration  (cardiac lipidosis),  myocardial necrosis  (necrotic
 cardiac lesions),  impaired mitochondrial function and enlarged pale adrenals
 in rats receiving high-erucic-acid rapeseed oil (Kramer and Sauer 1983  Sauer
 and Kramer  1983).

      Although the development of  rapeseed strains with low erucic acid
content abolished cardiac lipidosis in rats, this was not the case with
respect to myocardial lesions.  Subsequently,  however, it was shown that this
latter problem was not a unique toxic consequence of ingestion of low erucic


xWest Vancouver Laboratory, Department of Fisheries and Oceans,
 West Vancouver, BC, Canada

                                     88

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acid rapeseed oil but arose from an imbalance between primarily saturated and
monounsaturated fatty acids (Kramer and Sauer 1983).

      Besides high erucic acid content, most of the rapeseed produced in the
world before 1974 was characterized by a high content of glucosinolate com-
pounds.  The hydrolysis of these compounds by the enzyme myrosinase in poorly
processed meals or by intestinal microorganisms can yield several possible
products (e.g., isothiocyanates, 5-vinyloxazolidine-2-thione or goitrin,
thiocyanates, and nitriles) depending upon the conditions of hydrolysis
(Tookey et al. 1980).  Some of these, e.g., goitrin,  suppress thyroid func-
tion.  This  is manifested by thyroid hypertrophy, reduced levels of intrathy-
roidal iodothyronines, depressed thyroid hormone titres and altered affinity
of serum albumin for thyroxine (Higgs et al. 1979).  Other consequences may
include reduced conversion of thyroxine to 3,5,3'-triiodo-L-thyronine (T3) in
the liver and kidney and possibly alterations in the capacities and affinities
of nuclear T3 receptor sites.   Concomitant depression of food intake,
decreased food conversion, reduced growth, and histopathological changes in
the liver and kidney were reported (Higgs et al. 1979).

      These  adverse responses naturally restricted the use of high
glucosinolate rapeseed meal as a protein supplement in diets for poultry and
livestock.   Hence, genetical selection efforts were undertaken in Canada to
produce rapeseed having low levels of erucic acid and glucosinolates.  This
was accomplished successfully in 1974 when the world's first Brassica napus
variety of rapeseed with the foregoing traits was released.  In 1977, this
feat also was accomplished in a summer variety of Brassica campestris.  The
meals and oils derived from these new rapeseed varieties were shown subse-
quently to have dramatically improved nutritive value for poultry and live-
stock relative to  the old varieties  (Clandinin 1986).  To clearly distinguish
between the  two types of oil-bearing seeds, the new varieties were renamed
canola.

      Today,  Canada's production has switched almost entirely to canola seed.
The oil from this  seed, which contains less than 2% erucic acid versus 25% to
55% in high  erucic acid content rapeseed oils (Ackman 1977), now accounts for
60% of all edible  oil products such  as mayonnaise, salad dressings, margarine
and shortenings manufactured in Canada (Miller 1988).  Canola meal, by defi-
nition, now  contains less  than 30 /zmoles of glucosinolates per gram of air-
dried, oil-free meal.  The level of  glucosinolates in canola meals is about
eight- to ten-fold less than that in meals from high glucosinolate varieties
of rapeseed  (Clandinin 1986).


                   WORLD PRODUCTION  OF RAPESEED/CANOLA SEED

       The overall  assessment of the  potential for  including novel sources of
protein and  lipid  in animal diets includes considerations of supply,  cost,
and nutritive value.  Of  the 191.7 million tonnes  of oilseeds produced  on a
worldwide basis  in 1986/87, soybeans,  cottonseed,  rapeseed/canola seed, sun-
flower seed, and groundnuts  (shelled)  comprised, respectively, 51.4,  14.3,
10.3,  9.7, and 7.2 percent.  Thus rapeseed/canola  seed ranked third in  the
worldwide production of oilseeds.
                                      89

-------
       The major producers of the 19.8 million tonnes of rapeseed/canola seed
 in that period were Ohina (29.6%), Canada  (19.2%), India (13.9%), Poland
 (6.5%), France (5.4%), West Germany  (5.2%), the United Kingdom (4.8%), and
 Denmark (3.1%).  Other nations such  as Sweden, Czechoslovakia, East Germany,
 Hungary, Bangladesh and Pakistan each produced less than 500,000 tonnes
 (Canadian Gains Industry 1987).  Thus, considerable rapeseed/canola protein
 and oil are available on a worldwide basis for inclusion in animal foods,
 considering that canola seed typically contains 41% to 43% fat and 21% to 23%
 crude protein (Robblee et al.  1986).

       In Asia, however,  most of the rapeseed grown is high in glucosinolate
 and erucic acid content.   There the meal presently is used primarily as an
 organic fertilizer or a livestock feed supplement.  Moreover,  in the Asian
 countries of China, India,  Pakistan,  and Bangladesh,  the oil is used domes-
 tically for human consumption (Pigden 1983).   In Canada and Europe,  canola/
 rapeseed meals are employed totally as high protein dietary supplements for
 animal foods (Downey 1983) .                                            ':;


               COST AND NUTRITIVE VALUE OF  CANOLA/RAPESEED MEALS
                      AS PROTEIN SUPPLEMENTS IN FISH FOOD

       Per unit protein,  the  cost  of fish meal  is  about  twice that of canola
 meal  and soybean  meal  in North  America.   Therefore, both of these oilseed
 meals are  potentially  very attractive as  partial  or total". replacements, for
 fish  meal,  when considering  availability and cost only.  Unlike  soybean meal,
 however,  it is not practical to consider canola and rapeseed meals as  complete
 replacements for  fish  meal in circumstances where the fish  species have high
 protein  requirements and  fish meal  is providing most of the dietary  protein
 needs.   This is because canola/rapeseed products  have about 37% protein
 compared with 65%  to 72%  for fish meal and  50% for soybean  meal.

       By contrast,  this is not  true for canola/rapeseed concentrates that
 are prepared from  sound,  cleaned, dehulled  seeds  from which oil-  and water-
 soluble, nonprotein constituents have been  removed (Jones 1979).  These
 concentrates not only have extremely  low  glucosinolate  content, but  also
 higher protein and lower nitrogen-free extract (carbohydrate)  levels than
 other oilseed protein meals  (Table  1)  and lupin,  a potentially valuable
 legume for  inclusion in fish foods  (Hughes  1988).

      Moreover, the fibre content in  canola/rapeseed protein concentrates  is
 reduced considerably below that of  canola/rapeseed meals and other plant
 protein sources given in Table 1, except  soybean meal.  This is advantageous
 because fish cannot digest fibre and  dietary levels exceeding  10% have been
 observed to  depress growth,  diet digestibility, food conversion, transit time
 of intestinal  contents, and possibly mineral bioavailability in fish species
 (Higgs et al.  1988).

      The reduced carbohydrate content in canola/rapeseed protein concentrate
 also should potentially enhance nutritive value relative to that of the
 canola/rapeseed meals in salmonid species.  For instance, starch is digested
poorly in salmonids especially when dietary levels are high.  Moreover, starch
                                      90

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 has a low net energy value for rainbow trout, and this is true even for
 glucose, which is known to be highly digestible (Hilton et al. 1987).

       In regard to the mineral composition of the canola/rapeseed meals versus
 the fxsh meals, it is apparent that the former are lower in calcium,  phos-
 phorus,  and zinc than the latter.  When compared with soybean meal, however,
 canola meal'is known to be a better source of available calcium, iron,
 manganese, phosphorus, and selenium for poultry and livestock (Clandinin
 1986).  Further, both canola meal and canola protein concentrate have a better
 balance of indispensable (essential) amino acids than is noted in all of the
 other oilseed meals and in the lupin included in Table 2 for comparison.

       Here is should be mentioned that the nutritive value of animal  and plant
 protein sources is governed to a great extent by how well the available levels
 and balance of ten indispensable amino acids derived from protein digestion
 conform to species needs at the sites of tissue protein synthesis.  This can
 be examined by expressing each of the indispensable amino acids  in a  protein
 source,  including cystine and tyrosine,  as a percentage of the total  weight of
 indispensable amino acids (g/lOOg protein) and then comparing these percent-
 ages to  those derived from the indispensable amino acid requirements  of, for
 example, rainbow trout and carp in the manner described by Oser  (1959) for
 calculation of the essential amino acid index (EAAI).

       This approach has proven to be very worthwhile  for predicting the
 performance of carp (Murai et al. 1984)  and it assumes an adequate dietary
 energy intake as well as a balanced mixture of indispensable and dispensable
 amino acids.   Although the indispensable amino acid pattern in fish muscle and
 whole body protein shows the best correspondence to the requirements for carp
 and rainbow trout by this procedure (EAAI=97),  it also is apparent from Table
 2  that the indispensable amino acid balance in canola  meal shows  excellent
 agreement as well (EAAI=95).   Therefore,  canola/rapeseed meals and concen-
 trates have potentially high nutritive value for finfish if complete
 availability of the indispensable amino  acids  is assumed.

       Data on average and apparent protein digestibility coefficients  for
 canola and rapeseed meals  in rainbow trout provided in Table  3, however, show
 that rapeseed meal has  less  available  protein  than canola meal, full-fat
 cooked soybeans,  soybean meal,  or fish meal.  Moreover,  canola meal has  lower
 available  protein than  soybean meal  in this  species.   Also,  in Atlantic
 salmon,  the availability of  canola protein is lower than that  of  soybean meal
 and fish meal  but differences  between  the  protein  digestibility coefficients
 for these  protein sources are  less when  the  fish are in  seawater.  A trend
 similar  to  that  for protein was observed for the availability  of  energy  from
 these protein  sources in trout and salmon  (Table 4).

      The  causes  of the  reduced protein and energy digestibility  of canola/
 rapeseed meals probably  relate to  their higher content of  fibre compared to
 that in  soybean meal, and to their levels  of phytic acid,  tannins, and glu-
 cosinolates.  Phytic acid, the hexaphosphate of myoinositol, can  decrease
protein  and mineral bioavailability  (e.g., zinc) by mechanisms described by
Higgs et al. (1988).  It is clear that there is a need to remove  this  anti-
nutritional factor by novel processing procedures.  Tannins also are known
                                      92

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to impair protein availability (Sosulki 1979),  although their effect on the
nutritive value of rapeseed/canola proteins may be less than that of phytic
acid and the glucosinolates.

      Information regarding the nutritive value of rapeseed and canola meals,
flours, and concentrates for various finfish species is compiled in Table 5.
The acceptable dietary levels given were based on equivalent growth and food
conversion responses between test and control fish.   For rainbow trout, there
appears to be considerable controversy regarding acceptable dietary levels of
rapeseed or canola meals irrespective of whether this is judged on the basis
of percentage of dietary dry weight or protein.  This may reflect differences
between studies in the initial fish size (age), level of dietary
(glucosinolate) intake on a per body weight basis, and cultural conditions.

      Rapeseed protein concentrate was noted to be more acceptable to rainbow
trout  (Yurkowski et al. 1978) and to chinook salmon (Higgs et al. 1982) than
rapeseed and canola meal, respectively.  Generally,  the nutritive value of
canola meal for Pacific salmon species is consistently good, and acceptable
dietary levels range from 13 to 23% of the dietary protein.  For warmwater
species, the nutritive value of rapeseed meals of low or high glucosinolate
content and of canola meal appears to be higher than noted for coldwater
species.  For instance, in one study on tilapia (Jackson et al. 1982), close
to 50% of the dietary protein as high glucosinolate rapeseed meal was found to
be acceptable.

      Three studies conducted on salmonids (Higgs et al. 1983, Leatherland et
al. 1987, Fagerlund et al. 1987) provide conclusive evidence that glucosino-
lates are mainly responsible for depression of growth and protein utilization
when the diets contain high levels of canola meal.  In this situation, con-
current supplementation of the diets with T3 reinstates normal growth.  In
coho salmon, doses of 1.2 to 10 ppm of T3 are effective,  and dietary protein
quality is not compromised until canola meal exceeds about 27% of the dietary
protein (Figure 1).  Interestingly, hypertrophy of the thyroid follicle epi-
thelial cells was still noted in juvenile coho salmon as the dietary canola
meal levels were increased even though T3 was given orally at 9.45 ppm.
Moreover, dietary protein levels also influenced the response (Figure 2).

      The foregoing work serves to illustrate the importance of totally
eliminating the glucosinolates from canola meal through further genetical
selection and processing innovations.  Significant progress is being made in
both areas and one processing method developed by Rubin and Diosady (1984) and
Naczk et al.  (1986) not only almost entirely eliminates glucosinolates from
rapeseed or canola meals, but also concurrently elevates the protein content
to about 50% because of fibre, carbohydrate and lipid reduction.

      Thus the rapeseed and canola meals have high nutritive value for
warmwater species.  The use of canola meal as a protein supplement in diets
for coldwater species, especially rainbow trout, however, requires further
enhancement of its nutritive value through reduction of fibre, carbohydrate,
phytic acid and glucosinolate content.
                                      97

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              AVAILABILITY, COST AND NUTRITIVE VALUE OF RAPESEED
                      AND CANOLA OIL FOR FINFISH SPECIES

      In 1986/87, rapeseed/canola oils ranked third (6.79 million tonnes)  in
terms of the world production of oils and fats.  The production of rapeseed/
canola oils was exceeded only by palm (7.87 million tonnes)  and soybean oils
(14.5 million tonnes) (Canadian Grains Industry 1987).

      Generally, the prices of fish oils are less than those of edible
vegetable oils such as soybean and rapeseed/canola oils and  animal fats such
                                         -t	r
                     3O        35        40        45
                       PROTEIN (% of dry matter)
 i
50
         Figure 1.   Influence of dietary level  of  canola meal and
           protein  on final mean weight  of  juvenile  coho salmon
           (T3 = 10 ppm)  (Higgs  et  al.,  unpublished  data).
                                    98

-------
          c 35
         'o>
         I
          Q.
          >. 30
          D
         < 2O
         UJ
         <->  10
                               4.02
   - 1 - 1 - 1 - 1 - 1
20      25       30       35      4O      45
                    PROTEIN  (% of dry matter)
                                                                 50
          Figure 2.  Influence of dietary level of canola meal and
            protein on thyroid follicle epithelial cell height (pm;
            13 = 9.45 ppm) of juvenile coho salmon (Higgs et al. ,
            unpublished data) .
as lard (Bimbo 1987).  As Dosanjh et al. (1988) point out, however, it is
likely that within 10 years, the cost and availability of high quality marine
lipids for fish diets will be much higher and lower, respectively, than at
present because of increased utilization of these valuable lipids in the human
diet because of health concerns.

      Thus,  it may be prudent to identify alternate sources of supplemental
lipid to marine lipids for inclusion in fish diets in the near future.  The
selection of the appropriate alternate lipid source for a given species
depends upon its needs for essential fatty acids.  In this regard, the
essential fatty acid requirements of some of the important cultured finfish
species are provided in Table 6.

      Here,  it is readily apparent that considerable differences exist between
species in their essential fatty acid requirements.   Chum salmon, carp, and
eel perform well on balanced amounts (equal amounts) of linolenic acid
(C18:3w3) and linoleic acid (C18:2w6).  By contrast, tilapia requires solely
Cl8:2w6 or C20:4w6 (arachidonic acid) and rainbow trout and coho salmon have
more stringent needs for fatty acids of the linolenic series (w3 fatty acids).
Further, juvenile fall chinook salmon appear to metabolically adapt to high
dietary w6 fatty acid content (2.6%; Dosanjh et al. 1988) when the w3 fatty
                                      99

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        TABLE 6.  ESSENTIAL FATTY ACID REQUIREMENTS OF SELECTED FISH SPECIES
        Species
                            Source"
Essential fatty acid requirements
        Rainbow trout
        (Salnio gairdneri)
        Chum salmon            1,2
        (Oncorhynchus keta)
        Coho salmon             3
        (Oncorhynchus kisutch)
        Carp
        (Cyprinus earpio)

        Eel
        (Anguilla japonica)

        Tilapia
        (Tilapia zillii)
2: 20% and < 80% of the dietary lipid level
(>5%) as linolenic acid (C18:3co3) or > 10%
and < 40% as a combination of
eicosapentaenoic acid (C20::5u3) and
docosahexaenoic acid (C22:6o>3). Growth is
inhibited when C18:2u6 > 1%.

10% of the dietary lipid content as a  .
combination of C20:5w3 and C22:6w3
(w3 HUFAs).  1% C18:2t<>6 (linoleic acid)
+ 1% C18:3o>3.

1-2.5% of the diet as C18:3w3
Growth is inhibited when C18:2w6 > 1% or
when C18:3eo3 > 40% of dietary lipid content.
C18:2w6
              C18:3o>3
0.5% C18:2o>6 + 0.5% C18:36
        a (1) Watanabe  (1982),  (2) Takeuchi and Watanabe (1982), (3)  Yu and Sinnhuber
        (1979)
acid content is simultaneously high.  Rainbow trout and coho salmon show
reduced growth, however,  when the dietary level of Cl8:2w6 exceeds 1%.

      The percentages  of some of the  important fatty acids in canola and
rapeseed oil,  selected marine lipids, and soybean and  corn oil are given in
Table 7.   The most striking difference  in fatty acid composition between the
edible  vegetable oils  and the marine  lipids is the total absence of w3 highly
unsaturated fatty acids  (w3HUFAs; C20:5w3+C22:6w3) in  the former lipids.   The
wSHUFAs have higher essential fatty acid activity than CIS:3w3 in trout  and
their presence is essential in the diets of some cultured marine fish species
(Watanabe 1982).  Relative to soybean oil,  canola oil  has more Cl8:lw9  (oleic
acid) but less C18:2w6.   The percentages of C18:3w3 in these sources are about
equivalent (9%).  Corn oil, another commonly used lipid source in purified
fish diets,  is almost  devoid of linolenic acid and is  very high in linoleic
acid.

      In relation to the finfish fatty  acid requirements given in Table  6,
canola  oil would seem  to have the best  balance between linoleic and linolenic
acids to satisfy the needs of chum salmon,  carp,  eel,  and tilapia.  Moreover,
in species that are sensitive to high dietary levels of linoleic acid (e.g.,
rainbow trout and coho salmon),  this  should prove to be a better source  of
supplemental dietary lipid than either  corn or soybean oil.

                                        100

-------
      Several studies conducted on  salmonids  have shown that canola oil is an
excellent alternative supplemental  dietary lipid (Cho et al. 1974  Dosanjhet
al. 1984, Dosanjh et al.  1988).   By contrast, an unpublished study on rainbow
trout, (R.S. Parker and J.D. Hendricks)  reported by Dosanjh et al  (1984)
found pathological changes,  especially in the heart after *"*"* <^sf
a high erucic acid content.  It should be mentioned, however, that the fatty
acid proflle of  canola oil containing low erucic acid content has not been
found to induce  pathology in Pacific salmon (Dosanjh et al. ^84)   Further,
complete substitution of  canola oil for corn oil in tilapia diets did not
compromise  either growth  or food  conversion (Figure 3).

      Therefore  the potential for including canola oil in cultured finfish diets
as a  source of supplemental lipid  appears  to be excellent.although studies of
long  duration  are required to  confirm the preliminary findings
and ensure  the absence  of pathological changes.
       TABLE 7.  SOME IMPORTANT FATTY ACIDS IN SELECTED MARINE AND EDIBLE
                VEGETABLE OILS (g/100g)
Fatty Pacific1
Acid herring
oil
16:0
18:0
18:l(o9
18 : 2u>6
18 : 3w3
20:lc<>9
20 : 5w3
22:lco9
22:6w3
W6/W3
wSHUFAs
(20:5w3+22:
Unsat./Sat
13.3
1.8
14.1
2.9
1.4
5.3
9.0
17.1
9.6
0.12
18.6
6w3)
4.9
Chilean
anchovetta2
oil
(Talcahuano)
16.5
2.8
8.4
1.1
0.4
8.3
14.5
5.7
8.9
0.048
23.4

2.3
Rapeseed oil3 Canola1 Soybean3 Corn
(Echo; high oil oil oil
erucic, Canada)
2.5 4.4 15.3 11.5
1.0 1.7 4.2 2.2
32.5 58.3 23.6 26.6
18.8 21.7 48.2 58.7
8.9 9.4 8.7 0.8
12.0 1.6
---
23.5 0.8
...
2.1 2.3 5.5 73.4
...

23.4 15.0 4.1 6.2
          &S\Sl9ClLJ.J " ** * - 
         2 Ackman (1982)
         3 Downey (1983)
                                        101

-------
                                 ACKNOWLEDGMENTS

 v   tf-     a"thors gratefully acknowledge the information or assistance provided
 by  Eileen McGregor, Dr.  L.  Diosady,  Dr. W.C.  Clarke,  Dr.  Santosh Lall, Jane
 Higgs, Anna-Maria Hammons and Joan  Stewart.
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                                    102

-------
Cho, C.Y.,  S.J.  Slinger,  andH.S.  Bayley.   1983.   Bioenergetics  of salmonid
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                                     Canola
I   I  Growth rate
     Food conversion
                                                                uJ
6 r K**

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CM
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5 4

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Corn Oil Canola Oil
% of dietary
Lipid content 43 5
( 6.7% of
dry matter)

44.8


                     SUPPLEMENTAL   LIPID SOURCE

          Figure 3.   Growth rate and food conversion  of Tilapia
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            culated  27--29C filtered dechlorinated city water and
            fed 6 to 8X daily their prescribed diet to near maxi-
            mum ration for 57 days  (Higgs et al., unpublished data)
                                     103

-------
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 Downey  R.K   1983.  The origin and description of the Brassica oilseed crops
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                                     104

-------
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                                      107

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     MOBILIZATION OF BODY RESERVES DURING INDUCED GONADAL DEVELOPMENT IN THR
        JAPANESE EEL. ANGUILLA JAPONICA (TEMMINCK AND  SCHLEGEL') :   THE  ROLE
                OF THE PITUITARY GLAND AND CORPUSCLES OF STANWTTTS

                           D.K.O.  Chan1 and E.Y.L. Lau1

                                   INTRODUCTION

        The eel  is a terminal  breeder.   At the beginning of its seaward spawning
 migration, the animal stops  feeding altogether while the gonad is still very
 small  and immature.  The animal,  therefore, must rely on the mobilization of
 body reserves  for both gonadal development and for the long journey  to the
 spawning ground thousands of miles away.  The first successful induction of
 gonadal development, spawning and hatching was achieved by Yamamoto  et al
 U/4a, b) followed by a group of Chinese researchers in Xiamen in 1978 and
 Wang et al. (1980).  Induction of oocyte maturation and fertilization in vitro
 also has been  reported (Yamauchi and Yamamoto 1982).   Up to now, all hatched
 eels died at the prelarval stage, presumably due to insufficient yolk in the
 eggs to carry  embryonic development to the larvae stage when feeding would
 begin.   Furthermore,  the maturing eels were very fragile and many died before
 fooo?Xng^     S6XUal maturity (Ochiai et al.  1974,  Yamauchi and Yamamoto
 iy^;.   The present study was conducted to evaluate the status of the
 biochemical system(s)  responsible for intermediary metabolism and
 protein/purine catabolism as the gonads developed and to determine the
 endogenous source of the nutrients required for gonadal growth.   Concomittant
 changes in the pituitary and corpuscles of Stannius also were observed.


                              MATERIALS AND METHODS


 EELS

      A total  of 86 male  and 89  female eels were used over a  period of 5
 years.   They were caught  during  their  spawning migration  down the  Pearl River
 !!^f *" 1!te4^Umn;   In  the  ^oratory, the eels were transferred to sea
 water at 20 to  22C and maintained without food.  The water quality was
 controlled by circulating charcoal filters and flow-through UV-sterilizers.


 TREATMENT

      Eels were injected intraperitoneally once weekly without anaesthesia,
minimizing stress by raising  the net gently and injecting under water.

     Department of Zoology,  University of Hong  Kong,  Hong Kong.

                                     108

-------
Control eels were given saline.  Experimental eels received 1 i.u./g HCG
(Sigma) and 2 Mg/g acetone-dried powder of mature carp pituitary extracted
into saline and centrifuged at 5000 rpm for 5 minutes.  Some eels received 3
priming doses of 1 yug/g oestradiol-17fi per week.
SAMPLE ANALYSES

      Controls and homone- treated eels were sacrificed from 3 to 32 weeks by
spinal cord transection.  After taking a blood sample by cardiac puncture and
flushing the tissues clear of blood with heparinized saline, the gonad,  liver
and gut were taken and weighed.  Gonads and Stannius corpuscles were fixed in
Bouin and the pituitary gland in Bouin-Hollande for histochemical and
morphometric study.  Samples of liver, white epaxial muscle, and red muscle
were taken for water and lipid content by drying and defatting with petroleium
ether and, later, were extracted with 0.1M HN03-10% acetic acid for Ca and
phosphate determination.  Portions of liver and white epaxial muscle were
homogenized in 20- times its weight of ice-cold buffer (10 mMTris ,pH7 . 5 , ImMfi-
mercaptoethariol and lmMEDTA-Na2)  in a Potter -Elvehj em homogenizer and  a Teflon
pestle and run at 10,000 rpm for 1 minute, sonicated for 5 minutes and
centrifuged at 108,000 x g at 4C for 30 minutes.  The supernatant was used
for enzyme determination at a thermos tated temperature of 20  0.5C.
Conditions (pH, substrate and cof actor concentrations) for enzyme reaction
rates were pre- determined for each enzyme to yield optimal rates (cf .
Bergmeyer 1974).  Blood glucose, lactate, pyruvate, tissue glycogen, plasma
total lipid, free acid, protein, amino acid, phosphate and ammonia were
determined as in Chan and Woo  (1978) .  The carcass was then stored frozen at
20 C.  After thawing some months later, a 1- cm- thick segment of the truck at
the level of anterior tip of the dorsal fin was taken and divided according to
the skin, red muscle, white muscle and vertebra; the size of each compartment
was determined gravimetrically.  The vertebra then was extracted with an acid
mixture  for Ca and P determination.  Ca was determined by atomic absorption
spectrophotometry .
                                    RESULTS
 GONADS

      Male  eels  responded well  to  HCG  alone.  The  gonadosomatic  index reached
 >40%  in some  cases  by 18  to  20  weeks of  treatment.  Milt could be obtained by
 stripping and several animals spermiated spontaneously.  Mature  male eels
 appeared black with marked enlargement of the eye  and  shortening of the jaws.
 Male  eels were divided into  groups according to  the condition of the testis
 showing (I) spermatogonia, (II) spermatocytes,  (III) spermatids  and maturing
 sperms,  (IV)  fully  mature testis with  only sperms, and (V)  regressed testis
 with  sperm  discharged (Figure 1).   Only  a small  percentage  of male eels failed
 to respond  to gonadotropin treatment and these had a GSI of <0.01%.

       Female  eels responded  slowly to  HCG treatment alone but the response
 accelerated if carp pituitary extract  was added.   About 40% of  the eel did not
 respond to  the treatment, their ovaries  remained small (GSI of  3 to 5% at 30

                                     109

-------
            GONADOSOMATIC INDEX
                                                    SKIN
  Q
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30
20;
10;
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       M II  III IV V    F  II III IV V
              LIVER WEIGHT
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GUT WEIGHT
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              HAEMATOCRIT
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                     F  II III IV  V
20-
10-
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                                         M II III IV  V
F II  HI  IV V
                                                 WHITE MUSCLE
40"
n-
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                                         M II  III IV V
                                                        F  II III IV V
                                                 RED MUSCLE
zu
10-
n-
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                                        M n m iv  v
                                                        F II  III IV V
Figure 1.   Changes in gonadosomatic 'index, liver and gut weight,
  haematoprit and proportion of  skin/white muscle/red muscle in
  the trunk region of male and female eels during induced gonadal
  development (M=male control, P=female control; II,  in, iv, v=
  gonadal stages)  (mean+SEM).
                               110

-------
weeks) (data omitted here).  Of those that responded to the treatment, 4
animals reached GSI >60% by 20 to 25 weeks and these were invariably large
animals weighing about 700 to 900g.  These were not sampled until after
regression set in at about 30 weeks.  Priming with oestrogen also appeared to
be beneficial, especially for the initiation of vitellogenin synthesis in the
liver but it was not essential.  In male eels, oestrogen markedly blocked
testis development at the spermatocyte/spermatid state (n=5,  not shown in
figure).  As it was not possible to distinguish between the sexes at the early
stages, oestrogen was discontinued in later experiments.  When the GSI >30%,
some of the female eels took on a greyish silver color with the tip of the
snout and fins turning black.  Eye enlargement was noted.  They were divided
into groups showing (I) ovary with oogonia, (II) endogenous vitellogenesis
with lipid vesicles, (III) exgenous vitellogenesis with accumulation of yolk
platelets, (IV) maturing ovary with oocytes full of yolk granules, and (V)
atretic ovary.  Mature oocytes measured about 1 mm in diameter.


PITUATARY GLAND

      The somatotrophs (Luxol  fast blue acidophil, occupying the center of the
cell chords)  dominated the mesoadenohypophysis in control eels.  As the gonads
developed, gonadotrophs hypertrophied  (basophils: aldehyde fuchsin and PAS,
occupying the periphery).  The remaining  somatotrophs were degranulated with
small cytoplasmic volume and nuclei.   This  change began within 3 weeks after
hormonal treatment  and was pronounced  as  GSI  >10% body weight.  Differentia-
tion  of gonadotrophs was absent  in eels that  did not respond or responded
slowly  (GSI about  3-5% in  30 weeks)  to the  exogenous homone.  In the  proadeno-
hypophysis, as  the  eels were maintained in  seawater, the prolactin cells were
regressed in  the control eels  compared with eels maintained in freshwater.
Regression went further with gonadal development in both sexes so that fewer
prolactin follicles could  be seen in the  pituitary as GSI >20% body weight.
The thyrotrophs were  few and triangular in  shape in control eels and  showed
stimulation beginning at the early stages of  gonadal development  (stage II).
As GSI  >20%,  they  dominated the  proadenohypophysis and  the cells became round,
with large nuclei,  and were  loaded with glycoprotein granules.


CORPUSCLES OF STANNIUS

       In starved control  eels,  the cells  in the  Stannius corpuscles were
uniform in appearance and loaded with  large dense  granules  (aldehyde  fuchsin
 or PAS).   As  the gonads  responded to hormonal treatment, the  corpuscles showed
marked hypertrophy and hyperplasia in  both sexes.  Many large  cells became
 degranulate  especially in large  corpucles,  and many  smaller  cells  located near
 the lumen contained varying amounts of AF/PAS granules.   In male  eels,  the
 response began at early spermatogenesis.   In female  eels,  the  effect  developed
 slowly and peaked as exogenous vitellogenesis became  complete.   In both sexes,
 the corpuscles began to  re-accumulate  granules following gonadal  regression.
 Estrogen alone had limited effects on ovarian development  but suppressed  the
 testis.  Estrogen failed to affect the corpuscles  although plasma Ca  was
 raised markedly by this  treatment, especially in the  male  eel.
                                      Ill

-------
 BODY COMPARTMENT  SIZES
       The haematocrit values were maintained during 30 weeks of starvation but
 declined by 60% as  the gonads developed beyond GSI of 20% (Figure 1).  In eels
 with GSI >40%, the  haematocrit became so low in some cases that the animal
 showed signs of respiratory stress  (hyperventilation, air-gulping) even when
 ambient oxygen was  close to air-saturation.

       The liver size incresed significantly (up 50%) in both sexes as the
 gonads developed.   The gut weight remained unaltered up to 30 weeks of
 starvation but shrunk and lost more than half its weight as GSI >20%.  In the
 typical cross-section of the trunk region,  the proportion of skin and red
 muscle increased at the expense of  the white muscle compartment   Loss of
 white muscle drastically altered the shape of the tail, which became flat and
 thin.
 CALCIUM AND PHOSPHORUS METABOLISM
     _ In female eels, plasma Ca and P rose sharply as exogenous vitellogenesis
 was initiated, showing a 10- to 15-fold increase at the peak.  In the treated
 male eel, the plasma Ca and P rose slightly during the early phase of
 spermatogenesis (stage II) but levelled off later.  The amount of calcium
 deposited in the ovary was 10 times that of the testis of equivalent weight
 Loss of Ca and P from the vertebrae was detected as GSI >10% and progressed'at
 similar rates in both sexes as the gonads enlarged.  The rate of P removal
 from the bone was more rapid,  esppecially in the female, so that the ratio of
 Oa/P in the bone increased significantly from 1.8 to 2.0 as the GSI >20%.

       In male eels,  the white  muscles  began to show Ca deposition as GSI  >10%
 At maturity,  the amount of Ca  in white muscle was more than 8-fold that of the
 corresponding starved controls and the values returned to control levels  after
 sperm discharge.   In female eels,  this Ca deposition occurred much later when
 the ovary_>20% body  weight.  Eels  that showed little response to HCG treatment
 showed neither mobilization of bone minerals  nor hypertrophy of the Stannius
 corpuscles.
 CARBOHYDRATE METABOLISM
      Eels starved up  to  30 weeks were able to maintain normal blood glucose
and lactate levels.  Blood glucose was not affected by gonadal development but
lactate increased in both sexes as the GSI >20%  (Figure 3).  Blood pyruvate
decreased only in the  male eel as the testis developed.  The glycolytic key
enzymes, hexokinase and pyruvate kinase, were well maintained in liver and
white muscles during 30 weeks of starvation.  Liver hexokinase was elevated in
both sexes as the gonads  >10%, while liver pyruvate kinase increased in female
eel only   Muscle pyruvate kinase decreased with starvation but was maintained
in treated eels.  Muscle  lactate dehydrogenase tended to increase as
starvation progressed but showed no further changes as the gonads developed.

      The glycogen concentration in the ovary increased as the oocytes beean
exogenous vitellogenesis.   At a GSI of 30%,  the total ovarian glycogen was
                                     112

-------
             PLASMA CALCIUM
                                               PLASMA PHOSPHATE
   60




   40'




   20'




   0-
     M II HI IV V     F  II  III IV V

           TOTAL GONAD CALCIUM
30.
20;
10;
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                                      50
           TOTAL LIVER CALCIUM
                                    0
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      M II III IV V     F II  III IV V


             BONE CALCIUM
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     M II III IV V    F  II. Ill IV V


            BONE PHOSPHORUS
1.3-
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     M. II
III IV V F II HI IV V M II III IV V F II III IV V
WHITE MUSCLE CALCIUM . , RED MUSCLE CALCIUM

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          C. STANNIUS NUCL. AREA
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10;
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          C. STANNIUS LOB. DIAM.
BU-
40-
n_
I
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\

I
I
\
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I
      M II  III IV V
                                         M II III IV V
                      F  II  HI IV V
 Figure  2.   Changes  in plasma, gonad,  liver, muscle and bone cal-

   cium  and phosphorus and the corpscle of Stannius in male and

   female  eels during induced gonadal development (M=male  control,

   F=female control;  II,  III, IV,  V=gonadal  stage)   (mean+SEM).
                                   113

-------
  over 40-fold that of the control ovary.   Liver and muscle  glycogen store was
  maintained with starvation but became depleted in the  female  eel  as exogenous
  vitellogenetic activity began in the ovary.   As the animals were  starved  the
  gluconeogenic enzymes glucose-6-phosphatase  and fructose-1,6-bis-phosphatase
  were progressively elevated,  but gonadal  development had no further effect on
  these enzvmes (not- shown in -F-5 miv^
these enzymes (not shown in figure).
 LIPID METABOLISM
       The ovary accumulated  lipids during the endogenous and the initial phase
 of exogenous vitellogenesis, reaching peak values as the GS1 reached 10%
 (Figure 4).  At this time, the amount of lipid in the ovary was at least 60
 times that in the testis of  the same weight.  Thereafter, the ovaria lipid
 concentration declined and the total fat in the ovary levelled off.

       Free fatty acid levels in the plasma were elevated as the animals were
 starved.  Female control eels had much higher lipid content in the liver than
 did males.  Starvation caused a decline in liver total lipid towards levels
 found in the male by 20 weeks, but muscle fat content was unaffected by
 starvation in both sexes.  In the male, the liver lipid content rose markedly
 in all groups treated with HCG reaching peak values at GSI of 10%,  which was 4
 to 5_fold that of controls.  In the female,  the liver underwent fatty infil-
 tration during endogenous vitellogenesis and early exogenous vitellogenesis
 During this period of stimulated hepatic lipogenesis,  the pentose-shunt
 enzymes (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydro-
 genase), malic enzyme and cytosol NADP-isocitrate dehydrogenase in the liver
 increased significantly in both sexes.   As the gonads  increased beyond 15%
 body weight,  the amount of lipid in the liver and white muscle  began to
 decline  especially in the female eel;  lipid in the  red muscle  was  unaffected
 compared with that observed in the starved controls.


 PROTEIN AND  PURINE METABOLISM

  _     Plasma  protein increased  significantly in  female  eels  as  exogenous
 vitellogenesis was  initiated  due  to  the  vitellogenin released from  the  liver  
 (Figure  5).   Liver protein  levels  calculated on a  fat-free wet weight were
 unaltered  in  the male eel but increased  in the female.  White muscle protein
 content  declined significantly  as  GSI >10%,  especially  in male eels.  Both
 white  and  red muscle became hydrated.  Eels  that showed little gonadal
 response to the hormonal treatment still showed accelerated  loss of white
 muscle proteins.  Plasma-free amino acids increased progressively with
 starvation.   In male eels, the  levels increased further as the testis enlarged
 and was 3 to 4-fold higher than the control throughout testis enlargement.  In
 female eels,  the levels were unaltered.

      Glutamate dehydrogenase was' reduced markedly in the liver as gonads
 developed in both sexes but rose in white muscle of male eels only   AMP
 aminohydrase of white muscle was markedly elevated in both sexes.  The hepatic
 and muscle uricolytic pathway enzymes such as uricase,  allantoinase, allantoi-
case (only uricase was shown,  others had similar patterns) were markedly
elevated only in male eels.
                                     114

-------
              BLOOD GLUCOSE
   2-


                     I

      M II
III IV V    F  II III IV
  BLOOD PYRUVATE
   40'
   i
           \

   100
      M II  III IV V    F  II III IV
           TOTAL LIVER GLYCOGEN
   50-



      M II III IV V    F  II III IV
             LIVER HEXOKINASE
    4-

      M  II HI IV V    F  II III IV V
           LIVER PYRUVATE KINASE
 o
 a:
           I


   150:
      M  ii ra iv v    F  u m iv
             LIVER LACTATE-DH
 loop
   1
        I



                                1
      M  II III IV V
                      F  II  III IV V
                                       .60
                         o  .30-
                                                 BLOOD LACTATE

                                 1
  1
                                          M
II  III IV V     F II  III  IV  V
   TOTAL GONAP GLYCOGEN
100
50-
i
1
I

                               M  II III IV V    F  II III IV V
                                    WHITE MUSCLE GLYCOGEN
                                       0.1
                                                         i
                                       400
                               M II III IV V    F  II  III IV V
                                    W.MUSCLE HEXOKINASE
                              I
                               i
        \
\

                                                                 I
                                      4000
                                      2000
                               M ii in iv v     F  ii  in  iv v
                                    W.MUSCLE PYR. KINASE
                               i


     i
I

                                                                 I

                          30,000q
                               M II III IV V     F II  III  IV  V
                                     W.MUSCLE LACTATE-DH
                                    20,000-
                          10,000- |J
                               i
                i
        \
                                          M II III IV V
                                                           F II  III  IV  V
Figure  3.   Changes in carbohydrate metabolism in  male and  female
  eels  during gonadal .development: blood, glucose,.lactate and
  pyruvate; gonad, liver and white muscle glycogen  content;  liver
  and white muscle hexokinase, pyruvate kinase  and  lactate dehy-
  drogenase activities  (M=male control, F=female  control;  II,  III,
  IV, V=gonadal  stages)  (mean+SEM).
                                    115

-------
                                   DISCUSSION
 GONADAL RESPONSE

       Like other eel species (Boetius and Boetius 1967),  the testis of the
 Japanese eel responded well to HCG treatment.  In Anguilla anguL11la,  female
 eels typically reached a GSI of about 5 to 8% body weight after prolonged
 treatment with HCG (Boetius et al. 1962) or purified salmon gonadotropin
 (Dufour et al. 1984).  The failure of ovarian development beyond the iniital
 stages of exogenous vitellogenesis was due to the lack of gonadotropin release
 from the pituitary GTH cells.   Administration of the releasing hormone (GnRH)
 or the inhibiting hormone (GnlF)  inhibitor such as pimozide stimulated
 gondotropin release and ovarian development (Dufour et al.  1987).  but  the GSI
 still reached only 6 to 8% body weight.   Failure to respond to injected
 gonadotropin has been attributed to the puberty phenomenon with the GnRH-GTH
 axis being refractory to stimulation when the eel was  too small or too young.
 Carp pituitary extract was much more effective in inducing ovarian
 development, however, and GSI  >30% was obtained in this species after  several
 months (Fontaine 1964a).

       Using Japanese eels caught from the same source  as  the present study,
 Zhang (1988) conducted a series of experiments comparing  the effects of carp
 pituitary extracts,  HCG, LHRH-A,  and GnlF-blockers (dopamine antagonists)  on
 gonadal development.   Results  comparable to those reported in this study were
 obtained by a combined injection of carp pituitary/HCG/LHRH-A (GSI of45% in  30
 weeks),  although carp pituitary alone would bring about substantial response
 over the same period (GSI of 32%) .   LHRH-A or pimozide/domperidone givenalone
 was  not very effective.   Oestradiol-17 alone or in combination with LHRH-A
 increased pituitary and serum  GTH levels and serum vitellogenin levels,  but
 uptake  of yolk protein in the  oocyte did not occur and GSI  remained low.
 Differentiation of gonadotrophs during induced gonadal development has  been
 shown previously (Yamamoto and Naghama 1973).   Both oestrogen and  LHRH
 stimulate the pituitary GTH cells  showing that the positive  feedback system
 was  operative in the  eel (Olivereau et al.  1986)  but is inadequate to bring
 about full oocyte development.  The present study showed  that stimulation of
 gonadotrophs  in the pituitary was  a prerequisite  for the  gonadal response to
 exogenous gonadotropins.   Other pituitary hormones  present  in the  carp
 pituitary or  produced endogenously  (e.g.  TSH)  must  be  important also.
 Exophthalmia,  melanogenesis  and thicking of the skin seen in  maturing eels are
 all well  established  indexes of increased thyroid function in the  eel (Etienne



 CORPUSCLES OF STANNIUS, BONE DEMINERALIZATION AND Ca/P BALANCE

      Olivereau  (1961) first reported  stimulation of the corpuscles of
 Stannius  following the injection of carp pituitary extracts or TSH.  Fontaine
 (1964b) showed that surgical corpuscular removal  (stanniectomy) caused a
marked rise in plasma Ca and K levels and a decline in Na levels.  Because of
the osmoregulatory effects, much attention had been paid to the role of this
gland in calcium and iono-regulation.  In the anadromous eel, the immature

                                     116

-------
yellow eel had small corpuscles, but the migrating silver eels had bigger
corpuscles showing signs of stimulation (Hanke et al. 1969).  This was
interpreted as preparation in anticipation for the ionic challenge when the
fish entered seawater.  In the catadromous salmonids, stimulation of the
corpuscles also was noted at the beginning of the spawning migration and
degenerative changes were recorded after spawning (Heyl 1970).  Again, these
observations were attributed to a decrease in ionic challenge in freshwater
(Carpenter and Heyl 1974).  Plasma calcium, however, rose in both seawater and
freshwater stanniectomized eels but failed to do so in eels maintained in
distilled water (Chan et al. 1967).  The hypercalcemia was transient,
returning to normal within several weeks,  but plasma phosphate decreased
(Fontaine 1967; chan 1968, 1972).

      Stanniectomy did not raise dialysable plasma Ca levels in the European
eel and Japanese eel (Chan and Chester Jones 1978, Chan 1972) and a decline in
urinary Ca excretion was found in these species (Chan et al. 1969, 1972).  In
contrast, plasma dialysable Ca increased in the American eel in Ontario and
urinary Ca increased, which could be reduced by keeping eels in distilled
water (Butler 1969, Fenwick 1974a).  This apparent discrepancy probably arose
from the difference in Ca level in the environment because Sheffield and Hong
Kong tapwater is soft (Ca concentration about 100 uM) but Ontario tapwater is
hard.  The killfish Fundulus heteroclitus was much more sensitive to ambient
Ca levels, which directly affected the hypercalcemic response to Stanniectomy
(Pang et al. 1974).  Stanniectomy indeed increased Ca influx through the
gills, and the corpuscular hypocalcemic factor (hypocalcin or teleocalcin)
inhibited calcium uptake.  This was found to operate in salmonids as well as
in eels (Fontaine et al. 1972, Fenwick and So 1974).

      The inhibition of Ca fluxes and branchial Ca2+-AtPase  has been  used
extensively as a bioassay for the hormone (Copp et al. 1985).  Cultured
corpuscular cells responded to manipulations of the Ca level in culture
medium, discharging secretory products in high Ca media (Aida et al.  1980).
The evidence for the Ca-regulating role for the Stannius corpuscles hormone is
thus overwhelming.  Teleost fish require large amounts of Ca during somatic
growth because of the presence of Ca and phosphate mineral deposits in bone
and scales, and the Stannius corpuscles can function as a regulator of Ca
uptake from the environment, shutting down excessive Ca influx to adjust to
the internal demand.  Over the short term, intravenous injection of Stannius
corpuscles extracts could reduce plasma Ca without affecting plasma inorganic
phosphate concentrations (Kenyon et al. 1980).

      The-present study, however, demonstrated that large amounts of Ca were
needed for ovarian development, and plasma Ca levels remained high throughout
exogenous vitellogenesis but not so for testicular development.  Thus, if the
primary physiological role of the Stannius hypocalcemic factor is to suppress
plasma Ca leves, one should expect different responses in the two sexes.
Furthermore, the inhibition of branchial Ca uptake by the Stannius hormone
would limit the source of Ca from the water for the female eel.  Yet the
corpuscles of Stannius were extremely active (hypertrophy, hyperplasia and
degranulation) in both sexes during induced gonadal development.  Our previous
work on induced gonadal development in eels maintained in freshwater demon-
                                     117

-------
      60
              PLASMA TOTAL LIPI0
                                                 PLASMA FREE FATTY ACID'
      30-S
         I
    I
                  I
                                          1-
     40
           II III IV V    F  II III IV V
              TOTAL GONAD

I

 >-
 a
 o
 m
 o

 o
     20-
                                           M II  III IV  V
                                                 TOTAL
                                                    F  n in iv v



                                      O
                                      ^i
                                      X
                                      CD
      M II III IV V     F II  III  IV  V
            WHITE MUSCLE LIPID
     20
  o:
  u.
  i

1

                 i
                                 I
                                         50
                                            M II  III  IV  V    F  II III IV V
                                                  RED MUSCLE LIPID
      M  ii m iv v    F  n m iv v
            LIVER G-6-P-DHase
                                                             i
o
E
I
200
1 00
o:
771
I

1

                                        20&
                                           M II III IV V     F II  in  IV
                                                 LIVER -P-G-DHase
                                        10&
    50
       M II  III IV  V    F  II III IV V

             LIVER MALIC ENZYME
                                             f
                                             I
 O
 a:
 a.
E59

     M n in iv v
                       F II  III  IV  V
                                          M II  III  IV  V    F H III IV

                                              LIVER ISOCITRATE-DHase
                                           i

                                         M II III IV V
                                                           F II  III IV V
Figure  4.   Changes in lipid metabolism in male  and female  eels
  during induced gonadal  development;  plasma total lipid,  free
  fatty acid levels; gonad,  liver, white and red muscle  lipid
  content; and  liver NADPH-generating  enzyme activities  (M=
  male  control,  F=female  control; II,  III, iv,  V=gonalal stage)
  (mean+SEM).
                                  118

-------
strated that the Stannius corpuscle hypertrophic response was even more active
in freshwater where the environental Ca was low (Subhedar et al. 1981).

      From the present study, it was beyond doubt that the amount of Ca
available for the gonads was actually in excess, especially in the male where
deposition of Ca in the muscles began early in testis development, whereas in
the female eel this occurred when GSI >20%.  The picture is entirely different
if we consider the supply of phosphorus, which is required in enormous amounts
for nucleic acid and phospholipid synthesis (especially in the testis) and
vitellogenesis.  As the animal is starved, phosphorus is in extremely short
supply.  The present data clearly demonstrated that this large source of P
(along with Ca) was endogenous and derived from demineralization of the bone,
which contained at least 92% of the total Ca and 75% of the phosphorus in the
body (Fenwick 1974b).  Starved eels have been shown to decrease in length
suggesting mobilization of resources from bone.  Our own unpublished data show
that deposition of Ca in soft tissues was pronounced in eels starved for over
one and half years.  Like the maturing eels, the eel exposed to long-term
starvation must resort to bone demineralizttion for its phosphorus supply.
Again, the corpuscles of Stannius hypertrophied in these animals.  All these
data strongly suggest that the primary physiological role of the corpuscles of
Stannius is to regulate the phosphate supply from body reserve's.

      A possible role of the corpuscles of Stannius on bone remodelling has
been suggested by Lopez (1970) who showed that Stanniectomy cause the
disappearance of osteoclastic activity and loss of amorphous bone in the eel
(Lopez 1970b).  Using 45Ca  labels,  Lwowski (1978)  suggested that hypercalcemia
resulting from Stanniectomy probably arose from reduction in incorporation of
Ca into bone while release of 45Ca from pre-labelled old bone was unaffected.
Bone mineral  is deposited mainly as calcium phosphate complexes and part of
the deposit is in equilibrium with the plasma Ca and phosphate  levels  (Fenwick
1974b).  Newly formed bone mineral is amorphous and in the octacalcium
phosphate form (Ca8(P04)3) with a Ca/P ratio of  1.33  (Hirshman and Sobel
1965), but as the crytal grows CaC03, other stoichiometric forms of calcium
phosphate are added and the Ca/P increases.

       In the present study, control eel vertebrae contained a Ca/P of 1.8,
which  became  elevated to 2.0 as demineralization progressed.  This pattern is
in line with  the disappearance of amorphous bone from the mineral complex.
Corpuscles of Stannius from immature eels contained only one type of cells
containing dense cytoplasmic granules (Fujita and Honma 1967).  Based on
storage-granule staining and ultrastructure, two types of cells have been
described in  the Stannius corpuscles of other teleosts  (Nadkarni  and Gorbman
1966,  Krishnamurthy and Bern 1974, Wendalaar Bonga et al. 1975).  More recent
immunochemical study using anti-hypocalcin showed, however,  that  these
represented two stages of the same cell type (Kaneko et al.  1988).  The
present study supports the notion that only one cell type is present in the
Stannius corpuscles and that the enlarged cells devoid of dense storage
granules represent the active secretory state.
                                      119

-------
     80'
    40'
               PLASMA PROTEIN
                            1
                                        200
       M II III IV V
                       F  II  III IV V
    50
 o
 0}
 1
 X
 o
             TOTAL GONAD PROTEIN
       M II  III IV V
                       F  II III IV V
    30
   10:
            WHITE MUSCLE PROTEIN
I
                                 I
 I-
 o
  300%
HI
_I
o
t-
o
eg
a.

o
S
       M rr  m  iv v    F ii in iv v
           LIVER GLUTAMATE DH-ase
        I
             I
      M n in iv v
                       F II  III  IV  V
   so-
              LIVER AMPase

   20-
   10'
i
      M II III IV V    F  n III IV V
      	LIVER URICASE
           m
  i
      M II. Ill  IV  V
                      F  II III IV V
                          a.

                         'CD
                          
-------
MOBILIZATION OF BODY RESERVES

      Although data on changes in body weight and length and in body
compartment sizes (liver, gut) have been recorded for eels undergoing induced
gonadal maturation, not much is known about changes in biochemical composition
and the enzymes involved in mobilization of resources.  The migrating silver
eels certainly differed in biochemical composition from the yellow eel,
especially in their higher fat content, and these differences have been
attributed to natural gonadal development (Lewander et al. 1976).  Starved
eels lost between 0.1 and 0.2% body weight per day (Inui and Oshima 1966; Chan
et al. 1969, 1980) with about 30% of total energy derived from protein
catabolism and 70% from oxidation of fat (Fisher 1977, Chan and Woo 1978).
The bulk of the body reserves in support of metabolism came from mobilization
of protein and fat from white muscles (Inui and Oshima 1966, Aster and Moon
1981).  Starved eels have been shown to survive at least 3 years, by which
time about 70% of the initial weight had been lost (Boetius and Boetius 1967).

      With increased mobiliaation of tissue protein and lipid, plasma
aminoacid and free fatty acid levels increased as expected.  Loss of protein
from white muscle during starvation has been shown to originate mainly from
the insoluble fiber component (Moon 1983a,b).  The soluble protein fraction in
liver and muscle that contained the metabolic enzymes was unaltered.
Gluconeogenic enzymes (glucose-6-phosphatase, fructose-1.6-bis-phosphatase and
PEP carboxykinase) and aminoacid metabolizing enzymes (glutamate-pyruvate
transaminase, glutamate-oxaloacetate transaminase and leucine transaminase) in
liver tend to be conserved while muscle FDPase and GPT increased (Moon 1983a).

      Based on the NADPH-generating capacity, the liver appeared to be the
primary site of lipogenesis in the eel (Aster and Moon 1981).   In short term
starvation (1-3 weeks),  there was marked reduction in hepatic fatty acid
synthetase and acetyl-CoA carboxylase, but the NADPH-generating enzymes were
maintained (Abraham et al. 1982).  With prolonged starvation (26 weeks),
however,hepatic glucose-6-phosphate dehydrogenase was reduced while most other
enzymes were conserved (Aster and Moon 1981).  The rate of conversion of 14C-
acetate to fatty acid progressively decreaed with starvation up to 39 weeks
but significant levels of activity still remained up to at least 95 weeks of
starvation (Abraham et al. 1982).  Muscle lipid content actually increased
steadily during prolonged starvation (Moon 1983a).

      Blood glucose and carbohydrate supply was maintained (Dave et al. 1975,
Renaud and Moon 1980), but enzymes of the glycolytic pathway and the
tricarboxylic- acid cycle in the muscles decreased slightly (Bostrom and
Johansson 1972.).  Thus in a broad sense, starvation in eels evoked changes
that tended to preserve the metabolic enzyme machinery, whereas in other fish
species,  drastic decreases in liver and muscle enzyme activities would result.
The present data showed that the unique enzymatic adaptation to starvation in
the eel is put to important use during gonadal development, which takes place
without dietary nutrient supply.

      The nitrogen requirements of the developing testis differ substantially
from that of the ovary,  the former being rich in nucleic acids and the latter
in yolk proteins.  Differences in plasma protein levels in male and female
                                      121

-------
 eels  during  gonadal  development  reflected the hepatic  synthesis and release of
 vitellogenin in the  female  and its  absence in the male (Kara et al. 1980).
 Vitellogenin is a  phophoprotein  that  specifically binds Ca.  In male eels, the
 plasma-free  aminoacid levels  increased  drastically but did not rise in the
 female eel.   This  probably  represented  aminoacid in transit, much of which was
 removed by the  liver for vitellogenin synthesis in the developing female eel.
 Stimulation  of  glutamate dehydrogenase  and AMP aminohydroiase in white muscle
 during gonadal  development  was in line  with the elevated breakdown of muscle
 tissue.  Suppression of glutamate dehydrogenase in the liver was probably due
 to the need  to  conserve nitrogen.   In female eels treated with oestradiol-17,
 mobilization of protein without  concomittant gonadal development was
 accompanied  by  marked elevation  of  ammonia excretion and ammonia loss/oxygen
 consumption  ratio  in eels maintained  in freshwater (Chan and Cheung 1982).
 For purine catabolism, the  enzymes  of the  uricolytic pathway was elevated only
 in male eels suggesting marked increase in purine turnover.  This probably was
 related to the  marked elevation  of  nucleic acid synthesis in the developing
 testis.

      Substantial  increases in the  lipogenic enzymes (NADPH-generating
 systems) were recorded in both sexes  and this was probably due to the large
 demand on lipids for gonadal development.   Increased hepatic lipogenesis
 occurred early  in  gonadal development in both sexes,  but the large demand of
 lip id in the developing oocytes  during  exogenous vitellogenesis later depleted
 this store in the  female eel.  The  ultimate source of  the lipid was certainly
 the white muscle compartment,  which showed a marked decline in fat-store in
 female eels.  Unlike  other  teleost  fish, the eel has little abdominal fat and
 red muscle fat  content was unaffected during gonadal development.

      The present  study showed that the key enzymes in intermediary metabolism
were unimpaired and  in some cases elevated during induced gonadal development.
 In contrast, the marked decline  in haematocrit values placed the animal in
jeopardy and probably constituted the most important factor limiting survival.
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 Fenwick, J.C.   1974b.   The comparative in vitro release of  calcium from
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 Fenwick, J.C. and Y.P.  So.   1974.   A perfusion  study  of the effect "of
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 Fisher, Z.  1977.  Nitrogen changes in starved  eel  (Anguilla  anguLlla) P01.
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 Fontaine, M.  1964.  Corpuscules de Stannius  et regulation  ionique
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Fontaine, M. 1967.  Intervention des corpuscules de Stannius  dans  1'equilibre
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Fontaine, M., N. Delerue, E. Martelly,  J.  Marchelidon, and C. Milet.  1972.
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Fujita, H.  and Y. Honma.  1967.  On the fine structure of corpuscles of
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      77:175-187.

                                     124

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Hanke, W., K. Bergerhoff, and O.K.0. Chan.  1969.  Histological observations
      on pituitary ACTH cells, adrenal cortex, and the corpuscles of Stannius
      of the European eel.  Gen. comp. Endocr.  9:64-75.

Kara, A., Y. Yamauchi, and H. Hirai.  1980.  Studies on female-specific serum
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Heyl, H.L.  1970.  Changes in the corpuscles of Stannius during the spawning
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Hirschman, A. and A.E. Sobel.  1965.  Composition of the mineral deposited
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Inui, Y. and Y. Oshima.  1966.  Effect of starvation on metabolism and
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Kaneko, T., S. Harvey, L.. Lkine, and P.KXT. Pang.  1988.  Localization
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Krishnamurthy, V.G. and H.A. Bern.  1969.  Correlative histological study
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Lewander, K.G. Dave, M.L. Johansson, A. Larsson, and U. Lidman.  1974.
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Lopoez, E.  1970A.  Etude histophysiologique des corpuscules de Stannius
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Lopez, E.  1970.  L'os cellulaire d'un poisson teleosteen Anguilla anguilla L.
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Lopez, E., H.S.  Lee, and C.  Baud.  1970.   Etude histophysique de 1'os d'un
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 Moon, T.W.  1983b.  Changes in tissue ion contents and ultrastrueture of
       food-deprived immature American eels, Anguilla rostrata (LeSueur)
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 Nadkarni, V.B. and A. Gorbman.  1966.  Structure of the corpuscles of Stannius
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       Pacific salmon.  Acta Zool. Stockh.  47:61-66.

 Ochiai,  A., S. Umeda, and M. Ogawa.   1974.   On the acceleration of maturity
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 Olivereau,  M., P.  Dubourg,  P.  Chambolle,  and J.  Olivereau.   1986.   Effect of
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 Pang,  K.T., R.K.  Pang, and W.H.  Sawyer.  1974.   Environmental calcium and the
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 Renaud,  J.M.  and T.W. Moon.   1980.   Starvation and the  metabolism  of
       hepatocytes  isolated from the  American  eel,  Anguilla  rostrata. LeSueur.
       J.  comp.  Physiol.   135:127-137.

 Subhedar, H.A., A.  Cheung,  and D.K.O.  Chang.   1981.  Histophysiology  of  the
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 Wang,  Y., C.  Xhao,  Z. Shi, Y.  Tan, K.  Zhang,  Y.  Li, Y.  Yang,  and Y. Hong.
       1980.   Studies  on  the  artificial inducement  of reproduction  in  common
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 Yamamoto, K.  and Y. Nagahama.   1973.   Cytological  changes in  the
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Yamamoto, K.  and K. Yamauchi.  1974a.   Sexual maturation of Japanese eel and
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Yamamoto, K. , T. Morioka, 0. Hiroi,  and M. Omori.  1974b).   Artificial
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Yamauchi, K. andK. Yamamoto.  1982.  Experiments o,n artificial.maturation and
      fertilization of the Japanese eel (Anguilla. japonLca).  In:  Proc. Int.
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Zhang, M.L.  1988.  Studies on Hormonal Inducing Maturatin in the Eel
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                                   127

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                      UTILIZATION OF INORGANIC  NITROGEN  BY
                                TILAPIA NILOTICA

                                       by
                        Wang Yi  Qiang1 and Shu Xue Bao1

                                  INTRODUCTION

      The  development of  animal  culture throughout the world requires large
 sources  of protein.   Whether the protein source can be  supplied sufficiently
 will affect  this  development directly.   Thus to exploit protein sources is an
 outstanding  problem.

      The  use of  NPN  by organisms to  synthesize protein was examined in
 ruminants  in the  late 19th Century.   Urea and  other nitrogen compounds can be
 utilized by  ruminants.  These compounds  may be decomposed into ammonia by
 microorganisms  in the rumen of the animal; ammonia, then, becomes an effective
 protein  source  through synthesis by microorganisms.  So it is considered that
 urea can be  used  to replace a part of protein  in the diet.

      Urea-contained  nitrogen is about 2.8 times that of protein-contained
 nitrogen,  and the price of urea  is  low.   So several hundred thousand tons of
 urea are used each year in animal feed in the  USA.  Molimoto (1971) reported
 that urea  might be used to replace  about  20 to  30 percent of protein,
 resulting  in greater  economic efficiency.

      Urea was used by microorganisms  to  synthesize protein,  which was
 supplied to  the animal.  The urea was not utilized by the animal directly.

      Animals have different dietary natures requiring different levels of
 dietary  protein.  Some animals that require little protein grow rapidly.
 Obviously, a part of protein is  synthesized in  the animal body.   It may be
 that the non-protein  substance in the animal body, under the condition of
 existence  of nitrogen, transforms into protein.  Wang Yi-Qiang et al. (1987),
using an immunocytochemical method, confirmed  that the grass carp
 (Ctenopharvngodon idellus) and the black carp  (Mylopharvngodon piceus)  have
 different  levels  of insulin in their islets of Langerhans.   Evidently,  their
levels of metabolism of carbohydrates are different.   Grass carp use more
carbohydrates to  transform into protein and,  therefore,  they should be
provided with more nitrogen to increase growth.  So,  whether the animals  can
or cannot use NPN directly to synthesize into protein is a problem for
discussion.
    Shanghai Fisheries University,  Shanghai,  PRC

                                    128

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       Fishes have  different  dietary natures  and  require different protein
 levels in  diet  also.   Satia  (1974) proved  that rainbow trout require 40  to
 46%.   Ogino and Saito  (1970)  and Takeuchi  et al.  (1979) discovered  common carp
 require 31 to 38%.  Lin  Ding et  al. (1980) and Yang Guo-Hua et al.  (1985)
 reported grass  carp need 20  to 25%.  Garling and Wilson (1976) proved  catfish
 need  22 to 40%.  Jauncey (1982)  confirmed  that tilapia require 35%.  There are
 contrary opinions  among  scientists concerning whether fish can use  NPN
 directly.  Nilni and Keveren used a diet containing urea to feed carp  and
 catfish.   They  proved  that fish  cannot utilize inorganic N.  Fladofska and
 Valet used urea to replace the protein source to feed the carp and  mullet.
 They  proved that urea  can replace a part of  protein in the diet.  Chen et al.
 (1986) made a special  diet containing urea to feed carp.  They got  a high
 output.

       All  the different  scientific opinions  were obtained from studies of
 growth by  adding NPN to  the  diet.  The investigators examined whether  the
 fishes can utilize NPN directly  to replace the protein in diet.  None  of them
 investigated the physiological activity of the animal.

       This paper studied the amount of NPN directly through the digestive
 tract or peritoneal cavity of the fish.  The utilization ratio of absorbed NPN
 in fish was measured by  means of stable nuclide  15N-labeled NPN.   The results
 will  provide scientific  basis for fisheries  production.

                             MATERIALS AND METHODS

       Tilapia nilotica (spawned  juvenile)  were taken from the fish  farm  of our
 university and  were transferred  to a circulating,  filtrating aquarium  for
 provisional rearing.   The size of aquarium is 200 x 80 x 80 cm3.  The  length
 of fishes  was from 5 to  13 cm; the weight  was from 3 to 33 grams.

       The  pellet diet  was made containing  the high protein level and normal
 protein level by the materials according to  the  formula as Table 1.
                  TABLE 1.  THE DIET FORMULA  (percent)

                                                        Rape
               Fish        Rice          Plant          seed  Vitamin'
               meal  Bran  chaff  Flour  oil   Mineral  cake  compound Protein
High protein
level diet
Normal protein
level diet
53
15
8
30
5
20
4 
9
3
3
4
4
23
19
0.01
0.01
49.87
27.62
      15NHAC1 (made by Shanghai Chemical Industrial Institute),  abundance
95.44%,  was added to the diet.

      The fishes were fed a pellet diet containing 30% protein until they ate
it at once when the food was put in the water.   After acclimation to the diet,
the fish were divided into three groups:  a high protein level  diet group, a

                                    129

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normal protein  level  diet  group,  and a  starved group.   Before  the  experiment,
the diet of both  the  high  protein level diet  group  and  normal  protein level
diet group, which did not  receive 15NH4C1,  were used for 15  days.   The starved
group also was  fed with normal protein  level  diet for 8 days,  then starved for
7 days.  Then the test began.

      The fish  were divided  into  a feeding group and an injection  group  for
introducing 15NHAC1 into the  organisms.   In the feeding  group,  the  fish were
fed a high protein level diet and a normal protein  level diet, both of which
contained 15NHAC1.  The amount was 0.5 g/fish/day.   From the first  time of
feeding, three  fish were taken for sampling from each group.

      In the injection group, a  30% 15NH4C1 injection solution was  introduced
at a rate of 0.3  mg NH4C1  per gram of body weight through intraperitoneal
cavity  injection. After injection, three  fish were taken from each group for
analysis.  During the whole  experimental time,  the  fish were fed either  a high
protein level or  a normal  protein level diet  not containing 15NH4C1.

      The temperature of the water was  24  to  25C;  the  dissolved oxygen
content was 3.5 to 5.5 mg/L.

      Fish removed for sampling were dissected at once  and  all muscle weighed.
The amount of N in a  certain weight of  muscle was determined by Kjeldahl's
method.  Another  portion of  the muscle  was treated  twice with  5%
trichloroacetic acid  to precipitate the protein and then N  was determined
using Kjeldahl's  method.   The abundance of 15N in each sample was determined
by mass spectrometry.

      Formulas  used in the calculation  were:   AN1/N1=___RG^Ri	
                                 N%=AN/N -  SNX
                                        SN2

      where:  AN/N = the amount  of NH4C1-N absorbed (ANj/N^  or transformed
                     into protein (AN2/N2)  within each gram protein-N of fish
                     muscle
                Rg  abundance of 15N in muscle  sample
                Rf - abundance of 15NH4C1 in diet or injected solution
                Ri = the natural abundance of 15N  in fish muscle
               13$! = the total amount of N in fish muscle
               SN2 = the amount of NPN  (mg) induced by eating or injection

                            RESULTS  AND DISCUSSION

      Tilapia can absorb NH4C1-N through the digestive tract, as shown in
Figure 1.
      In the feeding groups, all the fishes in the high protein level diet
group, the normal protein level diet group, and the starved group, showed an
increase of N in muscle according to the continuous experimental time.  The
AN/N! rose from 0.052,  0.054, and  0.036 mg N/g N at 5 hours to 0.568, 0.380,
and 0.518 mg N/g N at 48 hours, respectively.
                                    130

-------
0.6-
z
 0.5-
z
O>
"o
*- 0.3-
X
5 0.2-
Z
< 0.1-
0-









^
mi ll
5 10


























-




















^



















24 36 48
Time, hours
                     Figure  1.  The  absorption of NI^Cl-N
                        through  digestive  tract at differ-
                        ent times.
      Tilapia can absorb the NH4C1-N through the peritoneal cavity, as shown
in Figure 2.

      Five hours after injection, the ANj/Nj. was 0.132,  0.224, and 0.155 mg
N/g N for the high protein level group, the normal protein level  group,  and
the starved group, respectively.  This means that, in tilapia, NPN was
absorbed through peritoneal cavity very quickly.  From  5 hours to 10  hours,
the ANi/Nj.  decreased,  meaning that,  at this time, a certain amount of NPN was
excreted.  From 10 hours to 48 hours, the  AN.,/^ continued to decrease slowly,
which means that NPN may take part in synthesizing into protein.
                  z  
                  z
                  m 0.3 -
                     0.2-
.p
\
}
5 10
J
I
24
Time, hours
m
36
Ito
48
                    Figure 2.  The absorption of
                      through peritoneal  cavity at  dif
                      ferent times.
                                     131

-------
       After the NH4-N was absorbed, does  the N  take part  in synthesizing into
 protein?   We used trichloroacetic acid to treat the muscle of fish in feeding
 group in  order to precipitate the protein in muscle.   We  then determined the
 abundance of  N and observed its variation and calculated the amount of NPN
 transferring into the muscle.   The result shows that  the  abundance of 15N in
 muscle  increased according  to digestion and absorption (Figure 3).
                     0.6-
                  Z
                  o>
                    0.5-
                    0.4-
                  o
                  *- 0.3-1
                  X
                  1H
                  < o.,J
10       24      36
       Time, hours
                                                      48
                    Figure 3.  The amount of NPN trans-
                      ferring into the protein of fish
                      muscle and the total amount of
                      absorbed NPN.


       In comparing the amount of NPN transferring into the protein of fish
 muscle (AN2/N2) with the  total amount of absorbed NPN  (AN^N^ ,  we see that the
 ratio (R)  has risen.   At 10 hours, the R values of the three groups are 54.1%
 56.1%,  and 55.5%,  respectively.   At 48 hours,  the R values rise to 75.5%,
 74.7%,  and 60.3%,  respectively.   It means that, in tilapia,  not only theW-N
 can be absorbed through digestive tract but also the absorbed NPN can be
 synthesized continuously into the protein of muscle.

      ^Using the feeding amount within 48  hours, the total amount of muscle
 protein in each fish,  its percentage in the total fish protein,  and other
 data, we established  the absorptivity of  NHAC1  in 48 hours by tilapia body and
 muscle  and its  transferability of protein from the  absorbed  NHA-N  (Table 2)
      A few papers Delate metabolism of fish studied with stable  nuclide 15
                                             'N.
  m                                ----  -  ---- -   > -n^-.b v w TT j. *,ii ta W-U.I_/ J-^r  J.II_4.*^ .L J. U-C  J.1 *
Yitou studied the juvenile  of common carp,  crucian carp,  loach, etc.  absorbing
the NH3-N and N02-N from water.  He confirmed that the absorbed NH3-N takes
part in metabolism entering various  tissue  and  organs by  biosynthesis.

      In this paper, we proved that  the  amount  of absorbed NPN  in muscle at 48
hours is ten times that at  5  hours;  that the  absorptivity of NPN  in  muscle
after injection at 5 hours  is 4 to 5  times  the  absorptivity after feeding at  5
hours; and that, by treating  with 5 mg NH4C1,  after treatment at  5 hours   the
absorptivity of NPN through the peritoneal  cavity is 3 times that through
digestive tract.  From the  R  value at 48 hours, we estimate that  most of the
absorbed NPN took part in the synthesis  of  muscle protein.
                                    132

-------
   TABLE 2.  THE ABSORPTIVITY OF NHA-N BY FISH BODY AND MUSCLE PROTEIN
             AND THE TRANSFERABILITY OF PROTEIN FROM NH4-N IN MUSCLE IN
             48 HOURS
                             Absorptivity of
                             Fish body   Fish muscle
Transferability
of NH4-N into
fish muscle, %
High protein level diet
Normal protein level diet
Starved group
3.27
2.54
1.77
1.97
1.53
1.06
75.5
74.7
60.3
      Now it is sure that,  in tilapia, the NHA-N can be absorbed directly and
be synthesized into protein.  It can be quantified, but the amount of
inorganic N replacing protein is very little.  In the ruminant, adding 2% urea
can replace 20 to 30% protein in the diet.  And the dietary coefficient can be
decreased to 1.3 by adding  2% urea into the diet of rainbow trout.

      Urea is the end excreta of metabolism in mammals.  After they are
decomposed they can be utilized to synthesize protein by microorganisms.  This
is a kind of indirect utilization but is not direct utilization as this
experiment.

                                  CONCLUSION

      In tilapia, the direct absorption of NH4-N can be both through its
digestive tract and the peritoneal cavity.  At 5 hours, the absorptivity
through the peritoneal cavity is higher than that through the digestive tract.
The absorbed NHA-N took part the synthesis of body protein.   In tilapia,  at 48
hours, the transferability  is 60.3 to 75.5% in the muscle.

      In tilapia, the absorptivity of 2% NH4C1 in diet is 1.77 to 3.27%.


                                  REFERENCES

Chen,  Mui Bin et al.   1986.   Studies of the application of urea in the pellet
      diet of carp.   Freshwater Fisheries. 3:6-9.
Garling,  D.  L.  Jr.,  and R. P. Wilson.   1976.  Optimum dietary protein to
      energy ratio for channel catfish fingerlings (Ictalurus punctatus).   J.
      Nutr.  106:1368-1375.
Jauncey,  K.   1982.   The effects of varying dietary protein level on the
      growth,  food conversion,  protein utilization,  and body composition of
      juvenile tilapias (Sarotherodon mossambicus).   Aquaculture.  27:43-54.
Lin Ding et al.   1980.   Optimum requirement of protein for juvenile of grass
      carp (Ctenopharyngodon idellus)  in growth phase.   Acta Hydrobiologica
      Sinica.   7(2):207-212.
Ogino, C.  and K.  Saito.  1970.   Protein nutrition in fish.   The utilization of
      dietary protein by young carp.   Bull.  Jpn.  Soc.  Sci.  Fish.   36:250-254.
                                    133

-------
Satia, B. P.  1974.  Quantitative protein requirements of rainbow trout
      Progress in Fish Culture.  36:80-85.
Takeuchi, T., T. Watanabe, and C. Ogino.  1979.  Optimum ratio of dietary
      energy to protein for carp.  Bull. Jpn. Soc. Sci. Fish. 45:983-987
Wang, Yi-Qiang et al.  1987.  Localization of insulin in grass carp (Ct-
      enopharyn^odon idellus) and black carp (Mvlopharvngodon piceusO with
      immunocytochemical method  (Unpublished).
Yang, Guo-Hua et al.  1980.  Fish nutrition and nutrient targets for several
      Chinese carp.  Scientific report of the Shanghai Fisheries Research
      Institute (No. 1) 3-14.
                                   134

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                 OXYGEN.  CARBON DIOXIDE AND AMMONIA TRANSFER
                          ACROSS  TELEOST FISH GILLS

                                      by

                               David J. Randall1


      Fish transfer oxygen, carbon dioxide and ammonia across their gills
between water and blood.  About equal amounts of oxygen and carbon dioxide are
transferred, but in opposite directions,  whereas ammonia excretion is only 10
to 30% of oxygen uptake.  This, of course,  is a reflection of metabolic
utilization and production of these compounds.  The carbon dioxide excretion:
oxygen uptake exchange ratio is usually between 0.7 and 1.0, whereas the
ammonia:carbon dioxide exchange ratio is between 0.1 and 0.3 (Randall and
Wright 1989).  Variations in the  exchange ratio outside these levels result
from changes in body stores of carbon dioxide and/or ammonia.

      Body stores of these two compounds represent about two or three times
the excretion rate per hour.  Oxygen stores, ignoring that in the swimbladder,
are only sufficient to maintain tissue requirements for about 5 minutes.  If
the swimbladder contains  only oxygen then, assuming the fish can utilize all
the oxygen present, swimbladder oxygen could maintain tissue oxygen require-
ments for a few hours  (see Randall and Daxboeck, 1984, for review).  It is
possible that swimbladder oxygen  stores supplement oxygen requirements during
exposure of fish to hypoxic environments (Smith et al. 1983).

      The gills consist of a series of gill arches, supporting rows of
filaments with lamellae,  forming  a sieve-like structure in the path of the
water flow.  The blood circulation is complex having both lamellar and
filamental  components.  The filament circulation is part of a secondary, low
hematocrit  circulation that serves superficial structures in the fish.
Surface structures  in  fish obtain oxygen from and excrete carbon dioxide and
ammonia directly into  the surrounding water  (Randall and Daxboeck 1984).

      Oxygen transfer  across the  gills into  the blood is to supply internal
organs only.  The  secondary circulation to the skin and gill filaments  is not
to exchange gases but  rather to deliver organic nutrients, remove waste-
products, and maintain osmotic balance in the tissues.  In resting fish, skin
and gill oxygen consumption represents about  20 to 30% of total oxygen uptake
      ^University of British Columbia, Vancouver BC, Canada.

                                      135

-------
 by the fish; that is, only 70% of oxygen consumed by the fish crosses the
 gills into the blood, the rest is supplied to superficial structures directly
 from the water.  The fraction of total oxygen uptake crossing the gills will
 increase with exercise because the increase in oxygen uptake goes almost
 entirely to the working muscles that receive their oxygen via the blood.

       Blood flows through the lamellae countercurrent to the water flow.
 Blood flows around pillar cells,  which act as posts, embedded with collagen
 that extends around the blood space,  holding the two walls of the lamellae
 parallel to each other (Figure la).   The blood sheet is about 9 to 12 urn thick
 and is very dependent on blood pressure.  The lamallae show no increase in
 length or height as blood pressure rises but the width does increase with
 blood pressure (Farrell et al. 1980).  Gas transfer occurs across the lamellar
 wall,  which consists of a pillar cell wall,  a basement membrane,  and two
 layers of epithelial cells.

       There are intracellular spaces  between epithelial cells but the outer
 layer of cells are bound together by  tight junctions,  ensuring a  low osmotic
 and ionic permeability of the respiratory epithelium.   Mucus covers  the apical
 surface of the epithelial layer and water flows  between the lamellae in a
 sheet about 25 urn thick.   Blood transit time through the lamellae is about a
 second whereas that for water is  about 100 milliseconds.

       Oxygen uptake across the gills  is both perfusion and diffusion limited
 (Randall 1982).   Increases in oxygen  uptake  are  achieved by increases in both
 blood and water flow and the  diffusing capacity  of  the gills.   Maximum oxygen
 delivery to the tissues  is determined by cardiac output and arterial oxygen
 content (Jones  1971).  Many teleost fish have blood with a marked Root shift,
 that  is,  a decrease  in pH reduces  the oxygen capacity  of the blood even at
 high Po2.  The Root shift plays an important role in unloading oxygen  into the
 swimbladder.

      Acidotic  conditions, therefore,  could  lead to  a  reduction in oxygen
 delivery to  the  tissues by reducing blood oxygen content.   This is
 ameliorated, however, by  the  release  of catecholamines  into  the blood.
 Catecholamines react with  beta-receptors  on  red blood  cells  and stimulate
 sodium/proton exchange.  This  causes  a  rise  in intracellular pH and  an
 increase  in  intracellular  sodium and  chloride levels, which  results  in
 swelling  of  the erythrocytes  as water  is  sucked  into the  cells  (Nikinmaa et
 al. 1987).  The elevation  in  erythrocytic pH, due to stimulation of
 sodium/proton exchange by  catecholamines, offsets the effect of a reduction in
plasma pH on erythrocytic pH and contributes to the maintenance of arterial
blood oxygen content (Primmett et al.  1986, Randall et al. 1987).

      Carbon dioxide is excreted as C02, but a small fraction (about  10% of
total^carbon dioxide excretion in resting fish) is converted to bicarbonate in
the gill epithelium and exchanged for chloride across the apical membrane
 (Figure 2).  There appears to be no functional carbonic anhydrase  activity in
the plasma or associated with the respiratory endothelium (Perry et al. 1982).
In trout, and probably other fish, there is no carbon dioxide excretion in the
absence of red blood cells.  Under conditions of controlled blood  flow and
carbon dioxide content, excretion is  proportional to hematocrit (Perry et al.

                                     136

-------
                                                             H
                                                             H  rH
                                                              tn H
                                                                 H
                                                             -P  tn

                                                              0  
-------
1982) .   Carbonic anhydrase activity in fish red blood cells is high and is
required to catalyse the bicarbonate dehydration reaction in blood in the
gills.


      The  erythrocyte membrane  also contains high  concentrations of the
protein associated with chloride/bicarbonate exchange  (Table  1)   That is   red
blood cells are required to ensure adequate rates  of bicarbonate dehydration
?UriS Si transit of the S111' and to maintain  low  levels  of carbon dioxide
in the blood.  The gill epithelium also contains high  levels  of carbonic
anhydrase, this enzyme appears  to function in maintaining adequate fluxes of
protons and bicarbonate, through the catalysis of  the  carbon  dioxide hydration
reaction   for anionic and cationic exchange processes  on the  apical surface of
the gill (Figure 2).
                                               c.a = carbonic anhydrase
                     Na
                  H
             CO
                                                   ERYTHROCYTE
                WATER
                             GILL
                         EPITHELIUM

            Figure 2.   Diagramme to illustrate the pattern of carbon
              dioxide  excretion across the gills  of teleost fish.  The
              dotted line represents possible but unlikely pathways
              of carbon dioxide excretion in fish.
                                   138

-------
              TABLE 1.   CHARACTERIZATION OF - CHLORIDE/BICARBONATE
              TRANSPORT SYSTEM (BAND III MOLECULES) IN TROUT RED
                   BLOOD CELLS COMPARED WITH THAT FOR HUMAN
                RED BLOOD CELLS  (from  Romana  and Passow 1984)

Band III molecules , cell"1
Cell surface , cm2
Band III molecules, cm"2
Half time for Cl" ion exchange, s:
0C
10C
15C
38C
Trout
8 x 106
2.67 x 10"6
30 x 1011

3.42
1.29
0.81

Human
1 x 106
1.42 x 10"6
7 x 1011

17 . 2
2.32
0.89
0.05
      Carbon dioxide and ammonia are the major metabolic endproducts excreted
across the gills of fish.  In general, the fish excretes about ten times as
much carbon dioxide as ammonia.  These compounds exist in the body in both an
ionized and unionized form.  The ratio of carbon dioxide (C02) to bicarbonate
(HC03~)  and ammonia (NH3) to  ammonium  ion  (NHA+) varies with pH,  but the pKs of
these reactions are very different.  The pK of the NH3/NHA+  reaction  is  about
9.5, whereas the apparent pK of the C02/HC03"  reaction is around  6.1
(Boutiller et al.  1984).  The C02/HC03" and NH3/NH4-+ ratios  are equal  at the pH
where the two lines cross in Figure 3A, at the midpoint between  the pK  of  the
NH3/NHA+ reaction and the apparent  pK  of the C02/HC03~ reaction.   The  pH at
this point is similar to the pH of fish blood over a range  of temperatures.
The midpoint between these two pKs varies with temperature  in much the  same
way as blood pH changes in fish.   This is of  functional significance, for  the
animal must maintain the transfer  of both ammonia  and carbon dioxide out  of
the body.   [The term carbon  dioxide refers to total  carbon  dioxide (C02 +
H2C03  + HCQ3~ + C03=) ,  and ammonia to  total ammonia (NH3 + NH^) ] .

      The gill epithelium is not very permeable to either HC03"  (Perry et al.
1982) or NHA+ (see review by Randall and Wright 1987), but is very permeable
to C02 and NH3.   Thus,  C02 and NH3  will be the predominant forms  excreted,  as
long as adequate blood-to-water C02 and NH3 gradients exist.  This will occur
for both metabolic endproducts if  blood pH is maintained at the  midpoint
between the pK of  the two reactions and environmental levels of  NH3 and C02
remain low.  The  fish must  maintain  C02 and NH3 excretion because increased
C02 levels result  in an acidosis and because  high  levels of ammonia  in  the
body  are  toxic,  resulting in convulsions  in  all vertebrates.  A blood
acidosis,  however, will  favour  CO2 excretion  whereas a blood alkalosis  will
augment NH3 excretion (Hillaby and Randall 1979).  Both blood C02/HC03" and
NH3/NHA+ ratios are very low because blood pH is nearly two orders of
magnitude away from the  point where  pH equals pK for either reaction.   Thus in
both  instances,  the  unionized form represents only 1 to 5% of the total and
there are large  stores  of HC03" and NHA+ in the body.
                                      139

-------
 A.
        15C
          1.4
CO2/HCO3
                                                            10.0
B.
  pH
         8.0 -
         7.8 -
         7.6 
         7.U
                     10
15
20     25
  T(C)
                                                30
                            35
 Figure 3.  A. The effect of varying  pH  on  the  C02/HC03~  and NH3/NH4+
   ratio in trout plasma at 15C.  B.  The  pH at which  the  ratios  are
   equal have been calculated at different  temperatures and added  to
   a graph of variations in plasma pH with  temperature  for  several
   fish (from Randall and Wright 1988).
                                140

-------
      Excretion of CO2 and NH3 will be  influenced by  the  composition of water
near the gill surface, which may have a different chemical composition from
that of the bulk medium.  The surface of the gills is covered by a mucous
coating and there is also a boundary layer of water next to this mucous^ layer.
Molecular C02 and NH3,  along  with some  HC03" (Perry et al. 1982) and NHA
(Wright and Wood 1985), are excreted into the mucous and boundary water layer
next to the gill surface  (Figure 4).  Bicarbonate ions represent only a small
portion of the carbon  dioxide excreted, probably less than 10%, and  the amount
of ammonia excreted as NH3 varies between 45 and 100% (see review by Randall
and Wright 1988).  As  long as the pH is above 6.1, then most of the  C02
excreted will form HC03" and  acidify the water.   Wright et al.  (1986) showed
that this reaction was catalysed by carbonic anhydrase in the mucous layer.

      The mucous layer contains a large number of damaged gill epithelial
cells, which are known to contain high  levels of carbonic anhydrase  (Lacey
1983)!  C02 entering this boundary layer will acidify the mucous and boundary
               Blood
 Boundary
water layer
    CO2

    11
   HCO;
                                                  H
Free flowing
  water
                                                             co2
                                                             1L
                                                            HCO3
               Figure 4.   Schematic cross-section through the gill
                 epithelium,  mucus and water boundary layer,  contain-
                 ing carbonic anhydrase ().  The thickness of the
                 arrows denotes the approximate magnitude of the
                 particular process illustrated (after Randall and
                 Wright 1987).
                                     141

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 layer of the gill  (Wright et al. 1986).  The extent of acidification will be
 determined by the  rate of carbon dioxide excretion in relation to water flow
 and the buffering  capacity and pH of the water.  Excreted NH3 will combine
 with a proton and  increase water pH, but because more C02 than NH3 is  excreted
 across the gills,  the overall effect usually will be an acidification of the
 boundary layer.  NH3 entering this acidified layer next to the gill surface
 will be converted  to NH4+ and will  diffuse  out  of  the boundary water layer
 into the bulk medium.

       The conversion of NH3 to  NHA+ and its subsequent diffusion from the
 boundary layer will reduce NH3  levels in the water and,  therefore, NH3 levels
 in the blood (Wright et al. 1989).  Thus, there is an interaction in the
 boundary layer between carbon dioxide and ammonia excretion.  Starving trout
 have elevated blood NH3 levels  (Hillaby and Randall 1979)  and this can be
 correlated with a decreased C02 excretion.

       When water pH is below the apparent pK of the C02/HC03~ reaction, C02
 excretion will^ have only a minor effect on water pH.   At a water pH of 4.00,
 almost no HC03  will form and, therefore, almost no further  acidification of
 gill water will occur.   In fact,  the opposite will occur,  for NH3  excretion
 will now cause an increase in boundary water pH (Figure 5B).  At a pH  of 5.1,
 at 10C,  only 10% of the C02  entering the water will be  converted  to
 bicarbonate,  but essentially all NH3  will be  converted  to NH4+.  It is  at
 about this pH that there is no  change in the pH of water as  it passes  over the
 gills (Figure 5B) ;  that is,  H+ production with  HC03~ formation is equal to  H+
 consumption by NH4+ formation.  This observation is consistent with a ratio of
 10:1 for C02  to NH3 excretion.  The real situation is more complicated than
 indicated above,  which  ignores  the  contribution of Na+:NH4+ and C1~:HC03~
 exchange  to total carbon dioxide  and ammonia excretion,  as well  as the
 excretion of  any other  compounds  that may affect the pH of gill  water.

      Acid conditions reduce  ammonia excretion  because  of an inhibition  of
 Na :NHA  exchange (Wright and Wood 1985)  resulting  in increased blood NH3
 levels.   Acid conditions,  in  themselves,  will favour NH3 transfer because any
 NH3 entering the water will be immediately converted to NH^, maintaining NH3
 gradients  across  the gill  epithelium.  This  effect, however,  is  not sufficient
 to offset the effect of inhibition  of Na+:NH4+ exchange,  and  blood  NH3 levels
 rise  (Wright  and  Wood 1985).  Under  less  acidic conditions (water  pH 6.64)
 when  there  is no  inhibition of Na+:NH4+ exchange, blood  NH3 levels  are reduced
 compared with fish  exposed to water of pH 7.85  (Wright and Wood  1985).

      The reverse is true under alkaline  conditions.  C02 excretion results in
 a  large acidification of the boundary layer,  as nearly all the C02 is con-
verted to bicarbonate, or even carbonate, in  the water  (Figure 5B)   This
 acidification, however, may not be sufficient to maintain NH3 excretion,  and
 alkaline conditions can lead to increased NH3 levels in the body (Wright and
Wood 1985, Randall  and Wright 1989).  Elevated ammonia levels stimulate urea
production via uricolysis in some fish, but not trout (Olsen and Fromm 1971).
Oreochromis alcalicus grahami lives in Lake Magadi, an alkaline lake, but
unlike other teleost fish, is completely ureotelic, producing urea via the
ornithine-urea cycle.  Thus, this fish avoids problems of ammonia buildup when
exposed to these very alkaline conditions by converting ammonia to the less
toxic urea (Randall et al. 1989).

                                     142

-------
                                   pH electrodes
                                              Open-ended
                                              block plastic box
             Valve-
                               . Latex mask
                               mounted on divider
                                  V0percular cannula
                              "Stirring bar
                        Slopped-flow
                        apparatus
                          ;upport
                         for box
         B
                  10-
                   9-
                   e-
                  o.
                  ! 7-
Vancouver dechlorinated water

Rainbow trout
                                          Exhalent
                                           water
                                        Exhalent water pH-
                                         pH of flowing water
                                        A Stop flow pH
                                                  10
                                   Inspired water pH
Figure 5.  AThe apparatus  for measuring inhalent  and exha-
  lent water pH in trout.  The exhalent water transit time
from the gills to the pH electrode was less  than 2  seconds.
The  pH of the flowing water  in the exhalent  chamber was re-
corded and then flow was stopped and the equilibrium pH
 (stop flow pH) was measured.   B---The relationship of exhalent
to  inhalent  water pH in trout breathing dechlorinated Vancou-
ver tapwater  (from Randall  and Wright 1989) .
                                 143

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                                    REFERENCES

 Boutilier,^R.G.,  T.A.  Heming,  and  G.K.  Iwama.   1984.  Appendix:
       Physiochemical parameters  for use in  fish respiratory physiology   In
       Fish Physiology, Vol.  10A.   W.S.  Hoar and D.J. Randall  (eds.).  Academic
       Press Inc., New  York.  pp. 403-430.
 Farrell, A.P., S.S. Sobin, D.J.  Randall, andS. Crosby.  1980.  Intralamellar
       blood flow  patterns in fish  gills.  Am. J. Physiol.  239.-R428-R436
 Hillaby, B.A. and D.J. Randall.  1979.  Acute ammonia toxicity and ammonia
       excretion in rainbow trout (Salmo gairdneri^.  J. Fish. Res  Bd  Canada
       36:621-629.
 Jones, D.R.  1971.  The effects  of hypoxia  and  anaemia on the swimming
       performance of rainbow trout (Salmo gairdneril.   J. Exp. Biol.  55:541-

 Lacy, E.R.  1983.  Histochemical and biological studies of carbonic anhydrase
       activity in the opercular  epithelium of the euryhaline teleost, Fundulus
       heteroclitus.   Am.  J.  Anat.  166:19-39.
 Nikinmaa, M.,  J.F. Steffensen,  B.L. Tufts,  and D.J. Randall.   1987.   Control
       of red cell volume and pH  in trout:  Effects of Isoproterenol, transport
       inhibitors,  and extracellular pH in bicarbonate/carbon dioxide-buffered
       media.   J.  Exp.  Zool.   242:273-281.
 Olson, K.R.  and P.O.  Fromm.   1971.   Excretion of urea  by two  teleosts exposed
       to different concentrations of ambient ammonia.   Comp.  Biochem  Phvsiol
       40A:999-1007.                                                  '
 Perry, S.F.,  P.S.  Davie,  C.  Daxboeck,  and D.J.  Randall.   1982.   A comparison
       of C02 excretion  in a spontaneously ventilating blood-perfused  trout
       preparation and  saline-perfused gill preparations:   contribution of the
       branchial  epithelium and  red  blood cell.   J.  Exp.  Biol.   101-47-60
 Primmett,  D.R.N.,  D.J.  Randall, M.M.  Mazeaud,  and  R.G.  Boutilier.  'l986  ' The
       role of  catecholamines  in erythrocyte  pH regulation and oxygen transport
       in rainbow trout  (Salmo gairdneril .  J.  Exp.  Biol.   123-139-148
 Randall,  D.J.  1982.  The control of  respiration and circulation in  fish
       during hypoxia and exercise.  J.  Exp.  Biol.   100:275-288
 Randall,  D.J.  and  C. Daxboeck.  1984.   Oxygen  and  carbon  dioxide  transfer
       across fish  gills.  In  Fish Physiology, Vol.  10A.  W.S. Hoar and D.J.
       Randall  (eds.).   Academic Press  Inc.,  New  York.  pp. 263-314
 Randall.^D.J., D.  Mense, and  R.G. Boutilier.   1987.  The effects of burst
       swimming on  aerobic swimming  in  chinook salmon (Oncorhynchus
       tshawvtscha").  Mar. Behav.  Physiol.  13:77-88.
 Randall, D.J., C.M. Wood, S.F.  Perry, H. Bergman, G!M.O. Maloiy, and P.A.
       Wright.^ 1989.  Urea excretion as  a strategy  for survival in a fish
       living in a  very  alkaline environment.   Nature.  337:165-166
Randall, D.J. and  P.A. Wright.  1987.  Ammonia distribution and excretion
       in fish.  Fish Physiol. Biochem.   3:107-120.
Randall, D.J. and  P.A.  Wright.  1989.  The interaction between carbon
       dioxide and  ammonia excretion and water.pH in fish.  Can. J. Zool   In
      press.
Romano, L. and H. Passow.   1984.  Characterization of anion transport system
       in trout red blood cell.  Am.  J. Physiol.  246:C330-C338
Smith,_D.G.,  W. Duiker,  andl.R.C.  Cooke.  1983.   Sustained branchial apnea
      in the Australian short-finned eel, Anguilla australis   J  Exp  Zool
      226:37-43.
                                   144

-------
Wright, P.A., T.A. Heming, and D.J. Randall.  1986.  Downstream pH changes
      in water flowing over the gills of rainbow trout.  J. Exp. Biol.
      126:499-512.
Wright, P.A. and D.J. Randall.  1987.  The interaction between ammonia and
      carbon dioxide stores and excretion rates in fish.  Annls. Soc. r. Zool.
      Belg.  117 (supplement 1):321-329.
Wright, P.A., D.J. Randall, and S.F. Perry.  1989.  Fish gill water boundary
      layer:  A site of linkage between carbon dioxide and ammonia excretion.
      J. Comp. Physiol.  In press.
Wright, P.A. and C.M. Wood.  1985.  An analysis of branchial ammonia
      excretion in the freshwater rainbow trout:  Effects of environmental pH
      change and sodium uptake blockage.  J. Exp. Biol.  114:329-353.
                                    145

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                         CONTROL OF VENTILATION IN FISH

                                       by
                        E. W. Taylor1 and D.  J.  Randall2

                                  INTRODUCTION

      Respiratory gas  exchange in fish takes place by diffusion over the
 surfaces  of the gills.   The  gill arches are  ventilated by muscular  pumps
 around  the  mouth and pharynx at a rate determined by an  oscillator  in the  CNS,
 which varies in its  activity with oxygen supply and  demand.   The generation  of
 the central respiratory rhythm and its modulation by peripheral
 mechanoreceptors and chemoreceptors are not  completely understood.   This
 review  concentrates  on some  recent evidence  of  a role for circulating
 catecholamines  in the  control of the ventilatory response to  hypoxemia.

                   RESPIRATORY RHYTHM GENERATION IN  THE  CNS

      In  fish,  water is  propelled continuously  over  the  gills in one  direction
 by the ventilatory muscles that operate around  the jaws  and skeletal  elements
 in the  gill arches,  lining the pharynx (Ballintijn and Hughes 1965, Hughes and
 Ballintijn  1965).  These muscles  are innervated by cranial nerves with their
 neuron  cell bodies located in the brainstem,  close to  the site  of the central
 respiratory pattern  generator (CPG).

      Rhythmic  ventilatory movements  continue in fish  following brain
 transection to  isolate the medulla oblongata, although' changes  in pattern
 indicate  that there  are  influences  from higher  centres (Shelton 1959).
 Central recording and marking techniques have identified  a longitudinal strip
 of neurons with spontaneous respiration-related bursting  activity, extending
 dorso-laterally throughout the whole  extent of  the medulla (Shelton 1970,
Waldron 1972).  These neurons  comprise  the trigeminal Vth, facial Vllth,'
 glossopharyngeal IXth and vagal Xth motor nuclei, which drive the respiratory
muscles,  together with the descending  trigeminal nucleus  and  the reticular
 formation (Ballintijn 1982).   The respiratory rhythm is thought to originate
 in a diffuse CPG in  the reticular formation,  which remains functional
 following anaesthesia (Ballintijn 1988).
England
             of Biological Sciences,  University of Birmingham,  Birmingham,
     ^Department of Zoology,  University of British Columbia,  Vancouver,  BC
Canada

                                     146

-------
        All the motor nuclei are interconnected and each receives an afferent
  projection from the descending trigeminal nucleus and has efferent and
  afferent projections to and from the reticular formation (Figure 1).  The
  intermediate facial nucleus,  which receives vagal afferents from the gill
  arches that innervate a range of tonically and phasically active
  mechanoreceptors,  projects to the motor nuclei (Ballintijn et ail. 1983).
  Finally, areas in the mid brain such as the meseneephalic tegmentum have
  efferent and afferent connections with the reticular formation (Ballintijn
  1982,  1988).  These meseneephalic neurons are responsible for initiating
  bursts of ventilation during the periodic or bout respiration shown by some
  inactive or hyperoxic fish (Ballintijn 1988).

        Studies of retrograde intraxonal transport of HRP along nerves
  innervating the respiratory muscles revealed that the neurons in the various
  motor nuclei are distributed in a sequential series in the brainstem of fish
  (Withington-Wray et ail. 1986, Levings and Taylor 1987, ithington-Wray et ail.
  1988).  Recordings of efferent activity from the central cut ends of the
  nerves innervating the respiratory muscles of the dogfish Scyliorhinus
  canLcula (Barrett and Taylor 1985) and ray RaLa clavata (E. W. Taylor and J.
  J. Levings, unpublished) have revealed that the branches of the Vth, Vllth,
  IXth and Xth cranial nerves fire sequentially in the order of the sequential
  rostro-caudal distribution of their moto-nuclei in the brainstem.
                         IFOREBRAIN
          SENSORY
 Respiratory
   muscle
proprioceptors
Trigeminal Vth
  Nucleus
Gill Arch
Receptors
IX & Xth
?

Facial IVth
Nucleus
                                 | MESENCEPHALON|
                                        CPG
                                         IN
                                         RF
                                                                  MOTOR
Motor V
Mandibular

Motor V11
Post-spiracular
Motor
Glosso 1X
Motor
Vagal X1
X2
X3
X4
Hypobranchial
Occipi tal
Spinal
1&2
->
 





h*
Jaw
Muscles

Opercular
or
Spiracular
Muscles

Gill
Arch
Muscles
Accessory
Respiratory
& Feeding
Muscles
        Figure  1.  Summary diagram of the possible functional connections
           involved in  the central nervous control of ventilation in fish.
           Abbreviations  are:  CPG, central pattern generator; RF, reticular
           formation.(Taken  from Taylor  1989)
                                       147

-------
       Experiments in which ventilatory movements were prevented with curare
 demonstrated that the CPG in fish continues to generate rhythmic bursts of
 activity that are slower than the unparalysed respiratory rhythm in teleosts
 (Ballintijn 1972) but faster in elasmobranchs (Barrett and Taylor 1985).
 These changes in rate following paralysis indicate that activity in the CPG
 normally is modulated by peripheral receptors.  This is consistent with the
 fact that the CPG receives afferent information from a range of
 mechanoreceptors, proprioceptors and skin stretch receptors around the gill
 arches and jaws (Ballintijn 1982) with their sensory (afferent) innervation in
 cranial nerves V, VII, IX and X.  Part of the population of motoneurons
 innervating the respiratory muscles is silent in the paralysed animal in both
 fish and mammals and may be stimulated to fire by artificially induced
 proprioceptive information (Ballintijn 1982).  The recruitment of these silent
 motoneurons may serve to increase the motor output and consequent amplitude of
 contraction of respiratory muscles.   In addition,  fish may recruit feeding
 muscles innervated by the hypobranchial nerve trunk,  which contains fibres
 from the occipital and anterior spinal motor nuclei (Figure 1).  These insert
 on the skeletal elements around the mouth and pharynx and can contribute  to
 active,  forced ventilation when respiratory demand is high.

       Thus,  rhythmic contractions of the respiratory muscles are determined by
 a central respiratory pattern generator that is  modulated by feedback on  the
 force of ventilatory contractions from mechanoreceptors in the respiratory
 apparatus and by inputs from elsewhere in the CNS.

                            CHEMORECEPTOR RESPONSES

       Fish pump water over their gills at rates  controlled with respect to
 oxygen supply or demand.   In teleost fish,  ventilation increases when oxygen
 supply is reduced by environmental hypoxia,  or when transport in the  blood is
 reduced either directly by anemia or indirectly  by  hypercapnia,  when  the
 resultant acidosis causes  a Root effect,  reducing oxygen carrying capacity
 (for references see reviews  by Randall 1982,  Shelton  et al.  1986).  Similarly,
 an increase  in oxygen demand during  vigorous  swimming or following the  stress'
 of experimental manipulation results in an increase in ventilation rate.
 Obversely,  an increase in  oxygen supply resulting from environmental  hyperoxia
 causes a decrease  in ventilation rate,  which  may result in hypoventilatory
 hypercapnia  (Figure 2).

       Most of these responses  can be interpreted as arising  from the
 stimulation  of peripheral  or  central chemoreceptors sensitive  to  changes in
 oxygen supply (i.e.,  oxygen  content  and blood flow, Randall  1982).  Evidence
 for  the  location and characteristics of the oxygen  receptors  is  largely
 circumstantial  and the mechanisms  by which reflex ventilatory  changes are
 initiated are not  clear  (Taylor  1985).  Both  the ventilatory  (Eclancher and
 Dejours  1975) and  cardiac  (e.g., Butler and Taylor  1971) responses  to sudden
 exposure  to hypoxia  or exercise  are  rapid in  onset  indicating  that nervous
 pathways  are  involved.  The motor  arm  of the  response is of course nervous as
 the ventilatory muscles are innervated by the efferent cranial nerves (Hughes
 and Ballintijn 1978) .  The afferent  arm has not been clearly identified
 (Taylor 1985) and neuropharmacology  of the central  connections implicated in
 these  reflex responses, and the possible roles for  circulating hormones are
not known.  Circulating catecholamines may be involved in the control of
ventilation and this review now concentrates on this point.
                                     148

-------
                 5OO -i
                 4OO H
                 30O -
              c
                 20O -
                  100
                         9.4
                          17.2
                                Hypoxia
                                     15.0
Hypercapnia
                           Anemia
                                       14.6
                                                 Normoxia
                                             33.3
              Hyperoxic
                  hypercapnia
                             i       i      i      i      i
                       45      6789
                      Arterial blood  oxygen content (Vol  %)
                Figure 2.   The relationship  between rate  of water
                  flow over the gills  and  the  oxygen content of
                  arterialised dorsal  aortic blood  from the
                  rainbow trout Salmo  gairdneri  (mean values
                   SE).   The number by each point  is the cor-
                  responding arterial  oxygen partial pressure
                  in kPa.  (Taken from  Randall  1982)
                          BLOOD CATECHOLAMINE LEVELS

      Teleost fish have low (<0.5 nmol I"1), but measurable, resting levels of
circulating catecholamines (Boutilier et al. 1988).   The levels are higher in
the elasmobranch fish ScylLorhinus canicula. (above 20 nmol I"1) , possibly
because these fish lack a sympathetic connection to the heart and branchial
(gill) apparatus, which may enhance the role of circulating catecholamines in
cardiovascular and ventilatory control (Butler et al. 1978).  Changes in
physiological state, particularly those induced by stressful stimuli such as
physical disturbance (Nakano and Tomlinson 1967) cause an increase in
circulating catecholamine levels.  Other stimuli include hypoxia (Boutilier et
al. 1988), anemia (Iwama et al. 1987), hypercapnia (Perry et al. 1988), acid
infusion (Boutilier et al. 1986), and violent exercise (Primmett et al. 1986).

      Circulating catecholamines.play a number of important roles in the
control of metabolism and respiratory gas exchange and transport, including
                                     149

-------
  stimulation of anaerobic glycolysis during exercise;  increased contractility
  of the heart,  vasodilation or vasoconstriction of peripheral blood vessels
  ?TT  /?!!? v  nt chanSes in blood Pressure and vascular resistance to blood flow
  (Wood 1975);  increased permeability of the gill respiratory epithelium (Isaia
  et al.  1978);  release of red blood cells from the spleen,  increased red cell
  volume and intracellular PH (Nikinmaa 1982).   In addition,  it is now clear
  that many stimuli causing ventilatory increases also  are accompanied by
  changes in circulating catecholamines (Figure 3).   The simplest interpretation
  of_this observation is that the ventilatory  changes are in  some way induced  or
  reinforced by  changes in circulating catecholamine  levels.

                          CATECHOLAMINES AND VENTILATION

       Measurable  increases  in circulating  catecholamine levels  in fish  are
  associated with environmental hypoxia in both teleosts  (Boutilier et  al   1988)
  and  elasmobranchs  (Butler et  al. 1978).  The  hypoxemia  resulting from anemia
  (Iwama  et  al.  1987)  and hypercapnia  (Perry et al. 1988) also  is  associated
 with  increased catecholamine  levels.  Thus many stimuli that  result in
  increases  in ventilation  (Figure 2) also cause increased levels  of circulating
 catecholamines (Figure 3).                                                   &

       Evidence for a direct link between ventilatory responses and changes in
 catecholamine levels have been obtained by acid infusion (K. Holmgren
 unpublished observations) and hypoxia (S. Aota, unpublished observations) in
 trout   Both cause an increase in circulating catecholamines and in gill   -
 ventilation.                                                         

       The -adrenergic blocking agent, propranolol,  inhibits the ventilatory
 infus? 5 n^ ^ incre?Se in blood catecholamines.   During hyperoxia,  acid
 infusion did not cause an increase  in circulating catecholamines nor was there
 any increase in .gill ventilation.  The simplest explanation of these data is
 isaLrt?VrfTa^  ^ yentilation durin hypoxia or following an acid injection
 is  mediatedjry  the release of catecholamines  into the  circulation.  The  site
 of  action of_these catecholamines could be  either peripheral or central,  but
 the pathway is  blocked by propranolol and so  involves ,9-adrenergic receptors.

                        SITE  OF ACTION OF  CATECHOLAMINES

       Peyraud-Waitzenegger  (1979) showed  that  intravenous injection of
 adrenaline  in the  eel caused an increase  in ventilation.  The  injection  of
 catecholamines  into  dogfish  (Scyliorhinus canicula and Squalus acanthius) 
 caused changes  in  central respiratory  drive measured as  the  efferent,  motor
 activity in a branchial branch (to a gill arch) of the vagus nerve  of
 paralysed fish  (i.e.,  in the absence of peripheral mechanoreceptor  inputs)
 The fish were force ventilated with hyperoxic  seawater to reduce  stress and
 decrease endogenous levels of  circulating catecholamines (E. . Taylor and D
J. Randall-unpublished observations).  Intravenous injection of adrenaline or
noradrenaline  after a short delay (20 sec), caused a transient but marked
increase in efferent activity  in S.  canicula,  which recovered to normal
activity, then after a slightly longer delay showed a further transient
increase.
                                    150

-------
           z
           o
           l-
           <
           a:
           H
           z
           LU
           U
           z
        to  p
        Ul  U
<  LU
_J  t/)
o  <
T  LU

LU  (J
H
              60
      20
              16
             12
           Z
           o
           P   8
           a:
           o
           a.
           s   *
           a.
                      A NA
                    HYPOXIA
                          A NA
                          ANEMIA
    A  NA
HYPERCAPNIA
  A  NA
  ACID
INFUSION
          Figure  3.  The  proportional  increase  in  the  plasma  levels  of
            circulating catecholamines in  the trout  Salmo  gairdneri
            elicited by exposure  to hypoxia  or  hypercapnia by experi-
            mentally induced  anemia and by acid infusion.  Abbrevia-
            tions are: A,  adrenaline;  NA5  noradrenaline.
      Our interpretation of this rather complex response is that the bolus of
blood containing high levels of injected catecholamines passed for two
circuits of the circulatory system over a receptive area that triggered an
increase in activity in the medulla.  Subsequently, the catecholamines would
be extracted from the blood and metabolized by the gills and other tissues
(Nekvasil and Olson 1986a).  The approximate location of the receptive area
can be deduced from our limited knowledge of circulation times in the dogfish
to lie efferent to the heart, possibly in the cerebral circulation.  These
effects of intravenous injection of high concentrations of catecholamines were
most marked for noradrenaline.  This may cross the blood-brain barrier in fish
more easily than adrenaline (Nekvasil and Olson 1986 b).

      When catecholamines were injected into curarised, hyperoxic S. canLcula
in which activity in a hypobranchial nerve, which innervates feeding muscles,
                                     151

-------
 was recorded simultaneously with that in a branchial branch of the  vagus,
 direct evidence of nervous recruitment was observed (J.  J.  Levings  and E. W.
 Taylor,  unpublished observations).   In the hyperoxic fish prior to  injection
 of adrenaline,  the branchial branch showed regular bursting activity,  but the
 hypobranchial nerve showed low levels of activity and only  fired .
 intermittently,  with a respiration-related activity,   injection of  adrenaline
 caused high levels of respiration-related, bursting activity in the
 hypobranchial nerve,  which resembled or exceeded that observed in the
 disturbed normoxic and fictively hyperpnoeic fish.   This result seems  to
 identify a role for circulating catecholamines  in the onset of forced
 ventilation.

       We currently are investigating the possibility that catecholamines exert
 their effects upon the central pattern generator or the  respiratory
 motoneurons by direct injection of  catecholamines into the  CNS.   Injection  of
 small volumes- (8-20 /*!)  of a 10"4 molar solution of adrenaline into  the fourth
 ventricle of  S.  acanthius caused a  marked change in the  pattern of  central
 respiratory drive  (Figure 4).   The  response was  complex  but stereotyped.  An
 initial increase in the  rate of bursting of respiratory  motor units was
 followed by a slowing of the rhythm accompanied  by a huge increase  in  the
 activity within each burst (Figure  4b),  apparently due to the recruitment of
 units having  larger recorded spikes,  implying larger fibre  diameters,  as all
 vagal fibres  are myelinated (Short  et al.  1977).   This implies that the
 increase in ventilation  brought about by injection of catecholamines may
 result from an increase  in stroke volume rather  than rate of contractions of
 the respiratory apparatus.   The response to injected catecholamines was
 blocked by simultaneous  injection of propranolol,  a j8-adrenergic  antagonist
 (Figure  4c).

       These data imply that areas in the CNS, accessible  from the fourth
 ventricle,  respond to an increase in catecholamine  concentration  with  an
 increase in central respiratory drive.   The vagal  respiratory motoneurons lie
 in the dorsal vagal motor nucleus,  which is situated bilaterally  in the
 medulla  in a  medial position close  to the  wall of  the fourth ventricle
 (Withington-Wray et al.  1986).   These neurons are rhythmically active,  under
 the influence of the  central pattern generator,  and supply  the efferent
 innervation to  intrinsic respiratory muscles  in  the  gill  arches.  Their
 location close  to  the wall  of  the fourth ventricle may have  caused  them to be
 directly affected by  the injection  of catecholamines  into this location.
 Identification of  the site  of  action of  catecholamines on the  respiratory
 rhythm generator and  associated respiratory motor neurons in the medulla must
 await  microelectrode  studies of single,  identified neurons.

                                  CONCLUSIONS

       Our  current knowledge of the  control  of ventilation in fish is
 incomplete at all levels.  The  respiratory  rhythm.originates  in a medullary
 central pattern  generator, which has yet to be clearly identified and
 characterised.   Its activity is modulated by inputs from elsewhere  in the CNS
 and from peripheral mechanoreceptors.  The  central location of respiratory
motor neurons, innervating the various respiratory muscles,  has been described
 in  detail for some fish, particularly elasmobranchs.
                                     152

-------
       adr-
                                             15 sec
         b control
           post adr-
                                     1 sec
       ADR
     ADR PROP
     ADR PROP
                                                                15 sec
Figure 4.  The effect of injection of adrenaline into the fourth ventricle
  of the dogfish Squalus acanthius upon efferent activity in the third
  branchial (respiratory) branch of the vagus nerve, ainjection of  20 ul
  of 10~^ molar adrenaline (adr.), after a 20-sec latent period, induced a
  transitory increase in the frequency of the bursting activity recorded
  from the nerve.  After'-;about 75 sec, this was replaced by a slowing in
  bursting rate accompanied by an increase in the amplitude of the recorded
  bursts,  bdetail of recording from trace in (a) to compare the rate
  and amplitude of the bursts recorded before and after injection of  adre-
  naline,  cinjection of adrenaline (ADR) into the fourth ventricle of
  another experimental animal induced a similar response to (a) that  was
  subsequently blocked by simultaneous injection of adrenaline and propran-
  olol (ADR PROP).                     3

-------
      We are still unclear, however,  about the link between the CPG and the
sequential firing of the motor neurons,  which result in coordinated
contractions of the respiratory muscles,  and about the mechanisms that result
in recruitment of feeding muscles into forced ventilation.  Ventilation is
matched to oxygen requirements by stimulation of chemoreceptors,  which seem to
respond to oxygen content or supply.   The precise location and characteristics
of these chemoreceptors are still not known.

      Chemoreceptor stimulation evokes a number of reflex changes in the
respiratory and cardiovascular systems of fish that are rapid in onset and
seem adaptive (e.g., increased ventilation and a bradycardia in response to
hypoxia).  Conditions that result in hypoxemia and the consequent ventilatory
changes also cause an elevation in circulating catecholamine levels.   We have
explored the possibility of a causal relationship between these levels and the
ventilatory response.  Strong evidence arises from experiments on hypoxia and
acid infusion, which trigger a ventilatory increase and a rise in circulating
catecholamines.  Both ventilatory responses are blocked by an injection of
propranolol despite an increase in catecholamine levels.

      The ventilatory response to hypoxia, at least, occurs very rapidly,
perhaps before any marked increase in circulating catecholamines and almost
certainly before any blood catecholamines could reach the respiratory neurons.
This argues for an immediate neuronal reflex based on chemoreceptors in the
gill region responding to hypoxia.  Clearly, circulating catecholamines also
affect ventilation through some action in the medulla and could act in concert
with a direct neuronal chemoreceptive drive during hypoxia. .The studies on
acid infusion during hyperoxia, where there is an acidosis but no increase in
ventilation or blood catecholamines,  would argue against any hydrogen ion
receptor, either peripheral or central,  being involved in the reflex
ventilatory response to acidotic conditions in fish.

      The release of catecholamines into the circulation, therefore,  seems to
be an absolute requirement for the ventilatory response to acidosis in fish.
Present evidence supports a role for ^8-adrenergic receptors on respiratory
neurons, stimulated by changes in the levels of circulating catecholamines, in
the control of ventilatory responses to changes in blood oxygen levels in
fish.

                               ACKNOWLEDGMENTS

      This work was supported by a grant from NATO which enabled EWT to visit
the University of British Columbia and funded the work at Bamfield Marine
Station.  We wish to acknowledge this support and the help provided by the
staff at Bamfield.  Our attendance at the Symposium was funded by the U.S.
Environmental Protection Agency and we are grateful to both the EPA and
Zhongshan University, Guangzhou,  PRC, for organizing this successful
international meeting.
                                     154

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                                  REFERENCES

Ballintijn, C. M.  1982.  Neural control of respiration in fishes and mammals.
      In: Exogenous and Endogenous Influences on Metabolic and Neural Control.
      A. D. F. Addink and N. Spronk (eds.).  Pergamon Press, Oxford.
Ballintijn, C. M.  1988.  Evolution of central nervous control of ventilation
      in vertebrates.  In: The Neurobiology of the Cardiorespiratory System.
      E. W. Taylor (ed.).  Manchester University Press, Manchester.
Ballintijn, C. M. and G. M. Hughes.  1965.  The muscular basis of the
      respiratory pumps in the trout.  J, Exp. Biol.  43:349-362.
Ballintijn, C. M., B. L. Roberts, and P. G. M. Luiten.  1983.  Respiratory
      responses to stimulation of branchial vagus nerve ganglia of a teleost
      fish.  Respir. Physiol.  51:241-257.
Barrett, D. J. and E. W. Taylor.  1985.  Spontaneous efferent activity in
      branches of the vagus nerve controlling heart rate and ventilation in
      the dogfish.  J. Exp. Biol.  117:433-448.
Boutilier, R. G., G. K. Iwama, and D. J. Randall.  1986.  The promotion of
      catecholamine release in rainbow trout, Salmo gairdneri, by acute
      acidosis:  Interactions between red cell pH and hemoglobin oxygen-
      carrying capacity.  J. Exp. Biol.  123:145-157.
Boutilier, R. G., G. Dobson, U. Goeger, and D. J. Randall.  1988.  Acute
      response to graded levels of hypoxia in rainbow trout (Salmo gairdneri):
      metabolic and respiratory adaptations.  Respir. Physiol.  71:69-82.
Butler, P. J. and E. W. Taylor.  1971.  Response to the dogfish (Scyliorhinus
      canicula L.) to slowly induced and rapidly induced hypoxia.  Comp.
      Biochem. Physiol.  39A:307-323.
Butler, P. J., E. W. Taylor, M. F. Capra, and W. Davison.  1978.  The effect
      of hypoxia on the levels of circulating catecholamines in the dogfish,
      Scyliorhinus canicula.  J. Comp. Physiol.  127:325-330.
Eclancher, B and P. Dejours.  1975.  Control de la respiration chex les
      poissons teleosteens:  existence de chemorecepteurs physiologiquement
      analogues aux chemorecepteurs des vertebras superieur.  C. R. Acad. Sci.
      Paris Ser. D 280:451-453.
Hughes, G. M. and C. M. Ballintijn.  1965.  The muscular basis of the
      respiratory pumps in the dogfish.  J. Exp. Biol.  43:363-383.
Isaia, J., J. P. Girard, and P. Payan.  1978.  Kinetic study of gill
      epithelial permeability to water diffusion in the freshwater trout,
      Salmo gairdneri:  Effect of adrenaline.  J. Membrane Biol.  41:337-347.
Iwama, G. K., R. G. Boutilier, T. A. Heming, P. A. Wright, D. J. Randall, and
      M. Mazeaud.  1988.  The interaction between gill ventilation, blood
      catecholamines and gas exchange in rainbow trout.
Levings, J. J. and E. W. Taylor.  1987.  Vagal, preganglionic innervation of
      the gut in the lesser spotted dogfish Scyliorhinus canicula.  J.
      Physiol.  394:99P.
Nakano, T. and N. Tomlinson.  1967.  Catecholamine and carbohydrate
      concentrations in rainbow trout (Salmo gairdneri) in relation to
      physical disturbance.  J. Fish. Res. Bd. Canada, 24:  1701-1715.
Nekvasil, N. P. and L. R. Olson.  1986a.  Extraction and metabolism of
      circulating catecholamines by the trout gill.  Am. J. Physiol.
      250:R526-R531.
Nekvasil, N. P. and K. R. Olson.  1986b.  Plasma clearance, metabolism and
      tissue accumulation of 3H-labelled catecholamines in trout.  Am. J.
      Physiol.  250:R519-R525.

                                     155

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 Nikirunaa, M.  1982.  Effects of adrenaline on red cell volume and
       concentration gradient of protons across the red cell membrane in the
       rainbow trout, Salmo gairdneri.  Mol. Physiol.  2:287-297.
 Perry,^S. L. , P. Kincaid, P. Fletcher, and D. J. Randall.  1988.  Factors
       influencing the release of catecholamines (in preparation).
 Peyraud-Waitzenegger, M.  1979.  Simultaneous modifications of ventilation and
       arterial Po2 by catecholamines in the eel,  Anguilla anguilla L. :
       participation of A and B effects.  J. Comp. Physiol.  129:343-354.
 Primmett, D. R. N.  D. J. Randall, M. Mazeaud, and R. G. Boutilier.  1986.
       The role of catecholamines in erythrocyte pH regulation and oxygen
       transport in rainbow trout (Salmo gairdneri) during exercise.  J  EXD
       Biol.  122:139-148.
 Randall, D. J.  1982.  The control of respiration and circulation in fish
       during exercise and hypoxia.  J. Exp. Biol.  100:275-288.
 Shelton, G.  1959.  The respiratory centre in the tench (Tinea tinea L/),.   I.
       The effects of brain transection on respiration.   J. Exp  Biol   36-191-
       202.
 Shelton, G.  1970.  The regulation of breathing.   In:  Fish Physiology,  Vol.
       4.  W. S.  Hoar and D.  J.  Randall (eds.), Academic Press, New York.
 Shelton, G., D.  R. Jones, and W.  K.  Milsom.  1986.   Control of breathing in
       ecothermic vertebrates.   In:   Handbook of Physiology-The Respiratory
       System,  S.  R.  Geizer,  A.  P.  Fishman,  N.  S.  Cherniack and J.  G.
       Widdicombe (eds.)  Section 3, Vol, II, pp.  857-909.   American
       Physiological Society,  Bethesda, Maryland.
 Taylor,  E. W.  1985.   Control and coordination of gill ventilation and
       perfusion.  Symp.  Soc.  Exp.  Biol.  39:123-161.
 Taylor,  E. W.   1988.   Cardiovascular respiratory interactions  in fish and
       crustaceans.   In:   Neurobiology of the Cardiorespiratory System, E.  W.
       Taylor (ed).  Manchester  University Press,  Manchester.
 Taylor,  E. W.   1989.   Nervous control of ventilation and heart rate in
       elasmobranch fish,  a model  for the study of the central  neural
       mechanisms  mediating cardiorespiratory interactions in mammals.  In:
       Nonmammalian Animal Models  for Biomedical Research,  A. D.  Woodhead (ed )
       CRC Press,  Florida.
'Waldron,  I.  1972.  Spatial  organisation of respiratory neurones in the
       medulla  of  tench and goldfish.   J.  Exp.  Biol.   57:449-459.
 Withington-Wray,  D. J., B. L. Roberts,  and  E.  W. Taylor.   1986.  The
       topographical organisation of  the vagal  motor  column in  the elasmobranch
       fish,  Scyliorhinus  canicula L.   J.  Comp. Neurol.  248:95-104.
 Withington-Wray,  D. J., E. W. Taylor,  and J. D. Metcalfe.   1988.  The location
       and distribution of vagal preganglionic neurones in the  hindbrain of
       lower vertebrates.   In:  The Neurobiology of the Cardiorespiratory
       System,  E. W. Taylor  (ed.).  Manchester University Press, Manchester.
                                     156

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                 ACCUMULATION OF CARBON DIOXIDE IN FISH FARMS
                           WITH RECIRCULATING WATER

                                      by
                     J. F. Steffensen1 and J. P. Lomholt2

                                 INTRODUCTION

      In northern Europe, intensive culturing of eels, Anguilla anguilla. in
heated fresh water at 25C is economically feasible in recirculated systems
provided with mechanical and biological filtration.  Because of the
consumption of energy for heating, the eels are grown in tanks with
recirculating water, located in well-insulated buildings.  The culture is
based on weaning of wild glass-eels to dry feed.

      The density of fish is high (75 to 150 kg/in2) in eel farms with
recirculating water.  Because the eels are fed continuously and the water is
warm, the oxygen consumption of the fish is high.  Some fish are reported to
grow slower at hypoxic conditions (pOo = 70 mmHg)  (Stewart et al. 1967).
Hence, it is common practice to enrich the water supplying the fish tanks with
pure oxygen (to 200 to 400% 02 saturation) to ensure that the eels are never
exposed to hypoxic water.

      In a typical eel farm with recirculating water (Figure 1), normoxic
conditions in the fish tanks is fulfilled by injecting pure oxygen, but carbon
dioxide produced by the fish is not removed effectively.  Because high carbon
dioxide concentration is reported to inhibit oxygen consumption, to reduce the
oxygen affinity and the oxygen carrying capacity of blood (Root 1931), or even
to induce a narcotic acidicosis, it may be profitable to focus on reducing
carbon dioxide in recirculating eel farms.

                                 THE EEL FARM

      A diagram of a typical Danish eel farm is shown in Figure 2.  From the
fish tanks, the water flows through an over-flow to a sludge separator and
pump sump.  The water then is pumped through an aerator to submerged and
trickle filters for nitrification and denitrification.  Finally the water is
pumped through an oxygenator, supplied with pure oxygen, and back to the fish
tanks.  In the figure, the measured pH and partial pressures of oxygen (pO)
are indicated, the latter in mmHG.  Water is maintained at 6.8 to 7.2 pH by
adding bicarbonate or another base.  Danish ground water has a high
bicarbonate concentration and a relatively high C02 concentration.

      It appears that the oxygen tension is normoxic or hyperoxic in the
entire system.  It also appears that carbon dioxide tension is considerably
      "-The Marine  Pollution Laboratory, The National Environmental Protection
Agency,  Charlottenlund, Denmark
      n
      'Department  of  Zoophysiology, University of Aarhus, Aarhus C, Denmark


                                     157

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                 Production
                 Standing stock
                 Number of fish tanks
                 Area  of fish tanks
                 Volume of fish tanks
                 Total  vol.  of system
                 Reclrculatlon flow
                 Density of fish
                 Food  - dry pellets
                 Water oxygen saturation
                 Oxygen supply
                 HCO3- supply
                 Production time
                 Temperature
                 Area  of filter
                 Current market value
100 ton/year
 60 ton  
 60
860 m2 (4.6 m dla.)
426 m3
660 m3
660 m3/hour
 60 -  100 kg/m2
660 kg/day (1.1 % b.w. pr. day)
300 %  at Inlet -  100 % at out!
300 kg/day
 76 kg/day
 24 months (from .36 to 260 g)
 26 DegC
26000  mZ
B US$/kg
             Figure I.  Typical  eel  farm with recirculating water.


elevated, hypercapnic,  in the entire system,  even though some CC>2 is "blown
off", especially at the trickle filter.  A cC02  of 32 mmHg.   For comparison,
CC>2 in the normal eel habitat rarely exceeds  5 mmHg.   Values of 20 to 30 mmHg
are far above  what eels normally are exposed  to.

                              THE EEL AND THE BLOOD

      Because  the excretion of carbon dioxide over the gills is a passive
diffusion process, pC02 in the blood is  somewhat higher than in the ambient
water.  Blood  pCO? in eels in natural conditions is 3 to 6 mmHg.  In the fish
farm with pC02 = 32 mmHg in the fish tank, the blood pC02 must be even higher.

      A pC02 above 30 mmHg for fish is far higher than normal.  An increase in
C02 will  cause a decrease in pH, which in fish will be compensated for by an
increase  in the blood bicarbonate concentration.   The coherence between pH,
pC02, bicarbonate concentration, and total C02 concentration is shown in the
Davenport diagram in Figure 3.

      Assuming a pC02 of 5 mmHg and pH = 7.7  as  normal values for eel,
bicarbonate concentration equals 10 meq/1, according to Figure 3.  In the eel
farm situation, however,  with a pC02 of  32 mmHg,  the blood will have a
slightly  lower pH.  At pH = 7.5 and 7.4, bicarbonate concentration will be 37
and 29 meq/1,  respectively.  These bicarbonate concentrations are far higher
than what hitherto has been considered physiologically normal for fish.

      Heisler  (1984 1986) suggested that the  maximal blood HC03- concentration
in hypercapnic fish,  which can be used for compensating a hypercapnic
acidosis, is 30 mmol~l.  Recently, however, Dimberg (1988) reported
bicarbonate concentrations of 55 to 66 mmol"-'- in rainbow trout exposed to
hypercapnic water with a pC02 of 26 to 34 mmHg.

                                   CONCLUSION

      Carbon dioxide has long been known to reduce the affinity of blood for
oxygen (Root 1931), to reduce the oxygen carrying capacity of the blood, and
to inhibit the metabolic rate of fish (Basu 1959, Saunders 1962, Beamish
1964).  Basu reported that the active oxygen  consumption of several freshwater
fishes decreased with increasing C02-  At higher concentrations, C02 depresses
ventilation and can even induce a narcotic acidosis (Dejours 1988).

      Only limited information is available on the effect of C02 and oxygen on
swimming  capacity.  Dahlberg et al.  (1968) reported that the prolonged
                                      158

-------
swimming speed  of largemouth bass did not decrease in response to
concentrations  up to  48  mg 1"1.   The performance of coho salmon, on the
contrary, decreased with increasing CC>2 concentrations between 2 and 61 mg
I"1.  The effect  of COg  was especially pronounced at high concentration of
oxygen  (lOmg  1  ).  This decrease of performance may be a result of the
decrease in blood oxygen affinity and carrying capacity, or an increased cost
of acid-base  regulation.          :

      In spite  of the unnaturally high CC>2 tensions in the fish-farms, the
eels seems to be  in good health  and growing well.  It is likely, however, that
these extreme CC>2 tensions have  some consequences for the well-being of the
eels.   Even though the fish are  growing well,  they may grow even better at
more normal COo conditions,  if,  for example, less energy has to be used for
acid-base regulation.  In one fish farm, it was observed that the eels
appetite increased after installing a trickle filter.

      The results_of  future research (growth studies, acid-base balance, etc.)
will determine  whether more effort should be aimed at decreasing the CC>2
levels  in intensive fish farms with recirculating water and oxygenators.
      If it is  desired to get more natural C02-conditions,  it is not
sufficient to adjust  the water pH by supplying bicarbonate, as practiced in
many fish farms.   Only by increasing the contact between the water and the
                                                                      AIR
    OXYGENATOR
                                                  BIOLOGICAL
                                                SUBMERGED FILTER
                                                        SLUDGE
                                                        & WATER
PUMP
Figure 2.  Diagram of eel farm with recirculating water.
                                     159

-------
                             aC02 = 0.04 mM/l : pKapp: 6.12-5.97
                                                 I	1	1	1	1
          6.7
                  6.9
                                        PH
    Figure 3.  Davenport diagram.
 atmosphere, for example with trickle filters, can the diffusion of CC>2 from
 the water to the atmosphere be facilitated, hence decreasing the COo-tension
 in the water.


                                 ACKNOWLEDGMENT

       Financial support from the Danish Natural Science Research Council and
 The University of Aarhus is gratefully acknowledged.
                                   REFERENCES

Basu, S. P.,  1959.  Active respiration of fish in relation to ambient
      concentrations  of oxygen and carbon dioxide.   J.  Fish.  Res.  Bd. Can.
      -LO * JL. / J "* ^ .L^ .
Beamish, F  W. H.   1964.   Respiration of fishes with special  emphasis on
      standard oxygen consumption.   Can.  J.  Zool.  42:847-856
Stewart, N. E. , D.  L.  Doudoroff,  and P.  Shumway.   1967.   Influence of oxygen
      concentration on the growth of juvenile  largemouth bass.   J.  Fish  Res
      Board.  Can. 24:475-494.
Dahlberg, M.  L. , D, L.  Shumway, and P.  Doudoroff.   1968.   Influence of
      dissolved oxygen and carbon dioxide on swimming performance  of
      largemouth bass  and  coho  salmon.  J. Fish. Res. Bd.  Can  25-49-70
Dejours, P.   1988.  Respiration in water  and air.   In:   Adaptations -
      Regulations-Evolution.  Elsevier, Amsterdam,  New York,  Oxford  p  179
                                     160

-------
Dimberg, K.  1988.  High blood CC>2 levels in rainbow trout exposed to
      hypercapnia in bicarbonate-rich fresh water--a methodological
      verification.  J. Exp. Biol. 134:463-466.
Heisler, N.  1984.  Acid-base regulation in fishes.   In:  Fish Physiology, Vol
      XA.  W. S. Hoar and D. J. Randall (eds.).  Academic Press, New York,
      London, p. 315-401.
Saunders, R. L.  1962.  The irrigation of gills in fishes.  II:  efficiency of
      oxygen uptake in relation to respiratory flow, activity and
      concentration of oxygen and carbon dioxide.  Can. J. Zool. 40:817-862.
Root, R. W.  1931.  The respiratory function of the blood of marine fishes.
      Biol. Bull. 61:441-462.
                                     161

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                     IMPACT OF ENVIRONMENTAL ACIDIFICATION
                            ON  GILL FUNCTION  IN  FISH

                                      by

                     Chris M. Wood1 and D. Gordon McDonald1
                                  INTRODUCTION


      Although environmental acidification resulting from industrial and
domestic S02 and NOX emissions  has been documented most strongly in north-
eastern North America and northern Europe, it is now recognized as a global
problem.  Zhao and  Sun  (1986) have reported that much  of China to the south of
the Yangtze River is subject to  "acid rain" due to a combination of intensive
combustion of high-sulphur coal  and low atmospheric buffer capacity (i.e., low
ammonia and alkaline particulates).  Soils are generally acidic, and some
major cities report annual rainfall pHs as low (-4.0)  as those in the most
seriously affected  areas of eastern Canada and Scandinavia.

      Damaging effects  on freshwater fisheries may occur anywhere in the world
where precipitation pH  is persistently below 5.6 (the  pH of distilled water in
equilibrium with atmospheric C02) and the buffer capacity of the watershed is
low.  Such "softwater"  regions are generally characterized by low calcium
carbonate alkalinity in soils,  bedrock, sediments, and the water column
itself.  The softwater  pH range  of environmental significance to fish is 4.0
to 6.0; pHs below 4.0 rarely; if ever, occur.  Most fish populations are not
adversely affected  at pHs above  6.0.

      Within this range, the deleterious effects of acidity are complex, and
include trophic degradation, reproductive failure, early life stage mortality,
and lethal and sublethal toxicity to juvenile and adult fish (Howells 1984).
Over the past decade, our research program has concentrated on this latter
area, investigating the physiological mechanisms of acid toxicity.   The
present paper provides  an overview of this research,  with emphasis  on recent
findings in the area of combined acid and aluminum toxicity,  sublethal
effects; and acclimation.
      Department of Biology, McMaster University, Hamilton ON, Canada.

                                     162

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                     PHYSIOLOGICAL RESPONSES TO PURE ACID
                      STRESS AND THE  INFLUENCE  OF  CALCIUM

      Figure 1 summarizes the major effects of acute exposure to pH = 4.0 -
4.5 on salmonids such as the rainbow trout (Salmo gairdneri).  For detailed
reviews of this earlier work on the lethal effects of acid alone, the reader
is referred to Wood and McDonald (1982),  McDonald (1983), and Wood (1988).   At
this pH range, disturbances in respiratory gas exchange.do not occur, although
they may contribute to toxicity when metals are present  (see below) or at more
acidic pHs where branchial epithelial swelling, delamination, and mucification
may occur.  Nevertheless, there are multiple toxic actions, including ionic
dilution and acidification of the blood and extracellular fluids (ECF),
demineralization of bone, electrolyte losses from the intracellular fluids
(ICF), especially white muscle, and disturbances of renal function.

      By far the most important toxic effects, and those ultimately
responsible for the death of the fish, however, are exerted on the external
surface of the gills.  This is a compound action, comprising an  inhibition of
active Na+ and Cl~ uptake through  the mitochondrial-rich "chloride  cells,"  and
perhaps more importantly, a stimulation of passive diffusive losses of Na ,
Cl", and other electrolytes.  The latter is thought to result from an
increased permeability of the paracellular diffusion channels in the branchial
epithelium.  The net  result is a large loss of electrolytes from the blood
plasma, and secondarily  from the tissues.  Osmotic pressure falls faster in
the ECF than in the ICF, and in compensation,  water shifts from  plasma into
cells, especially muscle and erythrocytes.  Hematocrit,  plasma protein
concentration, and blood viscosity all increase markedly as the  red blood
cells  swell and the plasma volume contracts.   These effects are  compounded by
adrenergically mediated  vasoconstriction of systemic resistance  vessels,
cardioacceleration, and  mobilization of additional red  cells from the spleen.
In the end, greatly elevated arterial blood pressures,  hematocrits as high as
70%, and plasma volumes  as  low as half normal  values result  in a circulatory
failure ultimately attributable to the branchial  ion loss.

       In  laboratory tests,  elevations in water calcium  protect against these
ion losses, and therefore against acid toxicity,  an observation  that  is
confirmed by  a wealth of field data  (e.g., Wright and Snekvik 1978, Howells et
al.  1983).  The mechanism of this protection  is thought to be the well known
effect of Ca2+ in limiting membrane permeability,  specifically in "plugging"
the branchial paracellular  diffusion channels  that are  permeabilized  by  H+.
Those  natural  softwaters most  sensitive to  acidification due to  their low
alkalinity, of course,  are  also low  in calcium (generally in the range 25  -
400 uequiv/L  =0.5  -  8 mg/L), which  exacerbates the problem for  the  fish.
 (Waters of higher calcium content are rarely  acidified,  except in  cases  of
mine acid drainage.)  The ameliorative effects of "liming,"  therefore, are due
to the increases  in water calcium, as well  as  in  pH and alkalinity.

       A point of  considerable  confusion is  the often-stated view that
 increased water calcium also  should  protect the fish  against H+  entry and
 internal  acidosis.  This is clearly  untrue,  as illustrated by the  data in
Figure 2.   Indeed at  a  given acidic  pH, the higher  the  water calcium level,
the greater  is  the depression in  arterial pH and  metabolic acidosis,  whereas
                                      163

-------
                                                          m
iS-o
X * X  +
+ + +  CM
+  +  +  O  'to !_
   X X O  2 O
                164

-------
the smaller Is the plasma Na+ and Cl" deficit.  This pattern has been     +
confirmed by the direct measurement of ion and "acidic equivalent" (net H )
fluxes across the gills.

      The explanation for this apparent dichotomy lies in simple physical
principles--the laws of electrical neutrality and the "strong ion difference"
relationships (Stewart 1983).  Stated very simply, the flux of strong cations
(mainly Na+ plus K+) minus  the flux  of  strong  anions  (mainly Cl") across a
biological membrane such as the gill will automatically constrain an equal
"flux" of acidic equivalents  in the opposite direction.  Water is an infinite
source or sink of acidic equivalents; net H"1" flux is  not an independent
variable, but rather one dependent upon differential strong cation and anion
flux.  Any resultant acid-base disturbance in the fish, therefore, is an
unavoidable consequence of ionoregulatory disturbance.

      Higher water calcium levels protect against strong electrolyte losses at
low pH, but are more effective in limiting Cl" efflux than Na+ or  K+ efflux,
probably because the diffusion channels become cation selective.  At lower
calcium levels representative of natural softwaters,  overall electrolyte
permeability increases, but anion permeability catches up to and may
eventually exceed cation permeability.  As a result,  there is negligible net
H+ uptake, or even a slight net H1" efflux across  the  gills.  Internal  acid-
base status is unchanged or may even become slightly alkalotic (cf. Figure 2).

      In  summary, under pure  acid stress, at environmentally realistic levels
of pH (4.0 - 6.0) and calcium (25 - 400 uequiv/L), toxicity is due to
ionoregulatory disturbance, not to acidosis, and not to respiratory failure.


                          THE IMPORTANCE OF ALUMINUM

      Unfortunately, pure acid stress  itself may not be an environmentally
realistic situation.  An increasing body of evidence argues for the
involvement of aluminum in acid toxicity in the field  (e.g. Schofield and
Trojnar 1980, Harvey and McArdle  1986, Johnson et al. 1987).  Aluminum is the
third most abundant element  in the earth's crust, occurring ubiquitously in
rocks, soil, and sediments.   Its  solubility increases exponentially as pH
falls below about 5.6  (Figure 3); consequently, it is readily mobilized into
acidified softwaters (Driscoll 1980, Lazerte  1984).  This mobilization has
been repeatedly suggested as  the  "missing link" explaining why mortality in
the  field is often much greater than that in  the  laboratory at the same pH and
calcium levels, although this point remains controversial  (e.g. Muniz and
Leivestad 1980, Schofield  and Trojnar  1980, Howells et al. 1983, Howells 1984,
Neville 1985, Schindler 1988).

      Much of the uncertainty surrounding the role of aluminum devolves from
its  very  complex aqueous chemistry.  A highly simplified speciation model is
shown  in  Figure'3, which deliberately  omits the fluoride,  sulphate, carbonate,
and  organic anion binding  reactions, all of which are thought to  lessen
aluminum  toxicity, and the various  polymerization reactions that may  occur
under  super-saturated  conditions.   The equilibria plotted  are based on the
dissolution of microcrystalline gibbsite, which is probably more  representa-
                                     165

-------
   +0.2


     0
     s --2
     E
       -0.4
     S  4'
       +8.0
_i  ~10
CT
UJ
E  -20
   -30


     0


_j  ~10
CT
UJ
E  -20


   -30
                                             ApH
                                             AH
                                                m
                                     x"" API
                                           asma
                                                ,.  _,
                                        APIasma [crj
               if'
                   Water
                                   (mEq/L)
Figure 2.  The relationship  between water calcium concentra-
  tion and the extent of various  acid-base and ionic distur-
  bances in the arterial blood of rainbow trout exposed to
  pH 4.3 for 3 days.   (A)  Changes in arterial pH. (B) Changes
  in blood metabolic acid  load.  (C)  Changes in plasma sodium
  concentration.  (D) Changes in plasma chloride concentration.
  Means 1 SEM  (n = 4 - 24) .   (from Wood 1988)
                             166

-------
  tive of field conditions (at least in eastern Canada, Lazerte 1984). A total
  solubility curve based on amorphous aluminum hydroxide also is presented.  The
  latter is representative of freshly prepared solutions used in the  laboratory.
  Figure 3 illustrates that the speciation, as well as the solubility, of
  inorganic monomeric aluminum is critically dependent upon pH.  As pH falls,
  the non-toxic anion A1(OH)A"  is  sequentially  replaced by  the  Al(OH)2+,
  Al(OH)2"1",  and finally A13+ cations.  All these cationic forms are thought to be
  toxic to fish, although A13+  could also  be protective through its ability to
  mimic the action of Ca2+ (Baker and Schof ield 1982) .  Much of the previous
  work on the  physiological responses of  fish  to  aluminum  has been done  in
  recirculating systems, often without precise control of  pH or water chemistry,
  or realistic water calcium levels.  The resulting divergence  in  conclusions
  may well  reflect changing solubility, speciation, organic and inorganic
  complexation, and calcium/aluminum interactions.
          106-K
Aluminum
          10
  TOTAL Al (amorphous AlDH)3)
x
 TOTAL Al (gibbsite)
             4-0   4-2    4-4   4-6   4-8   5-0   5-2    5-4   5-6   5-8   6-0

                                              PH

  Figure 3.   Speciation chemistry and  solubility of aluminum (omitting complexation
    reactions)  as a function of pH in  low ionic strength waters.   The Al/pH combin-
    ations tested in the present study are noted.
                                       167

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         PHYSIOLOGICAL RESPONSES TO ACID/ALUMINUM/CALCIUM COMBINATIONS

       The  goal of our own recent work has been to  study the  physiological
 responses  of juvenile and adult salmonids to various  environmentally
 representative combinations of pH (4.4,  4.8,  5.2),  calcium  (25,  400  uequiv/L),
 and aluminum (0,  111,  333,  1000 ug/L)  under defined artificial  softwater
 conditions (reverse osmosis water,  50 uequiv/L Na+, 0 DOC and fluoride, pH set
 with H2S04) .  The fish are previously acclimated to the artificial softwater
 at  pH - 6.5,  Al = 0 ug/L for at least 2  weeks.

       Our  approach has been to "set"  the water chemistry, using  freshly
 prepared solutions,  and  then to move  the water past the fish as  quickly as
 possible to  minimize pH  shifts,  speciation changes, and organic  complexation
 reactions  (1/2 replacement  time ~20 min.  in the experimental chambers).   Note
 that the Al/pH combinations tested  (plotted on Figure 3) are well below the
 solubility limits for amorphous aluminum hydroxide  (and except in one  case for
 gibbsite also), so supersaturated conditions  are avoided, at least in  the bulk
 water.   The  brook trout  (Salvelinus fontinalis)  has been studied as  it is the
 salmonid endemic  to  the  acid-threatened  regions of  North America, and  the
 rainbow trout as  it  is a widely distributed reference species.   For  more
 detailed information on  the brook trout  studies, the reader  is referred to
 Booth et al.  (1988), Wood et al.  (1988a,b,c),  McDonald  and Milligan  (1988),
 and Walker et al.  (1988), and on the  rainbow trout  studies to Goss and Wood
 (1988)  and Playle et al.  (1989).

      Upon exposure  to acidic  pH alone in the  absence of Al, brook trout
 exhibit transient and sublethal net losses  of  Na+ and Cl" across  the  gills in
 proportion to the severity  of  the pH  (Figure 4); zero balance is re-
 established  in 12 to 72  hours.   The addition of Al  to the exposures  greatly
 exacerbates  these losses and kills many  of  the  fish within 24 to 72  hours
 (Figure 4).   Terminal blood samples reflect these losses, with severely
 depressed  ECF ions and dramatic  hemoconcentration in lethal  exposures, as
 previously seen in rainbow  trout dying from pure acid stress.  The cumulative
 ion losses prior  to  death in acid/Al exposures  are  not  as large, however,
 suggesting that an additional  toxic mechanism(s) may be  involved (see below).
 In  some cases,  the brook trout do not die in the presence of Al, but rather
 return  to  zero  or even positive balance  and survive indefinitely.

      Measurements of unidirectional fluxes across  the  gills with 22Na
 demonstrate that  initial  losses  reflect  a combination of inhibited active
 uptake  and stimulated diffusive  efflux;  the latter  is quantitatively much more
 important.   This  constitutes an  initial  "shock"  phase (0 - 12 hours)  of heavy
 losses  that may eventually kill  the fish by inducing intolerable fluid volume
 and circulatory disturbances.  The extent of cumulative ion  loss during this
period  is a reliable predictor of eventual  survival or death, even though that
death may not occur  for  several more days.  In trout that survive,  there
follows a  "recovery" phase in which the efflux component is returned to
control levels or below by 24  to 72 hours.  Active influx remains depressed,
however, for up to 10 days.  The initial large efflux is thought to result
 from opening  of the paracellular diffusion channels in the gills, and the
 later adaptive response  from an effective closing of these channels,  as
 discussed subsequently.
                                     168

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      By 48 hours, the majority of the electrolyte loss that will occur has
already done so.  Cumulative 48-hour Na+ loss and mortality are strongly
correlated (Figure 4).  These responses increase with Al concentration at any
given acidic pH, and increase with pH at Al = 333 ug/L.  Thus Al toxicity
appears to be greatest at pH = 5.2, where total solubility is lowest and
A1(OH)2+  and A1(OH)2+ predominate (Figure 3).

      The higher water calcium level (400 uequiv/L, still in the softwater
range) generally reduces the extent of electrolyte loss at any particular
pH/Al combination and removes the clear relationship between cumulative ion
                                             18H NA+ LOSS- (1000 UEQ/KG)
                                                 Ca2'l"=25ueq/l

                                                     I
      PH
       Figure 4.   Cumulative net sodium losses of brook trout over the
         first 48 hours of exposure to various combinations of pH and
         aluminum in flowing softwater (Ca = 25 uequiv/L).   Means 1
         SEM (n = 6 in each treatment).  Percentage mortalities at the
         end of 10 days of exposure are indicated for each treatment.
         (modified from McDonald et al. 1988, Booth et al.  1988)
                                      169

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 loss  and mortality seen at lower calcium (25 uequiv/L;  Figure  4).   Neverthe-
 less,  a substantial number of fish still die,  even though their  ion losses  and
 plasma Na+ and Cl~ depressions are quite small.  Under these conditions,
 respiratory  failure,  the second toxic mechanism alluded to previously,  becomes
 the primary  cause of death.   This interference with 02 uptake  and C02 excre-
 tion  across  the  gills is signalled by a pronounced hyperventilation accom-
 panied by decreases in arterial blood 02 tension  (PaO2;  Figure  5C)  and 02
 content below typical venous  values,  and reciprocal increases  in C02 tension
 and blood lactate.   Although  this phenomenon is most marked at the  higher
 calcium level (Figure 5G)  and higher  pHs,  it occurs prior to death  in all
 treatments where Al is present (Figure 5B,E).

       Although the  responses  of the rainbow trout are qualitatively similar to
 those  of the brook  trout,  there are two important differences.   Firstly, under
 any particular acid/Al condition,  rainbows are far less  tolerant, showing
 greater ion  loss (Figure 6B),  hemoconcentration (Figure  6C), and respiratory
 disturbance  (Figure 6D),  and  earlier  and larger percentage mortalities.  These
 physiological results are  in  accord with field and toxicological data  indicat-
 ing that Salmo  gairderi is the most  sensitive,  and Salvelinus fontinalis the
 most resistant,  of  a range of salmonids (Grande et al.  1978).  Secondly, the
 contribution of  respiratory disturbance to toxicity is  greater in the  rainbow
 trout  under  all  conditions where Al is present, not just at the higher water
 calcium level.   Indeed,  unlike the brook trout,  there is no evidence that
 respiratory  disturbance  is exacerbated by  higher  water  calcium,  although it is
 worsened by  higher  pH.   These  differences  emphasize that generalization
 between species,  even within  the same  Family,  must proceed with  caution.


                         BRANCHIAL MECHANISMS OF ACTION
                        FOR ALUMINUM,  ACID, AND CALCIUM

       The preceding observations can be summarized by saying that acidity and
 aluminum both cause  ionoregulatory toxicity, that calcium protects  against
 ionoregulatory toxicity, that  aluminum alone also causes  respiratory toxicity,
which  may or may not  be  exacerbated by higher  calcium, and that the extent of
 aluminum's ionoregulatory  and  respiratory  effects  appears  to be greater at
higher acidic pHs.  Clearly the  situation  is complex, and any  explanation(s)
must take into account the chemistry of aluminum,  the response(s) of the gill
 epithelium,   and  the nature of  the  interaction between the  two.   The final
point  is  the  critical one, about which we know very little.at present.  The
ideas  that follow are, therefore, highly speculative.

       An important  starting point  is the observation that accumulation of
 aluminum on  the  gills (Figure  6A)  appears  to be directly  associated with
 toxicity, and correlates well with physiological  indices  of disturbance
 (Figure  6B,C,D).  Gill aluminum  levels  increase in a time-  and concentration-
 dependent fashion, are greater at higher pH for any particular water Al level,
are greatest  in  dying fish, and  are clearly related to differences  in species
 sensitivity  (Figure 6A).  We view  this  as a superficial uptake onto or into
 the gill  cells,   for neither we nor Neville  (1985)  can find any evidence of
aluminum entry into the  fish in  exposures lasting up to 10 days.
                                     170

-------
               120


                80


                40


                 0"


                80

                40


                 0


                80


                40


                 0

                80


                40


                 0

                80


                40
pH=4.8, Ca=25 uequiv/L, Al=0 ug/L
pH=4.8, Ca=25 uequiv/L. Al=333 ug/L
 pH=4.8,
 Ca=400 uequiv/L, Al=333 ug/L *
PH = 4.4. Ca = 25 uequiv/L, Al=0 ug/L
pH = 4.4, Ca=25 uequiv/L. Al=333 ug/L
                    C 0
                           10
         20    30

           Time(h)
40
Figure 5.  Changes  in the Q tension of arterial blood (PaQ2) in
  chronically  cannulated brook trout during  10 days exposure to five
  different pH/Ca/Al conditions in flowing softwater.  Means  SEM
  (n = 9 - 28  in each treatment).  Data are  shown from the control
  period until 48 hours for all fish that survived beyond this time in
  each exposure (i.e., the most resistant fish in each group). Addi-
  tionally, terminal data (T, cross-hatched  bars) representing the last
  measurements prior to death in fish  dying  at any time during the
  entire 10 days exposure are shown in bar graphs at right and compar-
  ed with  initial measurements  (I, open bars)  during the control
  period for  these  same fish.   In panel A, none of the fish  died,  so
  data at  10  days exposure have been substituted for terminal data.
  Asterisks indicate significant differences (p=<0.05) from  appropriate
  control  or  initial values,  (from Wood et al. 1988a)
                                  171

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       Why should aluminum accumulate in the first place?  The branchial
 surface is rich in organic anions' on mucus and cell surfaces, and these anions
 may avidly bind the cationic Al species that occur at low pH (Figure 3).
 Analysis of mucus fragments extruded by the fish or gently scraped from the
 gills of dead fish show very high aluminum contents.   Furthermore, at bulk
 water pHs below about 5.5, the branchial micro-environment is much more
 alkaline than the inspired water (Figure 7A) probably due mainly to the efflux
 of NH3 across  the gills.   As  inflowing  water encounters  this  more  basic
 milieu, the formation of aluminum hydroxides will be  favored (Figure 3),  and
 perhaps more importantly,  supersaturating conditions  may occur,  resulting in
 direct precipitation of polymers on the gills.   The exponential  relationship
 between pH and Al solubility is critical here (Figure 3).

       Figure 7B illustrates that an aluminum concentration (111  ug/L)  that
 would appear to be well below saturation at a bulk water pH of 4.8,  is
 effectively right on the solubility limit,  because the pH at the gills is
 raised to about 5.2 (Figure 7A).   In turn,  at a bulk  water pH of 5.2,  where
 this  concentration appears to be at the solubility limit,  it is  in fact
 several-fold saturated (Figure 7B),  because the pH is raised to  about  5.5 at
 the gills (Figure 7A).   This  may explain why aluminum is more toxic  at higher
 acidic pHs,  although it is also possible that the less charged species are
 intrinsically  more toxic;  the two explanations  are certainly not mutually
 exclusive.

       Figure 8 presents a  simple model  of the branchial  epithelium incor-
 porating some  of the ideas presented in this paper and recent histological
 work  (Karlsson-Norrgren et al.  1986,  Tietge et  al.  1988, M.E.  Mueller,  pers.
 comm.).   H+ by  itself (Figure 8B) inhibits  the active Na+ and Cl" uptake
 processes through the chloride  cells  and, more  importantly,  increases  their
 diffusive loss by opening  up  the  tight  junctions  of the  paracellular channels,
 perhaps  by displacing bound Ca2+; respiratory gas exchange is not affected.
 If  aluminum also  is  present (Figure  8B),  there  is  a precipitation  of Al com-
 plexes  and/or  binding of Al to  organic  anions on  the  gill  surface.  In turn,
 this  acts  as an irritant that stimulates  the production  of additional  mucus
 and induces  an inflammatory response  comprising edema, white  cell  invasion,
 lamellar clubbing and even fusion.  A general thickening and  distortion of the
 branchial  epithelium results, which  together with  the mucus/Al layer,  in-
 creases  the  transcellular  diffusion distance  from water to blood,  thereby
 reducing 02 and C02 exchange.   At the same time,  this  distortion  further opens
 the paracellular  channels,  increasing diffusive electrolyte loss, while the
 surface  coating or Al cations themselves  further inhibit active Na+ and Cl"
uptake.  Recovery, if it occurs, presumably  reflects a reduction in the
 inflammatory response,  a closing of the paracellular channels, and a reduction
 in the transcellular diffusion  distance for 02 and C02.   Higher water calcium
 levels  (Figure 8D) also help close the paracellular channels, thereby reducing
 ion efflux.  In the brook  trout, where calcium intensifies the gas exchange
 problem, we  speculate that it acts in some way to  also thicken the mucus/Al
 coat.

                      ACCLIMATION TO ACID/ALUMINUM STRESS

      It was noted earlier that some  individual trout may recover from an
 acid/Al challenge in the continued presence of the stressors, which raises the

                                      172

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                                             Gill Aluminum
                                             Plasma Na+
                                              Hematocrit
      70 -,
                                               Plasma Lactate
                 10
                         20       30
                         Time (hours)
Figure 6.  A  comparison of the responses in (A) gill alumi-
  num accumulation,  (B) plasma sodium concentration,  (C)
  hematocrit,  and (D)  blood lactate  betweenjuvenile brook
  trout  (BT)  and juvenile rainbow trout (RBT)  exposed  for
  48 hours to pH = 4.8, .Al =  111  ug/L, at Ca = 25 uequiv/L.
  Means  1 SEM (n = 10 - 20 at each  point). (D.G. McDonald
  and C.M. Wood, unpublished  results)
                          173

-------
     +0.6
     +0.4
     +0.2
  X  0.0
  <
  6>
    -0.2
    -0.4
    -0.6
         4.0
                    4.5
                                        V5.5
                                                    6.0
                               5.0
                                                  6.5
                                                 t
                                  -PHin-
                                                     V--,
   10000
    1000
  D)
     100
       10
\\
        4.0
       Al solubility

in bulk water
 at gill A
                  t  i  i  i I  i  .
                                  I  I I  I.
    4.5
 5.0
5.5
6.0
                                                   6.5
                             PH
                                in
Figure 7.  (A) The relationship between inspired bulk water PH and
  gill water PH in the rainbow trout in softwater.  Each point re-
  presents an individual measurement. (B)  The effect of this shift
  in water PH at the gills on the solubility of aluminum.  See text
                 detallS- (R--  Playle and C'M- Wood,. unpublished
                             174

-------
possibility that acclimation may  occur.   This  is further suggested by field
survey data (Schofield  and Trojnar  1980,  Kelso et al.  1986) from chronically
acidified lakes, indicating that  salmonids may survive in the field at
aluminum levels known to be lethal  in laboratory trials.  Of course an ^
alternate explanation is that  of  genetic differences and natural selection.
Acclimation is defined  as  increased resistance to a more severe exposure
acquired as a result of a  previous  sublethal exposure.  Therefore, we have
evaluated the physiological responses to long-term sublethal acid/Al exposures
and subsequent more severe challenges.  For more detailed information on these
acclimation studies, the reader is  referred to Wood et al. (1988b,c), McDonald
and Milligan  (1988), Audet et  al. (1988), and Audet and Wood (1988).
                                           B
                  Control
                                                        H +
         Water
               
-------
       There  is  no  evidence  that  salmonids have  any  ability  to acclimate  to
 pure acid.   Rainbow trout exposed to  sublethal  low  pH  (4.8)  in flowing
 softwater show  a gradual loss  of electrolytes and elevation of plasma glucose
 and cortisol levels, both of which are  sensitive stress indicators.  Although
 a plateau is reached after  4 to  7 weeks  in most parameters,  there is no
 recovery back to control levels.   When  these fish are  challenged with a more
 severe exposure (pH =4.0)  they  exhibit  further ion losses  and other
 physiological disturbances  that  are greater than those seen in previously
 unexposed trout.   Thus sensitization, rather than acclimation, occurs.

       The situation is very different for combined  acid plus aluminum
 exposure.  Brook trout held for  10 weeks in flowing softwater at pH = 5.2 plus
 75 or 150 ug/L Al  actually  appear  less stressed than those held in pH - 5.2
 alone,  based on plasma electrolytes, glucose, and cortisol,  and rates of
 branchial sodium uptake.  The mechanism is unknown but may be associated with
 a proliferation of branchial chloride and mucous cells seen in fish chroni-
 cally exposed to low levels of aluminum (Tietge et al.  1988).  When these fish
 are subjected to a more severe combined acid plus aluminum challenge (3 days
 at pH - 4.8,  Al = 333 ug/L,  which is lethal to the majority of previously
 unexposed trout), they exhibit 100% survival,  and much smaller physiological
 disturbance.   This acclimation appears to protect against both toxic mecha-
 nisms (ionoregulatory and respiratory distrubance),  and is reflected in stress
 indicators (glucose, cortisol), respiratory parameters  (ventilation,  blood
 gases,  lactate)  and ionoregulatory parameters.

      For example,  Figure 9  illustrates  that the cumulative  Na+ loss over  the
 first 48  hours of challenge  (pH - 4.8, Al - 333  ug/L)  is  approximately halved
 by pre-exposure  to  low levels  of aluminum at pH  = 5.2,  and the  depressions in
 plasma Na and Cl"  correspondingly  reduced or abolished.  Note that the Na+
 loss  is only  reduced to the  level that is caused by  challenge with acid alone
 (pH - 4.8) and that pre-exposure  to acid alone  (pH =5.2)  provides no  protec-
 tion  against  acid plus  aluminum challenge.   In agreement with the rainbow
 trout experiments,  this finding indicates that the acclimation  is not  to  acid
 but rather to aluminum.                                                      '

      What is/are the mechanism(s)  of  acclimation to aluminum?  With other
 metals, acclimation has been traditionally associated with internal processes
 such as the induction of metal-binding proteins  (e.g. metallothioneins) or the
 activation of excretion mechanisms.  Aluminum appears to be  entirely a
 surface-active toxicant, however,  so these explanations appear unlikely.

      As a first  step in attacking  this problem,  we  recently have followed a
variety of physiological and histological parameters over time in juvenile
brook trout exposed  to sublethal  aluminum.  The goal was to see which para-
meters,  if any, change as acclimation develops.   After an initial sensitiza-
tion,  a significant  and sustained increase in resistance (i.e. relative LT50)
to more severe aluminum challenge occurs  between days 10 and 13 of exposure to
low levels of Al  (75 or 150 ug/L) at PH = 5.2 (Figure 10A).  Again exposure to
acid alone (pH =5.2) is relatively ineffective in inducing acclimation.   Most
internal parameters  show little or no correlation with this time course,  but
there is a striking relationship between gill aluminum burden and the develop-
 ment of acclimation  (Figure 10B).  Despite continued low level exposure to
                                     176

-------
    -6000
  O)
  3
  cr
  0
  3
    -4000
    -2000
      -40
  I   -20
  03
  E
         0
              Cumulative 48 H Na+ Loss
              pH = 6.5
               AI = O
                 pH=6.5
                  Al = 0
pH=5.2
 Al = 0
pH = 5.2
Al = 150
pH=5.2
Al = 75
CU APIasmafNa"*]
^ A Plasma
              pH = 4.8
             i	1
               Al = 0
                               pH = 4.8
                               Al = 333
                                 Challenge
Figure 9.  The effects of 10 weeks of  sublethal exposure  to  condi-
  tions shown  (in flowing softwater, Ca =  25 uequiv/L)  on the
  responses of brook trout to subsequent challenge with a more
  severe low pH alone  (4.8) or low pH  plus Al  (333 ug/L).  (A)
  Cumulative net sodium losses over 48 hours of challenge.' (B)
  Changes in plasma sodium and chloride concentrations  over  48
  hours of challenge. Means 1 SEM. (n  = 6  in each group),  (modified
  from Wood et al. 1988b)
                              177

-------
 the metal, the gill is in some way able to "clean" itself of bound or preci-
 pitated aluminum compounds.

       A pronounced hypertrophy of branchial mucous cells occurs, so this may
 be associated with increased rates of mucus synthesis and sloughing as the
 cleansing mechanism.  As the gill aluminum burden is reduced, so may be the
 inflammatory response, the paracellular permeability, and the diffusion
 distance for respiratory gases.  Whatever the explanation,  the nature of the
 gill surface appears to change so as to become less reactive to higher levels
 of waterborne aluminum during challenge.


                                   CONCLUSION

       The unifying theme of the research summarized here has been that the
 toxic actions of environmental acidity and associated aluminum stress,  as well
 as the protective effects of calcium and the adaptive responses of recovery
 and acclimation,  can all be interpreted at the level of gill epithelium.
 Nevertheless, our understanding of the chemical,  physical,  and morphological
 characteristics of the branchial surface remains  rudimentary.  A much finer
 characterization of this complex and delicate epithelium,  together with its
 aqueous micro-environment,  is required.


                                ACKNOWLEDGMENTS

       The work reported here was supported by grants from the NSERC (Canada)
 Strategic Program in Environmental Toxicology,  and a contract ("Lake
 Acidification and Fisheries",  RP-2346-01;  Dr.  J.  Mattice, project manager)
 from the Electric Power Research Institute,  Environmental Assessment  Dept.,
 Palo Alto,  CA (U.S.A.),  through a subcontract from the  University of  Wyoming.
 We thank Dr.  H.L.  Bergman and the staff  of the Red Buttes Fish Physiology and
 Toxicology Laboratory,  University of Wyoming,  for extensive  collaboration,  and
 R.C.  Playle,  R. Rhem,  M.E. Mueller,  and  D.R.  Mount for  permission to  cite
 previously unpublished data.


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Audet,  C. and C. M. Wood.  1988.   Do  rainbow trout acclimate  to low pH?
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                                     178

-------
o
p
0)
tr
       2-0 -i
       1.6-
       1.2-
       0.8-
       0.4-
       0.0
                                           pH = 5.2. Al = 75 ug/L
                LT50
                                10           15

                                Time (Days)
                                                       20
                                                                  25
 0)
 
        400 -i
       300-
        200-
        100-
                                          Gili Al
                                         5.2, A! = 150 ug/L
                                       pH = 5.2, Al = 75 ug/L
                                             Control
                                      -I---J/0-	-J	-5
 f m. * 	 * 	 	  	 	 * 	 
c
0
5
10
15
i 	 , 	
20
' 	 1
25
                                 Time (Days)

 Figure 10.   (A) The effects  of sublethal exposure  to  pH = 6.5
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                              179

-------
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 McDonald, D.G.  1983.  The  effects  of  H+ upon the gills of freshwater fish.
       Can. J. Zool.  61:691-703.

McDonald, D.G., and  C.L. Milligan.   1988.  Sodium transport  in the brook
       trout, Salvelinus  fontinalis:  The effects of prolonged low pH exposure
       in the presence  and absence of aluminum.   Can. J. Fish. Aquat  Sci
      45:1606-1613.

McDonald, D.G., J.P. Reader,  and T.K.R. Dalziel.  1988.  The combined
      effects of pH and  trace metals on fish ionoregulation.  In: Acid
      Toxicity  and Aquatic Animals, Society for Experimental Biology Seminar
      Series.  R. Morris, D.J.A. Brown, E.W. Taylor, and J.A. Brown (eds.).
      Cambridge University Press.

                                     180

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Muniz  I P  and H. Leivestad.  1980.  Acidification-effects on freshwater
     'fish.  In: Ecological Impact of Acid Precipitation.  D. Drablos and A.
      Tollan (eds.).  pp. 84-92, SNSF-project.

Neville  C M   1985.  Physiological response of juvenile rainbow trout,
      Salmo gairdneri. to acid and aluminum - prediction of field responses
      from laboratory data.  Can. J. Fish. Aquat. Sci. 42:2004-2019.

Playle  R C   G G  Goss, and C.M. Wood.  1989.  Physiological disturbances
      in rainbow trout (Salmo gairdneri) during acid and aluminum exposures in
      soft water of  two  calcium  concentrations.  Can. J. Zool.  In Press.

Schindler, D..  1988.   Effects  of  acid rain on freshwater ecosystems.
      Science 239:149-157.

Schofield, C.L., and R.J. Trojnar.   1980.  Aluminum toxicity  to brook
      trout  (Salvelinus  fontinalis)  in acidified waters.  In: Polluted Rain.
      T.Y. Toribara, M.W. Miller, and  P.E. Morrow  (eds.).  Plenum Press,  New
      York,  NY.  pp. 341-366.

Stewart,  P.A.   1983. Modern quantitative  acid-base chemistry.  Can. J.
      Physiol.  Pharmacol.  61:1444-1461.

Tietge   J E   R D.  Johnson,  and H.L. Bergman..  1988.   Morphemetrie  changes
       in gill secondary  lamellae of brook trout (Salvelinus  fontinalis)_after
       long-term exposure to acid and aluminum.   Can.  J.  Fish. Aquat.  Sci.
       45:1643-1648.

Walker  R.L., C.M.  Wood, and H.L. Bergman.  1988.   Effects  of low pH and
       aluminum on ventilation in the brook trout (Salvelinus fontinalis).
       Can. J. Fish. Aquat.  Sci.  45:1614-1622.

 Wood, C.M.  1988.  The physiological problems of fish in acid waters.   In:
       Acid Toxicity and Aquatic Animals,  Society for Experimental Biology
       Seminar Series.  R. Morris, D.J.A.  Brown, E.W.  Taylor, and J.A.  Brown
       (eds.).  Cambridge University Press.

 Wood  C.M.,  and D.G. McDonald.  1982.   Physiological mechanisms of acid
       toxicity to fish.   In: Acid Rain/Fisheries.  R.E. Johnson (ed.).
       American Fisheries Society, Bethesda, MD.  pp.  197-226.

 Wood  CM   and D.G. McDonald.  1987.   The physiology of acid/aluminum
      ' stress in trout.  In: Ecophysiology of Acid  Stress in Aquatic Organisms.
       H. Witters and 0'. Vanderborght  (eds.).  Annls. Soc. r. Zool. Belg. 117
       (suppl. 1):399-410.

 Wood  C.M., R.C. Playle, B.P.  Simons, G.G. Goss, and D.G. McDonald
       1988a  Blood gases,  acid-base  status, ions, and hematology in  adult
       brook trout  (Salvelinus  fontinalis) under acid/aluminum exposure.  Can.
       J.  Fish. Aquat. Sci.   45:1575-1586.
                                      181

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Wood, C.M., D.G. McDonald, C.E. Booth, B.P. Simons, C.G. Ingersoll, and H.L.
      Bergman.  1988b).  Physiological evidence of acclimation to
      acid/aluminum stress in adult brook trout (Salvelinus fontinalis).   1.
      Blood composition and net sodium fluxes.  Can. J. Fish. Aquat. Sci.
      45:1587-1596.

Wood, C.M. , B.P. Simons, D.R. Mount, and H.L. Bergman.  1988c.  Physiological
      evidence of acclimation to acid/aluminum stress in adult brook trout
      (Salvelinus fontinalis).  2. Blood parameters by cannulation.  Can. J.
      Fish. Aquat. Sci.  45:1597-1605.

Wright, R.F., and E. Snekvik.  1978.  Acid precipitation:  Chemistry and
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      Verein. Limnol. Biol.  20:765-775.
Zhao, D. and B. Sun.
      15:2-5.
1986.  Air pollution and acid rain in China.   Ambio
                                    182

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                          AMMONIA TOXIGITY TO FISHES
                              Robert V.: Thurston1


                                 INTRODUCTION

     Ammonia is a naturally occurring product of metabolic degradation and, as
a consequence, is commonly found in most aquatic environments.  It also is
frequently added to aquatic systems as a waste product from industrial,
municipal, and agricultural processes.  Ammonia is extremely toxic to fishes
and other aquatic animals, and this toxicity is affected by several factors
including water pH, dissolved oxygen content, temperature, concentration
fluctuations, degree of salinity, presence of other chemicals, and prior
acclimation.  Short-term exposure of fishes to high concentrations of ammonia
causes increased gill ventilation, hyperexcitability, loss of equilibrium,
convulsions, and then death (Smart 1978, Thurston et al. 1981c) .  These
effects are most likely the result of a direct effect of ammonia on the
central nervous system.  Chronic exposure of fishes to lesser concentrations
of ammonia include damage to tissues, decreases in reproductive capacity  (egg
production, egg viability, spawning delay) , decreases in growth, and increases
in susceptibility to disease (Thurston et al. 1984, 1986).  Chronic exposure
also may cause progressive deterioration of other physiological functions, any
one of which may be the ultimate cause of death (Randall and Wright 1987) .


AQUEOUS AMMONIA EQUILIBRIUM

     Ammonia assumes two chemical forms in aqueous solution, shown by the
following equilibrium equation.

                  NH3 + nH20 = NH3'nH20 = NH4+ + OH" + (n-l)H20.

These forms are the un- ionized ammonia species (NH3) , hydrogen-bonded to at
least three  (n > 3) water molecules  (Butler 1964) , and the ionized species
(NH4+) .   The toxicity of ammonia is  generally attributed to NH3, which  is  an
extremely soluble gas.  Total ammonia is the sum of NH3 and NHA*, and it is
total ammonia that is most commonly measured in aqueous solutions; the
concentration of NH3 is then calculated.  Tables of NH3 concentration,  as  a
function of total ammonia concentration, over a range of pH values from 5 to
12 and range of temperatures from 0  to 40 C are available (Emerson et al.
1975, Thurston et al. 1979).
'Fisheries Bioassay Laboratory, Montana State University, Bozeman MT USA

                                      183

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      The relative concentrations of ionized and un-ionized ammonia in a given
 solution are a function of the pH, temperature, and ionic strength of that
 solution.  ,As pH increases, the concentration of NH3 increases, while that of
 NHA  decreases.   For example,  a pH increase from 7.0  to 8.0  in the temperature
 range of 0 to 30 C results in a nearly 10-fold increase in the concentration
 of NH3.   Temperature increase also favors the NH3  species, but to a lesser
 extent; a temperature increase of 5 degrees between 0 and 30 C at pH 7.0
 results in an NH3 concentration increase of 40 to 50%.   An increase in the
 ionic strength of a solution, at low concentrations, favors the NH4+  species.
 In natural waters with low to moderate amounts of dissolved solids (200-1000
 mg/L) this effect will slightly reduce the concentration of NH3,  and the
 magnitude of this effect will vary with the composition of the water (Thurston
 et al.  1979).

 RELATIONSHIP OF AMMONIA TO NITRITE AND NITRATE

      Ammonia is interrelated with nitrite (NO;,0 and nitrate (N03") through the
 process of nitrification,  the biological oxidation of ammonia to nitrate.
 Under aerobic conditions ammonia is readily oxidized to nitrite by Nitro-
 somonas bacteria,  and nitrite is then oxidized to nitrate by Nitrobacter
 bacteria.   In well-oxygenated aquatic systems,  the conversion of ammonia to
 nitrite is the rate-limiting step in the total process, and the conversion of
 nitrite to nitrate occurs  fairly rapidly.   Nitrite concentrations in most
 natural systems,  therefore,  are usually low,  which is fortunate for aquatic
 life because nitrite,  also,  is extremely toxic to  fishes.   Nitrate,  although
 generally present in all but the most oligotrophic surface waters,  is rela-
 tively  non-toxic  to  fishes.   Where it is present in high concentrations, any
 problem is usually one of  contributing to eutrophication rather than  to
 toxicity.

                           ACUTE TOXICITY OF AMMONIA

      There is  some variation in the  susceptibility of fishes  to the ammonia
 toxicity.  Representative  96-hour  acute toxicity values (96-hour  median  lethal
 concentrations, or LC50 values)  are  listed in Table 1.   From  available data,
 salmonids^appear  to  be among  the most  sensitive  to  acute ammonia  exposure, and
 centrarchids,  catfish, and some minnows the most tolerant.  In addition  to
 differences  in susceptibility  among  fish species,  there also  can  be differ-
 ences in susceptibility at different life  stages of a given species (Thurston
 and Russo  1983).  Moreover, several  factors can affect  the toxicity of
 ammonia, including environmental pH, dissolved oxygen,  temperature, salinity
 and ionic composition, previous acclimation and intermittency  of  exposure, and
 presence of other toxicants.

 EFFECT OF pH

      Some years ago researchers observed that the toxicity  of  ammonia
 solutions was greater at high pH values  (Chipman 1934, Wuhrmann et al. 1947,
Wuhrmann and Woker 1948),  and because the percentage of NH3 in total ammonia
 increases as solution pH increases, these authors concluded that NH3 was  the
 toxic form of ammonia in the water.  They also concluded that NH4+ was non-
 toxic or appreciably less  toxic.  More recently, however, several researchers
have reported that NH3 is more toxic at low pH,  separate from the effect  of pH

                                    184

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Table 1.  Representative acute toxicity values for un-ionized ammonia.
          (Values reported are ranges.)

Species
Pink salmon
(Oncorhynchus gorbuscha)
Mountain whitefish
(Prosopium williamsoni)
Brown trout
(Salmo trutta)
Rainbow trout
(Salmo gairdneri)
Largemouth bass
(Micropterus salmoides)
Smallmouth bass
(Micropterus dolomieui)
Common carp
(Cyprinus carpio)
Red shiner
(Notropis lutrensis)
Fathead minnow
(Pimephales promelas)
Channel catfish
(Ictalurus punctatus)
Bluegill
(Lepomis macrochirus)
9 6 -hour LC50
mg/L NH3)
0.08-0.1
0.14-0.47
0.50-0.70
0.16-1.1
0.9-1.4
0.69-1.8
2.2
2.8-3.2
0.75-3.4
0.50-3.8
0.55-3.0
Reference
Rice & Bailey (1980)
Thurston & Meyn (1984)
Thurston & Meyn (1984)
Broderious & Smith (1979)
Calamari et al. (1981)
Thurston & Russo (1983)
Roseboom & Richey (1977)
Broderius et al. (1985)
Hasan & Macintosh (1986)
Hazel et al. (1979)
Thurston et al. (1983)
Colt & Tchobanoglous (1976)
Roseboom & Richey (1977)
Arthur et al. (1987)
Emery & Welch (1969)
Roseboom & Richey (1977)
                                      185

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on the NHs/NH/i"1" equilibrium  in  the water.   This  greater toxic effect has been
demonstrated with prawn larvae and Daphnia. as  well as with  fishes  (rainbow
trout Salmo gairdneri. fathead minnow Pimephales promelas. coho  salmon
Onchorhvnchus kisutch. chinook salmon 0.  tshawytscha.  channel catfish Ictal-
urus punctatus. and green sunfish Lepomis cyanellus)  (Tabata 1962,  Sousa et
al. 1974, Robinson-Wilson and  Seim 1975,  Armstrong et al.  1978,  Hillaby and
Randall 1979, Tomasso et al. 1980, Thurston et  al. 1981c,  McCormick et al.
1984, Broderius et al. 1985, Sheehan and  Lewis  1986).   Results of one such
study (Thurston et al. 1981c)  showed that there was a decrease in the 96-hour
LG50 value for NH3 for rainbow trout (Figure 1) and fathead  minnows  as  pH
decreased, indicating that  fishes were more susceptible to NH3 at low pH.   In
longer exposures, Broderius et al. (1985) also  reported a  marked effect of pH
on the toxicity of NH3 in a study on smallmouth bass  (Micropterus dolomieui)
from late embryo through 32 days of exposure.   The 32-day  "no-observed-effect"
                    1000 R
                     100
                   10
                  x
                  o>
                  o   10
                  10
                  cc
                  o
                   I
                  (D
                  01   1.0
                      O.I
                                               TOTAL AMMONIA
                                           UN-IONIZED AMMONIA
6.5    7.0
7.5   8.0
   PH
                                                 8.5   9.0   9.5
              Figure 1.   Acute toxicityof ammonia (96-hour LC50
                values)  to rainbow trout at various environmental
                pH values.  Error bars are 95% confidence inter-
                vals.  (Reprinted with permission from Thurston
                et al.  1981c; copyright 1981, American Chemical
                Society)
                                      186

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concentrations radically decreased from 0.61 to 0.044 mg/L NH3 as  pH was  low-
ered from 8.68 to 6.60.  In a preliminary study, we have observed that the
toxicity ,of NH3 also increased at pH values above 9 (Figure 2) (Russo et  al.
1988).

EFFECT OF DISSOLVED OXYGEN

     Several researchers, working with a variety of fish species,  have
reported that the toxicity of ammonia increases when dissolved oxygen con-
centrations decrease (Wuhrmann 1952, Wuhrmann and Woker 1953, Downing and
Merkens 1955, Merkens and Dpwning 1957, Alabaster et al. 1979, Thurston et al.
1981b).  Discharges of ammonia frequently are associated with reduced oxygen
concentrations in the receiving water, so the effect of dissolved oxygen on
ammonia toxicity can be important.

EFFECT OF TEMPERATURE

     Many researchers have reported that ammonia is less toxic to fishes at
temperatures near the higher end of their normal environmental range than near
the lower end, but not all studies support this.  Colt and Tchobanoglous
(1976) reported a decrease in acute toxicity of ammonia to channel catfish
with an increase in temperature over the range 22 to 30 C, and Roseboom and
Richey (1977) reported that bluegill (Lepomis macrochirus), largemouth bass
(Micropterus salmoides), and channel catfish were more susceptible to ammonia
toxicity at 22 C than at 28 to 30 C.  Arthur  et al.  (1987)  conducted 96-hour
ammonia toxicity tests on white sucker (Catostomus commersoni), walleye
(Stizostedion vitreum vitreum), rainbow trout, fathead minnow, and channel
catfish at temperatures ranging from 3.4 to 26 C.  Except for channel
catfish, none of the tests showed a progressive increase in LC50 with increas-
ing water temperature, and no clear relationship was found between ammonia
toxicity and temperature; the 96-hour LC50 values for channel catfish were
0.50, 0.98, and 1.29 mg/L NH3 at 3.5, 14.6, and 19.6 C.

FLUCTUATING CONCENTRATIONS AND ACCLIMATION

     Fish are frequently subjected to fluctuating concentrations of  ammonia as
a consequence of diurnal changes.  Also, episodic  "slugs" of  ammonia, as a
result of accidental spills or intentional discharges into rivers and lakes,
are common.  Thurston  et al.  (1981a) studied the acutely toxic effects of
fluctuating concentrations of ammonia on rainbow trout and cutthroat trout
(Salmo clarki) and reported that fish were able to withstand  short-term
excursions slightly above acutely toxic concentrations without any apparent
long-term adverse effects, provided the high ammonia concentrations were
followed by periods of low ammonia concentration.  However, they also reported
that fish were better  able to withstand steady concentrations of ammonia, over
96 hours, than they were fluctuating concentrations having mean values
comparable to those of the steady concentration for the same  length  of time.
Increased resistance to acute toxicity from ammonia as a consequence of prior
exposure to low ammonia concentrations has been reported by Vamos (1963),
Malacea  (1968), Schulze-Wiehenbrauck  (1976), Redner and Stickney  (1979),
Thurston et al. (1981a), and Alabaster et al.  (1983).  Soderberg and co-
                                    187

-------




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~t^
CD
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O
CD
e
1






3uu
200



100
80


60
50

40

30

20



in
" o i
0
o
D 8
 s : * s o
- M "
W A
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A
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 0~5%NaCI
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	 nf\ 	 1 	 1 1 1 1 1 1 1 1
                             6.0  6.5  7.0 7.5 8.0 8.5  9.0  9.510.0.10.5

                                            PH

              Figure 2.  Effect of pH and different water chemistry
                variables on the acute toxicity of un-ioniniaed
                ammonia to coho salmon alevins. (Reprinted with
                permission from Russo et al. 1988; American Fisheries
                Society)
workers  (1983), however, have  reported that  tissue  damage  in rainbow trout
reared for 4 months  in earthen ponds was  correlated with ammonia concentration
extremes, as opposed to ammonia averages.

MIXTURES OF AMMONIA  AND OTHER  CHEMICALS

     Information on  the toxic  effects  of  ammonia as a result of  exposure  in
combination with other chemicals is limited, but an increase in  ionic strength
appears to reduce ammonia toxicity, separate from the effect of  ionic activity
on the NH3/NH4+ equilibrium.  Increased calcium  concentrations in the water
have been reported to decrease ammonia toxicity to  channel catfish  (Tomasso et
al. 1980), and an increase in water salinity has been reported to reduce  the
toxicity of ammonia  to Atlantic salmon (Salmo salar) (Alabaster  et  al. 1979).

                                     188

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A decrease in the toxicity of ammonia to juvenile coho salmon with the
addition of sodium chloride to the test solution also has been observed in our
laboratories (Figure 2).

     Wuhrmann and Woker (1948) and Broderius and Smith (1979) studied combina-
tions of ammonia and hydrogen cyanide and reported synergistic effects of the
two toxicants.   The toxicity of ammonia and phenol combined was reported to
approximate the toxicity of either ammonia or phenol when tested separately
(Herbert 1962)    Research has also been conducted on the effect of ammonia in
combination with copper (Herbert and Vandyke 1964),  zinc (Ministry of Technol-
ogy, U.K. 1962), and combinations of these (Brown et al. 1969); the toxicity
of ammonia in combination with these other toxicants appears to be simply
additive.

                          CHRONIC  TOXICITY OF AMMONIA

     The longest ammonia exposure study reported in the literature is a life-
cycle test on rainbow trout, conducted over a 5-year period (Thurston et al.
1984).  Fish were tested at five exposure concentrations over the range 0.01
to 0.07 mg/L NH3.   Parental fish were exposed for 11 months,  the first filial
generation (Fx)  for 4 years, and the second filial generation (F2)  for 5
months.  The parental fish spawned of their own volition, and Fx fish were
manually spawned at 4 years of age.  Blood ammonia concentrations in Fx fish
were positively correlated with ammonia concentrations in the test waters, and
histopathological lesions were common in parental and F1 fish at 0.02 mg/L NH3
and higher.  No other major effects were observed.  Viable eggs were produced
by both generations at all ammonia concentrations tested, and there was no
significant correlation between ammonia concentration and numbers of egg lots
spawned, total numbers of eggs produced, numbers of viable eggs, growth of
progeny, or mortality of parents or progeny in any of the generations, tested.

     Effects of ammonia on fish reproduction and survival were observed,
however, in two life-cycle tests on fathead minnows (Thurston et al. 1986).
Fish were tested at five exposure concentrations for 1 year over the range of
0.07 to 0.96 mg/L NH3.  Growth and survival of,  and egg production by,
parental fish were all affected at 0.96 mg/L NH3; no effects were observed on
growth or survival of parental fish at 0.44 mg/L, or on egg production or
viability at 0.37 mg/L.  Growth and survival of F-^ larvae were not affected at
0.36 mg/L NH3 (the highest concentration at which they were tested),  and egg
hatching success was not affected at 0.19 mg/L, but was at 0.37 mg/L.  Brain
lesions were common in parental fish at all stages of their development at
exposure concentrations of 0.21 mg/L NH3 and higher, but not at 0.11 mg/L.   In
summary, the chronic effects threshold concentration for these tests on
fathead minnows, based on survival, growth and reproductive success, was
estimated to be 0.27 mg/L NH3; based on histological damage,  it was estimated
to be only half that, 0.15 mg/L NH3.

     Other studies at sublethal concentrations of ammonia, lasting from 1 week
to 3 months, have been reported for both salmonid and non-salmonid species
(Department of the Environment, U.K. 1972, Robinette 1976, Schulze-Wiehen-
brauck 1976, Burkhalter and Kaya 1977, Thurston et al. 1978, Rice and Bailey
1980).  At exposure concentrations as low as 0.002 to 0.15 mg/L NH3,  fish
                                     139

-------
 showed reduced food uptake and assimilation,  accompanied by growth inhibition.
 In the range of 0.04 to 0.4 mg/L NH3, effects reported for  a variety of fish
 species,  under a wide range of water test conditions,  included leucopenia and
 diminished numbers of red blood cells,  inflammation and  degeneration of gills
 and kidneys,  and lowered resistance to  disease  (Reichenbach-Klinke 1967,  Flis
 1968,  Smart 1976,  Thurston et al.  1978,  Peters  et  al.  1984,  Soderberg  1985,
 Dabrowska and Wlasow 1986,  Lang et al.  1987).

     Carline et al.  (1987)  tested the effects of domestic wastewater on the
 survival,  growth,  swimming performance,  and gill tissue  of  brown  trout (Salmo
 trutta) for 12 months at concentrations  from 0.005  to 0.066 mg/L  NH3.   No
 significant effects  were reported for survival, .growth,  or  swimming perform-
 ance,  but degree of  damage  to the  gills  was directly related to effluent
 concentrations.  Mitchell and Cech (1983)  have  implicated chlorine as  con-
 founding  ammonia-caused gill  damage.  Burrows (1964) tested rainbow trout  for
 6 weeks at concentrations as  low as  0.003  mg/L  NH3  (recalculated from the
 author's  original  data)  and reported extensive  gill damage, but Daoust and
 Ferguson  (1984)  tested that same species at concentrations up  to  0.4 mg/L  NH3
 for  90 days  and did  not observe  "characteristic" gill damage.  Meade and
 Herman (1986)  reported that lake trout  (Salvelinus  namaycush), reared for  8
 weeks  in  a water re-use  system,  showed gill and kidney damage  of  the kind
 reported  to be  caused by ammonia,  and yet  the ammonia concentrations to which
 these  fish were  exposed averaged 0.001 mg/L NH3  or less within each 2-week
 period, and  the  highest  recorded value was 0.003 mg/L.  Their  conclusion was
 that "parameters other than ammonia  concentrations  are, significant  factors in
branchial  irritation"  of fish under  culture.

     In an extensive  review article, Meade (1985) states as part of his
 summary:  "The accumulating  evidence  indicates that  gill hyperplasia, reported
 as characteristic  of  ammonia poisoning,  is probably not caused by un-ionized
 ammonia.   ...  All end products  of metabolism probably have not been identi-
 fied,  and  certainly their interactions in water of various qualities and in
various fish culture  systems.have not been determined."
                                LITERATURE CITED


 Alabaster, J.S., D.G. Shurben, and G. Knowles.  1979.  The effect of dissolved
      oxygen and salinity on the toxicity of ammonia to smolts of salmon,  Salmo
      salar L.  Journal of Fish Biology 15:705-712.

 Alabaster, J.S., D.G. Shurben, andM.J. Mallett.   1983.   The acute lethal
      toxicity of mixtures of cyanide and ammonia to smolts of salmon,  Salmo
      salar L- at low concentrations of dissolved oxygen.   Journal of Fish
      Biology 22:215-222.

 Armstrong, D.A.,^D. Chippendale,  A.W. Knight,  andJ.E.  Colt.   1978.   Interac-
      tion of ionized and un-ionized ammonia on short-term survival and growth
      of prawn larvae, Macrobrachium rosenbergii.   Biological Bulletin of  the
      Woods Hole Institute 154:15-31.
                                      190

-------
Arthur, J.W., C.W. West, K.N. Allen, and S.F. Hedtke,  1987.  Seasonal
     toxicity of ammonia to five fish and nine invertebrate species.   Bulletin
     of Environmental Contamination and Toxicology 38:324-331.

Broderius, S., R. Drummond, J. Fiandt, and C. Russom.  1985.   Toxicity of   '.
     ammonia to early life stages of the smallmouth  bass at four pH values.
     Environmental Toxicology and Chemistry 4:87-96.

Broderius, S.J., and L.L. Smith, Jr.  1979.  Lethal and sublethal effects of
     binary mixtures of cyanide and hexavalent  chromium, zinc, or ammonia to
     the fathead minnow (Pimephales promelas') and rainbow trout (Salmo
     gairdneri).  Journal of the Fisheries Research Board of Canada
     36:164-172.

Brown, V.M.,  D.H.M. Jordan, and B.A. Tiller.  1969.  The acute toxicity to
     rainbow trout of fluctuating concentrations and mixtures of ammonia,
     phenol and zinc.  Journal of Fish Biology 1:1-9.

Burkhalter, D.E., and C.M. Kaya.  1977.  Effects of prolonged exposure to
     ammonia on fertilized eggs and sac fry of rainbow trout (Salmo
     gairdneri).  Transactions of the American Fisheries Society 106:470-475.

Burrows, R.C.  1964.  Effects of accumulated excretory products on hatchery-
     reared salmonids.  Bureau of Sport Fisheries and Wildlife, U.S.  Fish and
     Wildlife Service, Research Report 66, 12p.
Butler, J.N.  1964.  Ionic equilibrium.
     Inc., Reading, Massachusetts, USA.
Addison-Wesley Publishing Company,
Calamari, D.,  R. Marchetti, and G. Vailati.   1981.   Effects of long-term
     exposure to ammonia on the developmental stages of rainbow trout (Salmo
     gairdneri Richardson).  Rapports et Proces-Verbaux des Reunions, Conseil
     International pour  1'Exploration de la Mer 178:81-86.
Carline, R.F., A.J. Benson, and H. Rothenbacher.
     treated domestic wastewater on brown trout.
         1987.  Long-term effects of
         Water Research 21:1409-1415.
Chipman, W.A.,  Jr.  1934.  The role of pH in determining the toxicity of
     ammonium compounds. Ph.D. thesis, University of Missouri,  Columbia,
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Colt, J., and G. Tchobanoglous.   1976.  Evaluation of the short-term toxicity
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                                     191

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                                     194

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                                     196

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                    ENVIRONMENTAL TOLERANCE OF SOME MARINE
                 FISH:  APPLICATIONS  IN MARICULTURE MANAGEMENT

                                      by

                                  R.S.S. Wu1

                                 INTRODUCTION

      Most studies of environmental effects on marine fish have confined
themselves to evaluating physiological responses of a few species.  Few
studies compare the environmental tolerances of different species.  Moreover,
differences in experimental protocols make it difficult to compare tolerances
and responses between studies (Davis 1975).

      Although it has long been recognized that the success or failure of
mariculture operations may hinge on the choice of species (Avault 1986), in
the absence of scientific data, the suitability of a species for a particular
hydrographic condition in a culture site can only be determined by experience
or by trial and error.  Hydrographic conditions, however, vary and fluctuate
greatly both spatially and temporally.  As a result, knowledge of environ-
mental tolerances and requirements of marine fish provides a valuable
scientific basis for selecting appropriate species to suit different culture
conditions and is of obvious importance from the viewpoint of mariculture
management and development.

      The tolerance of a species to a particular environmental factor may be
assessed by mortality and the behavioral and physiological responses of the
fish.  In this context, the time for-50% of the experimental population to
show mortality (LT50 value) ,  and the  time  for  50%  of  the  experimental
population to exhibit abnormal behavioral changes (BC50 value)  can serve as
useful tolerance indicators with which the relative tolerance of different
species in relation to a particular environmental factor can be assessed and
compared.  This paper presents experimental data on oxygen, salinity, and
temperature tolerances of some common mariculture fish in the Southeast Asia
regions, and the results are related to fish kill statistics collected for 10
years in Hong Kong.  The application value of these tolerance data in
mariculture management also are discussed.
                             MATERIALS AND METHODS

      All fish used for the experiment had been cultured in sea cages
(salinity, 30 to 32%; dissolved oxygen, 5 to 8 mg 02 L"1; water temperature,

     "City Polytechnic of Hong Kong,  Kowloon Tong,  Hong Kong.

                                     197

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 25 to 28C) for about a year.  Experimental fish weighing in the range of 150
 to 250 g were selected and acclimated in the laboratory (salinity, 30%;
 dissolved oxygen, 8 to 8.5 mg 02 L"1; temperature, 25  1C) for at least 48
 hours prior to the experiment and were not fed throughout the entire
 acclimation and experimental period.


 OXYGEN TOLERANCE

       A continuous flow system that provided a constant,  controlled level of
 ambient oxygen was set up for the hypoxic tolerance experiment (for detailed
 design of the system, see Wu and Woo 1984).   Twenty individuals of each of the
 nine species listed in Table 1 were acclimated in the experimental system for
 24 hours prior to experiments, during which period normoxic water (25  1C)
 was provided.  The levels of oxygen were adjusted to 4,  2.5, 1.0 and 0.5 mg 02
 L"1 within 15 minutes and  the  fish were  exposed to the desired dissolved
 oxygen level for 7 hours.   The time at which any fish began to show any stress
 symptom (i.e. loss of balance, abnormal swimming pattern,  jerking) was noted
 and the time of death of any fish was recorded.
 TABLE 1.   TIME (minutes)  FOR 50% OF VARIOUS EXPERIMENTAL FISH TO SHOW
 MORTALITY (LT50) AND ABNORMAL BEHAVIOR (BC50)a-b
Species
Ghrysophrys major
Rhabdosarga sarba
Siganus oramin
Lutlanus ruselli
Epinephelus akaara
Mylio macrocephalus
Epinephelus awoara
Lates calearifer
Epinephelus tauvina
0.5 mg 0
BC50
TQC
TQC
TQC
TQC
70
30
150
373
(10%)
' r1
LT50
TQC
TQC
7
14
92
100
200
393
(10%)
1.0 mg 0
BC50
20
130
10
78
110
NBC
190
NBC
NBC
'2 I"1
60
225
177
(30%)
160
NM
270
NM
NM
Actual mortalities are shown in parenthesis for mortality-behavior changes
 occurring in less than 50% of the population throughout the experimental
 period.

bTQC,  too quick to count;  NBC, no behavioral changes;  NM,  no mortality.

                                     198

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      To test whether there is any correlation between hypoxic tolerance and
metabolic rate of different species, the standard oxygen consumption rate of
each species at 25C (ambient oxygen level) was measured.  Five of each
species were placed individually in sealed Plexiglas chambers (20 x 12 x 12
cm) and were placed in experimental tanks of the continuous flow systems.
Fish were allowed to adapt to the experimental condition for 24 hours before
measurement.  Water was allowed to flow into the chamber through the inlets
and siphoned out from the outlet of the chambers.  The flow rate was adjusted
so that there was a measurable difference of about 0.5 mg 02 IT1 between  the
inflow and outflow of the sealed chambers.  An empty chamber identical with
that containing the animal was set up as a control, and the oxygen uptake of
the fish was calculated using the following equation.
q - (X0
                                   -  (X0-X2)(dV/dt)2
where q is the oxygen uptake, X0 is the oxygen concentration at the inflow, Xx
is the oxygen concentration at the outflow of the experimental chamber, X2 is
the oxygen concentration at the outflow of the chamber,  (dV/dt^ is the flow
rate of the experimental chamber, and  (dV/dt)2 is the flow rate of the control
chamber.  The oxygen consumption rate  then is calculated and expressed in
terms of mg 02 g body wet wt.'1 min."1


SALINITY TOLERANCE

      After acclimation, twenty individuals of each of the 13 species listed
in Table 3 were  transferred abruptly to 3 , 5 and 10% sea water  (temperature =
25  1C) for 14 days in the  laboratory.  In addition, twenty individuals  of
S . oramin E . akaara . and m. macrocephalus were transferred to freshwater  (0%) .
The time at which any fish began to show any stress symptom was noted and  the
time of death of any fish was recorded.


TEMPERATURE TOLERANCE

      A continuous  flow system in which a constant water temperature of 25 
0.2C was set up for the temperature tolerance experiment.  Twenty individuals
of each of  the eight species  listed in Table 4 were acclimated  in  the system
for 24 hours prior  to experiments.  The temperature of the system  was
gradually brought to 12  0.2C within 1 hour, and the fish then were
subjected to  the experimental temperature  for  7 hours.   The time at which any
fish began  to  show  any  stress symptom  was noted  and  the  time  of death  of  any
fish was recorded.
 FISH KILL STATISTICS

       Since 1976,  all major fish kills in Hong Kong have been reported to the
 Agriculture and Fisheries Department.   In most of these cases,  investigations
 were carried out to ascertain the cause of the kill and to assess the total
 loss as well as the species affected.   Over 40% of the major kills recorded
 was attributable to oxygen depletions  resulting from red tides and algal
                                      199

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  blooms; the total'loss between 1976 and 1988 was estimated at 124 tonnes,
  valued at approximately US $1 million.
                                     RESULTS
  OXYGEN TOLERANCE
        No observable stress symptom can be found for all species at 2.5 mg 02
  L"  and above.  Most of the species turned into a noticeable lighter color
  when oxygen dropped below 2.5 mg 02 L"1, and this phenomenon vraS particularly
  marked for the Sparidae.   Time required for 50% of each species to show stress
  symptoms and mortality are shown in Table 1.   Large variations  were found in
  hypoxic tolerance amongst the nine species  tested.   For example,  50% of _..
  major exhibited abnormal  symptoms within 20 minutes at 1 mg 02  L"1, whereas L^
  calcarifer and E.  tauvina did not show any  abnormal symptoms within the 7-hour
  exposure.   Similarly,  all C.  major and R. sarga died before the oxygen level
  dropped to 0.5 mg 02 L"1,  whereas only 10% of E.  tauvina showed  abnormal
  symptoms and mortality under  the same condition throughout  the  7-hour
  experimental period.

        Oxygen consumption  rates for the nine  species tested  are  shown in Table
  2.  The  metabolic  rate of intolerant  species  (e.g.  C.  major and R.  sarba) was
  more  than two times higher than those of tolerant  species (e.g. E.  tauvina  and


TABLE  2.   OXYGEN CONSUMPTION RATE OF VARIOUS  SPECIES AT 25C AND NORMOXIA
(Species  arranged in ascending order according to  their LT50 values at 0.5 mg
2 i"1)
Species
Oxygen consumption rate, (/^g 02 g"1 min.
Chrysophrvs major

Rabdosarga sarba

Siganus oramin

Lutjanus ruselli

Epinephelus akaara

Mvlio macrocephalus

Epinephelus awoara

Lates ealcarifer

Epinephelus tauvina
            4.65  0.81

            4.88  0.33

            9.73  1.42

            5.93  0.21

            3.48  0.12

            2.84  0.39

            2.20  0.40

            1.98  0.14

            1.99  0.53
                                      200

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L. calcarifer).   A Spearman rank correlation test showed a significant
correlation between the rank orders of the LT50  and  BC50 values at both 1.0 and
0.5 mg 02 L"1  (r=0.986 and 0.790, respectively).  A  significant negative
correlation was found between oxygen consumption rate and LT50 and BC^, values
of the species tested at 0.5 mg 02 L"1  (r=  -0.732 and -0.673,  respectively).


SALINITY TOLERANCE

      LT50 values  for each  species  at different  salinities  are shown in Table
3.  Except for Caranx eauula. no mortalities were found for any species at the
end of 14 days of exposure to 30 and 10%.  No mortality was found  in 6 species
exposed to 5%.  Three species survived without  any  abnormal behavior in 3%,
and 10% of M. macrocephalus survived without any abnormal behavior  at the end
of the 14 days of exposure to freshwater.


TEMPERATURE TOLERANCE

      Except  for S.  oramin. which  exhibited a 20% mortality,  no mortality was
found for  the other  7 species in a 7-hour  exposure  to  12C.   Time  for  50% of
experimental  animals  to exhibit abnormal behavior is shown  in Table 4.  EL.
oramin and Lates calcarifer showed a very  quick behavioral  response to  the
lowering  of .water temperature, followed in order by E.  tauvina. R.  sarba and
C. major.  No behavioral changes were  found for E.  akaara.  E.  aworara.  and M.
macrocephalus during a 7-hour exposure to  12C.


FISH KILL STATISTICS

      Species involved in  fish kills  due to oxygen  depletion  in Hong Kong  from
1976 to  1986  are  shown in  Table 5.   C. major and R. sarba were involved in 17
and 11 incidents  out of 19 major fish  kills, respectively.  L. calcarifer  and
E. tauvina were never reported in  any  fish kills caused by  oxygen depletion
although they also are commonly cultured.  Table 6  shows the  stock and
percentage killed of each  species  in a fish kill in which a dissolved  oxygen
level of 0.8  mg 02 L"1 was  recorded.  In  this  incident,  only C. major.  R.
sarba. L.  ruselli. and E.  akaara were  killed.   M. macrocephalus and E.  tauvina
were not affected although they also were  cultured  at  the same site.

      Statistics  of  fish kills caused by cold spells  (with  water  temperature
<15C for more than  7 days)  in Hong Kong are shown  in  Table 7.  L.  calcarifer.
E. tauvina were found to be  involved in all the five recorded incidents.   E.
akaara.  E. awoara. and M.  macrocephalus were never  reported although these
species  also  were cultured.


                                   DISCUSSION

      Most warm water fish survive under a wider range of  environmental
conditions than do cold water species  (Parker and Davis 1981).  In this study,
the species  tested showed  large variations in their tolerance to  hypoxia,
temperature,  and  salinity, and  several species  showed  remarkable  tolerance to

                                      201

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  TABLE 3.   LT50 VALUES (hours) OF VARIOUS MARINE FISH KEPT UNDER DIFFERENT
  HYPO-OSMOTIC SALINITIES FOR 14 DAYS (n=20).a-b
Species 0%
Slganus oramin 1.2
Caranx eouula
Parapristjpoma trilineatum
Chrvsophrvs major
Lethrinus nebulosus
Epinephelus akaara 2.5
Plotosus anguillaris
Pomadasvs hasta
Epinephelus awoara
Rhabdosarga sarba
Therapon iarbua
Lutianua russelli
Mylio macrocephalus 60.0
Salinity
3% 5%
2.3
3.1
4.4
4.8
9.1
31.3
40.0
70.0
76.0
96.0
NM
NM
NM
NM
42.5
38.0
11.1
86.0
NM
55.0
(30%)
NM
(10%)
NM
NM
NM
10%
NM
126.0
NM
NM
NM
NM
NM 
NM
NM
NM
NM
NM
NM
 "Actual  mortalities  shown in parentheses  for  mortalities  of less  than 50%
  within the experimental period (Wu and Woo 1982).
 bNM, no mortality.
one or more parameters.  For example, the black sea bream, Mylio
macrocephalus, was very tolerant to all three environmental parameters tested.

      Results of hypoxic tolerance experiments show that Lates calcarifer.
Epinephelus tauvina and Mylio macrocephalus are tolerant of, whereas
Chrvsophrvs major and Rhabdosarga sarba are particularly susceptible to,
hypoxic conditions.  Hypoxic tolerance is negatively related to the metabolic
rate^of the species.  It appears that species that have a lower metabolic rate
survive better in a hypoxic environment because less oxygen would be required
to sustain essential biological functions.  The hypoxic tolerance of fish
species derived from laboratory results agrees closely with these established
from analyses of fish kill statistics.  C. major and R. sarba always suffer

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TABLE 4.  TIME (minutes) FOR 50% OF EXPERIMENTAL ANIMALS TO EXHIBIT ABNORMAL
BEHAVIOR (BC50) AT  C  (n=20)
 Species
BC
                                                  50
Mortality, %
Siganus oramin

Lates calcarifer

Epinephelus tauvina

Rhabdosarga sarba

Chrysorphyrs major

Epinephelus akaara

Ep inephelus awoara

Mvlio macrocephalus
TQC

  1

 20

 50

 89

NBC

NBC

NBC
      20
aNBC,  no behavioral changes;  TQC,  too quick to count
heavy loss in fish kills caused by oxygen depletions; other species normally
are affected to a much lesser extent.

      Conversely, L. calcarifer and E. tauvina. which have never been reported
in any fish kills due to oxygen depletion, were found to be highly tolerant to
hypoxia in the laboratory experiments.  Detailed biochemical studies on
Ephinephelus akaara and Mylio macrocephalus exposed to hypoxic conditions by
Woo and Wu (1984) revealed no increases in serum and tissue lactate and only
slight changes in other tissue metabolites and electrolytes at 4.0 to 2.5 mg
02 L"1 for these two species, indicating that  they can obtain enough oxygen to
prevent anaerobiosis in this regime.  Marked  elevations of serum lactate and
serum Na+,  K+ and Ca2+ were found and osmoregulation failure occurred,  however,
when oxygen values fell below 1 mg 02 L"1.

      The overall results suggest, therefore, that hypoxic tolerant species
such as Lates calcarifer and M. macrocephalus may be cultured in eutrophic
waters in which oxygen depletion is more likely to occur and that culturing
sensitive species such as C. maj or and R. sarba should be avoided in the same
environment.  Nevertheless, the quick, abnormal behavioral response to hypoxia
exhibited by C. maj or and R. sarba may serve  as an effective indicator with
which culturists could detect the onset of oxygen depletion at an early stage
and aerate their cages to prevent loss of their stock.  Such a "biological
indicator" system has been proven to be successful in Hong Kong.
                                     203

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 TABLE 5.  NUMBER OF INCIDENTS IN WHICH A PARTICULAR SPECIES WAS INVOLVED IN 19
 MAJOR FISH KILLS CAUSED BY OXYGEN DEPLETION IN HONG KONG, 1976 to 1987
       Species
No. of incidents involving
species in 19 fish kills
 Chrysophrvs malor

 Rhabosarga sarba

 Epinephelus akaara

 Seriola purpurascens

 Lutianus ruselli

 Mylio macrocephalus

 Mvlio latus

 Lutianus sanguinensis
            17

            11

             6

             5

             4

             2

             2

             2
      Results  of the  salinity tolerance  experiments  show that 10 out of the 13
 species  tested are  euryhaline and survive without any abnormal behavior and
 tissue hydration for  more  than 2  weeks in salinities above 10% (Wu and Woo
 1983), suggesting that  such  salinities are normally not an important limiting
 factor for  culturing  most  of these species.  Further experiments carried out
 on E. akaara and M. macrocephalus showed only transient disturbance of various
 electrolytes and metabolites in the case of the latter at salinities above
 12%, suggesting that  physiological disturbance is unlikely to occur in
 salinity above  this regime (Woo and Wu 1982).  Dendrinos and Thorpe (1985)
 also found  that the bass (Dicentrarchus  labrax") survives in salinities between
 5 and 33% for  over  12 months,  and food and protein conversion efficiencies
 were maximal at 25  and  30%.   It may be hypothesized further that, because less
 energy would be required for osmoregulation in an iso-osmotic environment,
 energy saved might be channelled  to tissue production.  The possibility of
 increasing mariculture  productivity by culturing eurhaline species in an
 environment of  reduced  salinity (e.g. estuary) should be explored.

      Results from temperature  tolerance experiments indicated that S.  oramin.
 L. calcarifer,  and E. tauvina are  relatively sensitive to cold temperature,
whereas  E. akaara. e. awoara and M. macrocephalus are more tolerant.   Again,
 the laboratory  findings were clearly supported by fish kill statistics in Hong
Kong.  High mortality of the former three species were found during cold
 spells when water temperatures  fell below 15C for a prolonged period.
Conversely,  Mvlio macrocephalus. Epinephelus akaara.  and Epinephelus  awoara.
which showed no abnormal behavior at 12C in the laboratory,  have never been
                                     204

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reported upon in fish kills caused by cold spells.  The results, therefore,
indicate over-wintering problems for L. calcarifer and E.  tauvina in Hong
Kong.  Shortening of the grown-out period by, for example, importing larger
fingerlings might minimize the risk of fish kills for these two species in a
severe winter.

      Menasveta (1981) found that the range of upper lethal temperature for 24
species of marine fish in the Gulf of Thailand is fairly narrow (34C to
37.5C).  The upper lethal temperatures of Chrysophrys major and Mylio
macrocephalus were found to be 32 and 36C, respectively  (Woo and Fung 1980,
Woo per. com).  The low tolerance of C. major to high water temperature
suggests that this species is less suitable for culturing in shallow waters
(i.e. < 3m, the shallowest thermocline in the coastal waters of Hong Kong)
where water may easily be heated by solar radiation in the summer to a
temperature beyond the lethal limit of this species.
TABLE 6.  INVOLVEMENT OF VARIOUS SPECIES IN FISH KILLS AT PO TOI 0 HONG KONG
IN WHICH A DISSOLVED OXYGEN LEVEL OF 0.8 mg 02 I"1 WAS RECORDED
      Species
Stock (t)   Loss (t)    Loss (%)
Chrysophrys major
Rhabdosarga sarba
Lut janus russelli
Epinephelus akaara
Sub -total
Epinephleus awoara
Epinephleus tauvina
Mylio latus
Mylio macrocephalus
Lut janus argentimaculatus
Others
Sub -total
8.1
20.3
2.8
0.8
32.0
3.0
2.4
1.3
9.0
1.2
2.1
22.5
0.1
0.8
0.1
0.1
1.1
0
0
0
0
0
0
0
1.2
3.9
3.6
12.5
3.4
0
0
0
0
0
0
0
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 TABLE 7.   NUMBER OF INCIDENTS IN WHICH A PARTICULAR SPECIES  WAS  INVOLVED IN
 FIVE MAJOR FISH KILLS CAUSED BY COLD SPELLS (water temperature less  than 15C
 for more  than 7 days) IN HONG KONG,  1982 to 1987
       Species
No. of incidents involving species
      in five fish kills
 Lates calcarifer

 Epinephelus tauvina

 Lutjanus  sanguinensis

 Rhabdosarga sarba
                  5

                  5

                  4

                  1
                                   REFERENCES

 Avault,  J.W.,  Jr.   1986.   Which species to culture -  A check list.
       Aquaculture  Magazine.   12:41-44.

 Davis, J.   1975.   Minimal dissolved oxygen requirements of aquatic  life
       with emphasis on Canadian species:   A Review.   Journal of Fisheries
       Research Board Canada.   32:2295-2332.

 Dendrinos,  P.  and  J.P.  Thorpe.   1985.   Effects  of reduced salinity  on  growth
       and  body composition in the  European bass (Dicentrarchus  labrax  (L.'n
       Aquaculture.   49:333-358.

 Hughes,  G.M.   1973.   Respiratory responses to hypoxia in fish.   American
       Zoologist.   13:475-489.

 Menasveta,  P.   1981.  Lethal  temperature of marine fish of the  Gulf of
       Thailand.  Journal  of Fish Biology.   18:603-607.

 Woo, N.Y.S. and C.Y.A.  Fung.  1981.  Studies on the biology  of  the  red sea
      bream Chrsophrys  major.   IV Metabolic  effects of  starvation at low
       temperature.   Comparative  Biochemistry and Physiology.  244:1-5.

 Woo, N.Y.S. and R.S.S.  Wu.  1982.  Metabolic and osmoregulatory  changes in
      response  to reduced salinities in the red grouper, Epinephelus akaara
       (Temminck and  Schlegel), and the black sea bream Mylio macrocephalus
       (Basilewsky).  Journal of  Experimental Marine Biology and  Ecoloev
      65:139-161.                                                    &y

Woo, N.Y.S. and R.S.S. Wu.  1984.  Changes in biochemical composition  in the
      red grouper,  Epinephelus akaara (Temminck  and Schlegel), and the black
      sea bream, Mylio macrocephalus. during hypoxic exposure.  Comparative
      Biochemistry and Physiology.   77A:475-482.

                                    206

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Wu, R.S.S. and N.Y..S. Woo.  1982.  Tolerance of hypo-osmotic salinities in
      thirteen species of adult marine fish:  Implication for estuarine fish
      culture.  Aquaculture.  32:175-181.

Wu, R.S.S. and N.Y.S. Woo.  1984.  Respiratory responses and tolerance to
      hypoxia in two marine teleosts, Epinephelus akaara (Temminck and
      Schlegel) and Mylio macrocephalus  (Basilewsky).  Hydrobiologia.
      119:209-217.
                                      207

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                   STUDIES ON PHYSIOLOGY OF PHOTOTAXIS  OF FTRH
                           AND MARINE ANIMALS  IN CHINA

                                       by

                                    Daren He1
                                  INTRODUCTION

      LIghtfishing has a long history in China.   It can be traced back to
 ancient times when fishermen used pine torches  to lure cuttle fish and squid
 in the sea and used lamps to lure crabs and torches as an aid to catching
 cormorants.   As early as 1930s.,  the fishermen of Dongshan,  Fujian,  Nanao,  and
 Guangdong used gaslamps to lure  and catch round scads and sardines.   With the
 depletion of demersal fish resources,  many countries in the world have began
 to exploit the rich resources of pelagic fish.   At present,  lightfishing is
 an important means of catching pelagic fish.  With the development of science
 and technology and the improvements in lighting technology,  lightfishing has
 become an advanced means of production that plays an important role  in marine
 fishery in many countries.   China's coastal waters abound in varieties and
 quantities of pelagic fish,  of which chub mackerel,  round scads,  horse
 mackerel,  round herring,  sardines,  etc.  have  already become  the major obiects
 for purse  seine fishing with light.

      In the  early 1960s,  we  carried out  successful experiments  in underwater
 lamp fishing with the fishermen  of  Xiamen,  Dongshan,  and  other  areas    From
 the late 1960s to the early  1970s,  the fishermen of Xiamen,  Dongshan,  etc
 made tremendous strides in seine fishing using  lights  installed on motorized
 junks.   In the early  1970s,  Shanghai Fishery  Company and  other  units  began to
 develop  purse seine fishing  with lights,  furthering  the exploitation  of  the
 marine pelagic fish resource of  our country and raising the  proportion of the
 pelaghic fish yield in the total.  To  raise the  light  fishing industry
 further, we  must  solve the problems that arise  in  production.   Several
 questions  are involved.  What  is  the optimum  color light  source  for round scad
 and chub mackerel?  What  is  their optimum illumination?  Why is  the gathering
 effect of  light under  moonlight not so ideal?  Why is  the phototaxis  so bad
 during the breeding season?

     With  these questions in mind, we,  together with our colleagues in the
 Shanghai Institute of  Physiology and the East China Sea Fisheries Research
Department of Oceanography,  Xiamen University,  Xiamen,  PRC

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     With these questions in mind, we, together with our colleagues in the
Shanghai Institute of Physiology and the East China Sea Fisheries Research
Institute, made comprehensive studies of the phototactic character of the main
marine pelagic fish such as round scads and chub mackerel using ethological,
electro-physiological and visual-pigment-biochemical methods in the early
1970s.  We also tested new light sources such as thallium-indium lamp on the
sea and achieved excellent results.  Our achievements won the China National
Science Conference Prize in 1978.  Later we continued with our studies on the
theory and application of the mechanism of phototaxis of fish and marine
animals, vision physiology, and ethology.  This paper gives a summary of the
study in this  field in China.

     He Daren, Yu Wentsao, and others have studied  the phototactic behaviour
of round scad  CDecapterus maruadsi),  chub mackerel  (Pneumatophorus japonicus),
sardine  CSardinella perforata), silverside  (Atherina bleekeri), needlefish
 CAblennes anastomella") ,  cardinal  fish (Apogon lineatus) , Mugil carinatus and
squid  CLoliog  duvaucelii") using the  photogradient method; Zheng Meili, He
Daren,  and  others have  studied the behavioural physiology of  cuttlefish
 fSepiella maindroni); Luo Huiming and others have studied that of young  eel
 CAnguilla japonica)  and swimming  crab (Portunus  trituberculatus).  Their
 studies  included threshold  of reaction to  light, optimum illumination,
 reaction to colored light,  and the influence of  background  light  on
phototactic behaviour.   The  exposition of  the  above-mentioned characteristics
 of animals  can provide  a scientific  basis  for  the  selection of the optimum
 artificial  light source in  production,  the determination of effective
 attraction range,  and the calculation of gathering  rate.

      There were three major findings.  First,  young fish and adults  of round
 scad and chub mackerel showed positive phototaxis  under the horizontal
 photogradient of between 1CT1 and 103|x.  Second, the phototaxis of young fish
 was stronger than that of adults.  Third,  the  optimum illumination of young
 chub mackerel was 1CT2 to 14  Ix in water temperature of 24.5  to 27C.  In both
 single and group experiments, the results of response of chub mackerel are
 basically the same, and the response of chub mackerel are basically the same,
 and the response for the group experiment is even more stable.

      We also did comparison experiments on needlefish, cardinal fish and Mugil
 carinatus. the results are:  (1) needlefish were always distributed^in the
 relatively stronger illumination region of each series of photogradient; (2)
 under the same conditions,  cardinal fish were constantly distributed in the
 relatively weaker illumination region of each series of photogradient;  (3) the
 phototaxis of the young fish and adults of round scad and chub mackerel and
 the young of Mugil carinatus remained between the  former two.  Chub mackerel
 and. round scad both belong to the type of fish that show positive reaction to
 light but do not tend to the strongest light.  When a comparison was made
 between these two, round scad showed stronger phototaxis than chub mackerel.
 Therefore, both fish can be  the objects of light fishing.  The optimum       ^
 illumination  regions of squid and cuttlefish were  10"1 to ICPlx and  10   to 10
 Ix, respectively.  Both belong to the  type that tends to weak light.  The
 optimum illumination of swimming  crab was 10"3 to  10"2lx.  With growth      ^
 development,  the optimum illumination  of young  eels changed  from 10"  to 10 Ix
                                      209

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 to 1CT2 to  lO^lx.  This shows that the character of phototaxis differs in
 different stages of development.

      Some fish and marine animals can show selective response and color vision
 to colored light.  Study in this field is of great importance to light source
 color selection in production.  Under dark adaptation, the phototactic rate of
 young and adult round scad is highest to blue-green light while that to red
 light is the lowest.  After the transition from dark to light adaptation, the
 phototactic rate to yellow-green light is the highest.  Chub mackerel show the
 highest phototactic rate to violet and red lights under both dark and light
 adaptations.  The peak value of its spectral sensitivity remains to blue and
 green lights.  The author points out that the optimum light color is different
 from the sensitivity light color.   The squid shows the greatest phototactic
 rate to blue light.

      Moonlight,  functioning as background light,  poses a practical problem in
 production.   Our tests showed that background light invariably reduced the
 phototaxis  of the above-mentioned animals;  the influence of background light
 could be restrained by increasing the light intensity of illumination;  the
 background  light had a stronger influence on the  animals of weaker phototaxis
 than on those of stronger phototaxis.   Within the range of 101  to  103lx,
 however,  squids  showed a higher phototactic rate  with background light 'than
 without it.   Such an unusual phenomenon was associated with the sexual
 maturity and moonlight night reproduction of squids.   Therefore,  contrary to
 the common  phototactic fish,  the yield of squids  can be raised if we fish with
 a  hook and  a line at moonlight night during .their breeding season with
 properly increased light source intensity.   Water temperature can influence
 the phototaxis of young round scads  and increase  their phototaxis  within a
 relatively  lower optimum temperature range.

      He Daren and others (1983)  also have studied the feeding intensity of
 juvenile  mullets (Mugil sp_..)  under different illumination and probed into  the
 role  of vision during a fish's feeding.   The results  show that  the
 illumination intensity has  a very strong influence on the feeding  intensity of
 juvenile  mullet  to  daphnia.   Its feeding intensity reaches  the  maximum  under
 the illumination of 102lx; the feeding rate reaches the highest under 102lx
 and reaches  the  maximum within 20 minutes.

      The  peak of feeding activity of young mullet  appears  only  under  certain
 illumination conditions  and  is closely  related to  the  feeding activity  under
 natural conditions  and circadian rhythm.  Our tests have  confirmed the
 important role of vision in  their feeding activity and provided a basis for
 determining  the  environmental  brightness  and bait  quantity  at the  time  of
 throwing  in  the bait.

      Since 1984, He Daren and  others have studied  the optomotor reaction of
 fish.  They  studied the  optomotor reaction characters of the young fish of
 gray mullet  (Mugil cephalus") ,  perch  (Lateolabrax laponicus'), porgy (Sparus
latus), carp  (Cyprinus carpio'),  grass carp (Ctenopharvngodon  idellusl , etc.
Liu Lidong and others  (1986) also have made an overall study on the optomotor
reaction  of Nile tilapia and its influencing factors.  Their study included
the influences of environmental  illumination, the screen speed of rotation,
                                     210

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water temperature, structure of visual field, body length, etc., on the
optomotor reaction of tilapia.  They also described the characters of reaction
of individual, population, and monoculars.   The young fish of this kind has
typical optomotor reaction under the illumination of 1CT5  to 104lx.  Within a
certain range, the optomotor reaction of fish increases with rising
environmental illumination and water temperature and declines with increasing
screen rotation speed and body length.  The fish reacts most effectively to
black-and-white vertical stripes, less effectively to oblique stripes, and
none to horizontal stripes.  The reaction of optomotor reaction intensifies
with the increasing width and number of vertical stripes within a certain
range.  There is no obvious difference in the reaction of individuals and
populations.  The reaction of monocular fish is evidently weaker than that of
normal-binocular ones, and the former has an obvious orientation.  He Daren
and Zhou Shijie have studied the sensibility of young prawn (Penaeus
orientalis) to moving objects and its relationship with environmental
illumination.  They also have studied the optomotor reaction of young porgy
under conditions of colored light.


                         ELECTROPHYSIOLOGICAL STUDIES

     For the past dozen years and more, much has been done in the field of
electrophysiology of fish and in marine animal vision.  As the phototactic
character of fish is related directly to  its vision function, the
determination of the electro-activity of  different levels of peripheral and
central neurons is one of the ideal indexes  in the study  of physiology of
phototaxis.  The main work in China is to study the ERG character with rough
electrodes.  After an overall study on the ERG of round scad and chub
mackerel, Young Xiongli and others have found that their  ERG had the character
of the retina of mixed type and had obvious  off-response  and that its b-wave
was highly  sensitive to the oxygen deficiency;  Background light could
noticeably  raise the threshold of retina  sensitivity.  Zheng Weiyun and others
have found  through their study on red grouper (Epinephelus akaara) that its
retina has  a two photoreceptor system of  rod and cone, but they have not found
any typical character of mixed retina in  the ERG.  They believe that the cone
of such fish has degenerated and is adapted  to weaklight  vision without the
ability of  color discrimination.

     Yang Xiongli and others  (1978) have  determined the spectral sensitivity
of the ERG  b-waves of round scads and chub mackerel and found that the peaks
of spectral sensitivity curves of the two fishes under dark vision are 490 and
480nm, respectively.  The peaks of spectral  sensitivity curves under light
vision shift to 520 and 525 nm, respectively.  They also  have studied the
induction potential of the tectum of chub mackerel.  With the latent period of
induced potential as the  index, the spectral sensitivity  curves thus obtained
basically  correspond with  those under ERG dark vision.  Therefore, it is
believed that the spectral sensitivity remains unchanged  in the visual center.
Zheng Weiyun, Chai Minjuan, Chen Zhong, Liu  Lidong, and others have studied
the ERG character of cuttle fish, swimming crab and prawn (Penaeus penillatus)
and showed that the ERGs  of these invertebrates are cornea negative wave one
positive off-response.  This  is  identical with the structure and arrangement
of their retina.  Through  analyzing the ERG  spectral sensitivity of mud crab
                                      211

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 (Scylla serrsta) under different adaptation, Yang Xiongli and others have
 suggested that its compound eye has only a single receptive system and
 revealed the circadian rhythms of its ERG sensibility.  They also have drawn a
 schematic diagram for control of circadian rhythms in the ERG of the crab.
 Chai Minjuan and others have made some comparative studies on the ERG
 circadian rhythms of several kinds of crustaceans (mud crab,  tiger head crab
 Orithyia mammillaris,  swimming crab and prawn).   In recent years, scientists
 of our country also have begun to study micro-electrodes.   Yang Xiongli and
 others have recorded the horizontal cell potential of retina on the body of
 fresh water fish.

        STUDIES  ON BIOCHEMISTRY OF VISUAL PIGMENT  AND  HISTOLOGY  OF RETINA

      As visual pigment forms the material basis  of photoreception of retina,
 great importance has been attached to its study.   Not much work has been done
 in the field of biochemistry of visual pigment;  however,  Chen Ming and others
 have determined the rod visual pigment of round  scad, chub mackerel,  black
 carP (Mylopharyngodon piceus) grass carp, silver carp (Hypophthalmichthvs
 malitrix),  bighead (Aristichthvs hobilis),  tilapia,  and others.  Black carp,
 grass carp,  silver carp and bighead have pigment of simple retinene2;  the peak
 values of their spectral absorption are 530,  528, 525 and 527 nm, respective-
 ly.   Chub  mackerel show the pigment of retinenel and the  peak value of its
 absorption is 500nm.   Round scad contain two kinds of rhodopsin and their peak
 values of  absorption are 488 and 510nm,  respectively.  Tilapia contains the
 mixed pigment of retinenel and retinene2 and their peak values  of absorption
 are  500 and 522 nm,  respectively.

      Zheng Meili and others (1980)  have studied  the histological structure  of
 the  retina of cuttlefish.   Xu Yonggan and others  (1986) have  made histology-
 physiological studies  on the retina of gray mullet, porgy,  red  grouper,  round
 scad and sardine (Sardinella auritusl.   They also have studied  submicroscopic
 structure  and probed into  the relationship  between the retina morphology and
 the  function of the  five fishes and the  ecological environment.   Xu Yonggan,
 He Daren,  and Zhang Houquan have made  further studies on the  retinomotor
 reaction of  the young  fishes of porgy  and gray mullet.

      The paper  has so  far  given a brief  account of the research work that has
 been done in China for  the past, dozen years or more in the  fields of the
 phototactic  physiology  of  fish and  other  marine animals.  There  is  still much
 room for further  study.  For example, the varieties of fish that have been
 studied  are  still just  a few.   Round scads and chub mackerel have been studied
 through  various methods, but most of this fishes have been studied  only  in
 some  particular aspects.   Few  studies have been made  on the biochemistry of
 visual pigment; a systematic study on the factors  that influence the photo-
 taxis of fish is still  lacking.  It also  is necessary  to probe deeply into the
 mechanism of phototaxis.  As regards test methods, in  the past, more work was
 done  in  the laboratory  than actual observation on  the sea.   Experimental
 techniques must be improved--for example, automatic record-analysis equipment
 should take the place of naked-eye observation in  the study of ethology and
micro-electrodes and ultramicroelectrodes should  be used in electrophysioloey
All these must be improved in future work.
                                    212

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                                  REFERENCES

Chai, M.J. and W.Y. Aheng.  1982.   Spectral sensitivity of ERG of cuttlefish.
     Acta Oceanologica Sinica.  4(6):784-787.

Chai, M.J. and W.Y. Aheng.  1983.   Preliminary study on colour vision of
     cuttlefish (Sepiella maindroni de Rechebrune).   Marine Sciences.  5:36-
     39.
Cheng, C. and L.D. Liu.
     penicillatus.
                         1982.  A preliminary study on color vision of Penaeus
                                                                     1979.
He, D.R., J.H. Xiao, H.M. Luo, M.L. Zheng, J.X.  Xu,  andR.S.  Huang.
     Study on the phototactic behaviour of squid (Loligo duvaucelii
     d'Orbigny).  Journal of Xiamen University (Natural science) 3:99-104.

He, D.R., H.M. Luo, and M.L. Zheng.  1980.  A study on the phototactic
     behaviour of sardine (Sardinella perforata Cantor) and silverside
     (Atherine bleekeri Gunther).   Response to diffused white light and the
     process of adaptation to iso-illuminative spectral colour.  Universitstis
     Amoiensis Acta Scientiarum naturalium.  19(2):81-88.

He, D.R., H.M. Luo, and M.L. Zheng.  1983.  The feeding intensity and its
     dynamics of juvenile mullet under different illumination.  Transactions
     of The Chinese Ichthyological Society 3:21-27.

He, D.R., S.J. Zhou, L.D. Liu, H.C. Cai, and W.Y. Zhen.  1985.  Study on the
     optomotor reaction of some young fishes.  Acta Hydrobiologica Sinica.
     9(4):365-373.
He, D.R., L.D. Liu, and W.Y. Zheng.
      reaction of Tilapia niloticus.
      267.
                                     1987.  Factor influencing optomotor
                                     Acta Hydrobiologica Sinica.  11(3):255-
 Liu,  L.D., D.R. He,  and W.Y.  Zheng.   1986.  Electrophysiological studies on
      visual  characteristics of Mugil  cephalus and M. carinatus.  Journal of
      Xiameti  University  (Natural  Science)  26(2):227-232.

 Liu,  L.D., D.R. He,  and W.Y.  Zheng.   1986.  The characteristics of optomotor
      reaction of  young  Nile tilapia  (Tilapia nilotica).  Journal of Xiamen
      University  (Natural  Science).   25(5):580-587.

 Luo,  H.M., D.R. He,  and M.L.  Zheng.   1981.  Discrimination of  sardine  and
      silverside  to spectral colours.  Acta Scientiarum Naturalium Univer-
      sitatis Amoiensis.   20(2):237-242.

 Shanghai Institute of Physiology,  Department of Oceanography,  Amoy University,
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                       IDENTIFIED SEX PHEROMONES IN FISH:
                        ENDOCRINE AND BEHAVIORAL EFFECTS
                                       by

                    Norman E. Stacey1 and Peter W.  Sorensen2
      Teleost fish use pheromones  for a variety of functions--identifying
 individuals,  kin groups,  and species;  attracting  conspecifics  (i.e.,  school-
 ing);  triggering alarm or fright  reactions  that signal  the  injury  of  a
 conspecific;  and synchronizing reproductive activities  both between and  within
 the  sexes  (see  reviews by Colombo et al.  1982;  Lambert  et al.  1986; Liley
 1982;  Liley and Stacey 1983;  Stacey et al.  1986,  1987).  In nearly all cases,
 the  evidence for such pheromonal  activity comes from  studies in which in-
 dividuals  are exposed to  an unpurified conspecific  odor (holding water or
 tissue preparation).   Although such an experimental approach may convincingly
 demonstrate pheromonal activity,  until a  pheromone  is identified chemically
 and  available for testing,  it is  impossible to  conduct  carefully controlled
 experiments to  investigate  the mechanism  of pheromone action.

      In  the past few  years,  we have been  fortunate  in identifying  several sex
 pheromones  released by female goldfish that trigger physiological  and be-
 havioral responses in the male (Sorensen  and Stacey, in press).  The  fact that
 these  pheromones are  commercially available sex hormones has greatly  facili-
 tated  our  studies.  More  importantly,  our findings  raise the possibility that
 fish commonly use sex hormones and their  metabolites as sex pheromones;  if
 true,  both  fundamental and  applied aspects  of fish  sex pheromone research
 could  expand rapidly  in the near  future.  Indeed, there now seems no  doubt
 that sex pheromones soon  could be applied effectively to the problems of
 controlled  reproduction of  cultured fishes.
                       FEMALE SEX PHEROMONES IN GOLDFISH

     Ovarian growth and vitellogenesis in goldfish occur during fall and
winter (Stacey 1987).  In the spring, increasing water temperature and aquatic
vegetation (spawning substrate) trigger ovulation in females with post-
vitellogenic ovaries.  Females typically spawn only a few times during the
late spring spawning season.  As is typical of many cyprinids,  goldfish are
Department of Zoology,  University of Alberta,  Edmonton AB,  Canada
Department of Fisheries and Wildlife, University of Minnesota,  St.  Paul MN
  USA
                                     216

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group- or gang-spawners, many males following or chasing an ovulated female so
as to be close when she releases her oocytes in floating vegetation.  It is
highly likely that male goldfish have evolved adaptive responses to the odors
of periovulatory females as a result of selective pressures exerted by two key
aspects of goldfish reproduction--the existence of interacting mixed-sex
groups prior to spawning and the intense male competition for females during
spawning.  Because many cultured cyprinids display reproductive activities
comparable to those of goldfish, they also may have evolved similar sex
pheromone systems.

     The sex pheromones we have identified in female goldfish are released
during the brief periovulatory period that commences with the start of the
preovulatory gonadotropin (GtH) surge and ends with the completion of oviposi-
tion approximately 15 hours later.  This ovulatory process is closely
synchronized with the light:dark cycle, the GtH surge commencing in mid-
photophase, ovulation (follicular rupture) occurring in the latter half of
scotophase, and oviposition extending over several hours during early morning
(Stacey et al. 1979).  Between the onset of the GtH surge and ovulation,
females release a preovulatory pheromone with "primer" effects on the male
reproductive endocrine system (Stacey et al. in press).  Later, between
ovulation and the completion of oviposition, females release a postovulatory
pheromone with "releaser" effects on male sexual behavior (Sorensen et al.
1988).  A schematic model of the sequential function of these two pheromones
is presented in Figure 1.


PREOVULATORY PRIMER PHEROMONE

     Evidence of a preovulatory primer pheromone in goldfish was first
provided by Kobayashi et al. (1986a) who showed that, in males placed in
contact with preovulatory females, blood GtH rises in synchrony with the
female's GtH surge.  Because the odor of preovulatory females was sufficient
to trigger a GtH increase in males and because cutting the olfactory tracts
blocked the response, Kobayashi et al. (1986b) suggested that the GtH increase
in males was triggered by a female sex pheromone.  Our studies have confirmed
these  findings and demonstrated that the preovulatory pheromone is  17a, 20/3-
dihydroxy-4-pregnen-3-one (17,20/3-P), the ovarian steroid that induces' oocyte
final  maturation  (migration and breakdown of the nucleus; Nagahama  et al.
1983).  We have shown that preovulatory females release 17,20/3-P to the water
where  it then functions as a pheromone to rapidly increase circulating GtH
levels of males  (Stacey and Sorensen 1986, Dulka et al. 1987, Stacey et al. in
press).  This increase  in male GtH then stimulates an increase in sperm
production.  Males exposed to the preovulatory female pheromone thus benefit
from increased stores of releasable sperm (and therefore increased  fertility)
by the time the female  ovulates and begins to spawn.

      Blood levels of 17,20/3-P begin to increase several hours after the start
of the preovulatory GtH surge, reach peak levels 4 hours prior to ovulation,
and  return to basal levels near the time of ovulation  (Kobayashi et al. 1987,
Stacey et al. in press); peak levels are at least 30 times those preceding and
following the periovulatory surge.  Because the periovulatory profile of
17,20/3-P released to the water  is essentially the same as that in the blood
                                      217

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  FEMALES
                          (Maturation)
         PGFs
        (Ovulation)
   1200
              2000
        preovulatory
           17,206-P
          pheromone
                                          Female Sex
                                           Behavior
postovulatory
       PGF
   pheromone
   MALES
                                                    Spawning
                                                    Synchrony
                                (Milt)
         T
       Male Sex
       Behavior

 1200
             2000
                             0400
                          Time of Day
             1200
Figure 1.   Schematic model depicting actions of two hormonal sex
  pheromones produced by periovulatory female goldfish.  Preovula-
  fcory primer pheromonea preovulatory surge in blood gonado-
  tropin (GtH), triggered by environmental cues, stimulates ovarian
  synthesis of 17a20g-dihydroxy-4-pregnen-3-one (17,203-P), which
  induces  oocyte final maturation  (resumption and completion of
  meiosis).  17,203-P also is released to the water qhere it exerts
  a primer pheromone ennect on the male endocrine system, a rapid
  increase of blood GtH that stimulates testicular 17,203-P syn-
  thesis,  leading to increased releasable sperm within 2 to 4
  hours.   Postovulatory releaser pheromonemature follicles
  rupture  (ovulate) 10 to 12 hours after the GtH surge has begun.
  Ovulated oocytes in the reproductive tract then initiate and
  maintain the synthesis of F prostaglandins (PGFs), which enter
  the  circulation and act within the brain to trigger female
  spawning behavior.  PGFs and PGF metabolites also are released
  to the water where they function as a pheromone-triggering
  male  sexual arousal.
                              218

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(Stacey et al. in press), release of the steroid must be very rapid, an
important feature if 17,20/3-P concentrations in the water are to serve as an
accurate indicator of impending ovulation.  Nothing is yet known regarding  the
mechanism(s) of 17,20/3-P release, although studies of other teleost species
(Colombo et al. 1982) suggest that release in urine is likely.

     Extracellular electrical recording from the goldfish olfactory epithelium
(electro-olfactogram, EOG) has demonstrated that water-borne 17,20/3-P is an
extremely potent olfactory stimulant (1 pM threshold, Sorensen et  al. 1987  and
unpublished).  EOG experiments testing more than 30 steroids have  identified
three types of steroid that are goldfish olfactory stimulants (Sorensen et  al.
1987 and unpublished)--bile salts such as taurocholic acid; androgens such  as
androstenedione and testosterone; and "progestogens" related to 17,20/3-P.
Cross-adaptation experiments, in which the olfactory epithelium is first
adapted to one odorant and then stimulated with another, indicate  that the
olfactory receptors responding to 17,20/3-P are different from those responding
to bile salts and androgens.  Both the bile salts and androgens are con-
siderably less potent than 17,20/3-P, and the magnitude of the response induced
by androgens  is relatively small.

     Of the progestogens tested, the three most effective are 17,20/3-P,
17a,20/3,21-trihydroxy-4-pregnen-3-one (17,20/3,21-P) , and 17a-hydroxyproges-
terone (17-P), in decreasing order of potency.  Because any other  change to
the 17,20/3-P  structure,  or to its pregnane (A-ring-reduced) derivative, almost
completely destroys activity (Sorensen, unpublished results), it seems likely
that ovarian  17,20/3-P does not require further metabolism in order to function
as a pheromone.  Because ovulatory goldfish release equivalent preovulatory
surges of free 17,20/3-P  and conjugated 17,20/3-P (likely 17,20/3-P glucuronide) ,
however, the  possibility remains that conjugated 17,20y3-P also may have
pheromonal activity, (Stacey et al. in press).  Unfortunately, 17,20/3-P
glucuronide is not available for testing.  The goldfish olfactory  system does
not appear to detect glucuronides of estradiol, testosterone, or etiochol-
anolone  (a 5/3-reduced androgen)  (Sorensen et al. 1987), despite the finding of
Colombo et al. (1982) that the latter steroid attracts male goldfish.

     When 17,20/3-P is added to aquarium water to create supra-threshold con-
centrations ,  the blood GtH levels of males increase rapidly  (within 15 min)
and milt  (sperm and  seminal fluid) volumes increase within 2 to 4  hours  (Dulka
et al. 1987 and unpublished).  Blood 17,20/3-P levels of pheromone-exposed
males also increase  over this time period  (Dulka et al. 1987), supporting  the
proposal that testicular 17,20/3-P mediates GtH-stimulated sperm production
 (Ueda et al.  1985).  Of  a variety of steroids tested by addition to aquarium
water, 17,20/3-P is the most potent in inducing the GtH and milt increases,
although related progestogens also are'effective at higher concentrations.
Androgens are completely ineffective in stimulating either GtH or  milt volume
increase, indicating their olfactory activity  (as shown by EOG) is related to
a different function (Stacey and Sorensen  1986 and unpublished).   Both  the GtH
and milt responses to water-borne 17,20/3-P are abolished either by complete
section  of the paired olfactory  tracts or by selective section of  the medial
tracts  (Stacey and Sorensen 1986, Dulka unpublished).  These findings are
consistent with a number of studies showing that olfaction is the  dominant
sensory mode  for pheromone detection in fish and. that pheromonal  information
                                     219

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 is  conducted in the medial olfactory system (Stacey et al.  1986,  Kyle et al
 1987).

     Although simple addition of 17,20/3-P to aquarium water consistently
 evokes  rapid and dramatic increases  in GtH level  and milt volume  (Stacey and
 Sorensen 1986,  Dulka et  al.  1987), it is  likely that this "artificial"
 stimulation is  not  strictly comparable to that produced by  natural  interaction
 with a  periovulatory female.   For example,  the GtH  increase induced by  chronic
 17,20/J-P exposure in aquaria reaches peak levels  within 30  minutes  and  does
 not change  for  at least  several  hours (Dulka et al.  1987),  whereas  GtH  levels
 of  males exposed to the  odor of  females throughout  the periovulatory  period
 continue to increase until ovulation (Stacey et al.  in press).  Although there
 are many possible explanations for this difference  between  the  effects  of
 artificial  and  natural 17,20/3-P  exposure,  we suspect that during  artificial
 (chronic) exposure,  adaptation of olfactory receptors  makes the pheromone
 ineffective after a short period of  time,  whereas during natural  exposure,  the
 male is  repeatedly  stimulated by transient wisps  of pheromone,  each of  which
 could independently lead to  GtH  increase.   Indeed,  we  have  calculated (Soren-
 sen and Stacey  in press)  that the amount  of 17,20/3-P released by  a  female
 prior to ovulation  is sufficient to  produce only  a  small and very short-lived
 detectable  odor plume, in which  case males  would  not be chronically exposed to
 stimulatory 17,20/?-P concentrations.

     Also of concern in  comparing the effects of  artificial and natural
 17,20/3-P exposure is whether  behavioral interaction with the female,  or
 exposure to steroids other than  17,200-P,  normally  contribute to  the  male's
 endocrine response.   It  seems unlikely that behavioral interaction  with the
 preovulatory female  is an important  factor,  because  direct  contact  with
 periovulatory females induces no greater  increase in GtH levels and milt
 volumes  than does exposure to female odor  (Stacey et al. in press).   The
 potential involvement of steroids other than 17,20/3-P  is problematical,
 however, because periovulatory females  also are known  to release  large
 quantities  of 17-P  (Van  Der Kraak et al.  1989), which,  although less  potent
 than 17,20/3-P,  is capable of  stimulating both GtH and  milt  increase (Stacey
 and Sorensen 1986 and unpublished).

     Recently we have found that the male's  response to water-borne 17,20/3-P
 is  affected by  simultaneous exposure to water-borne  androgens (unpublished
 results).   The milt  increase  induced by 17,20/3-P  exposure is inhibited  in a
 dose-dependent manner by water-borne testosterone and  androstenedione.
Androgens probably exert  this  effect by inhibiting GtH  increase, but  this has
not yet been examined.   The biological  function for  a potential inhibitory
 androgen pheromone is not clear  because we  do not understand the patterns of
 androgens released by male and female  goldfish.  At present, the simplest
 explanation would be  that water-borne  androgens inhibit inappropriate respon-
 ses to the minor amounts  of 17,20/3-P  that likely  are released by males  and
nonovulatory  females.  Thus,  only the preovulatory females  release  sufficient
 17,20^-P to  overcome  the  inhibitory  effect  of released androgens.
                                     220

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POSTOVUIATORY RELEASER PHEROMONE

     As shown in females of many  teleost  species  (Liley  1982,  Liley  and Stacey
1983, Stacey et al. 1986), ovulated  female  goldfish  release  a  pheromone that
rapidly stimulates male courtship behaviors (Partridge et  al.  1976,  Sorensen
et al. 1986).  Our recent work  (Sorensen, et al.  1988) indicates  that  this
postovulatory releaser pheromone  is  comprised of  F2a- series prostaglandins
(PCFs).

     A variety of experiments (see Liley  and Stacey  1983,  Stacey  1987,  Stacey
and Goetz 1982) indicate that prostaglandin F2Q!  first functions as a hormone
that synchronizes spawning behavior with  ovulation and,  then,  is  released to
the water where it acts as a pheromone.   The presence of ovulated eggs  in the
reproductive tract stimulates synthesis of  PGF2o!,  which then enters the
bloodstream and is carried to the brain to  trigger female  sexual  behavior.
When oviposition is completed, PGF2o!  synthesis decreases, PGF2o, is quickly
removed from the circulation, and female  sexual behavior ceases.

     Sorensen et al. (1986) provided the  first evidence  that PGFs could
function as female sex pheromones by showing that male goldfish exhibit
similar behavioral responses to the odors of ovulated and  PGF2Q;- injected
females.  However, because males  exhibited  no response when  the PGF2os was
simply added to the water of the  testing  aquaria, they suggested  that  PGF2Q,
either was metabolized to an active form  prior to release  or stimulated
production of an unrelated product with releaser pheromone effects .  Further
studies using EOG responses to evaluate the olfactory activity of PCs
(Sorensen, et al. 1988) support the former  possibility.

     When a variety of prostaglandins  (PCs)  were  tested  as olfactory
stimulants (Sorensen, et al. 1988), the most potent  were found to be PGF2a
(100 pM threshold) and 15 -keto -prostaglandin F2o, (15K-PGF2o!; 1 pM  threshold), a
PGF2Q! metabolite  recently  identified  in goldfish (Goetz et al.  1987).  Cross-
adaptation studies indicate that  these two  PGFs stimulate  separate classes  of
olfactory receptors, which are different  from those  that respond  to  17,20/3-P,
bile salts, and amino acids (Sorensen, et al. 1988).  PCs  other than PGF2a! and
15K-PGF2o!  also  are  effective  in  stimulating  EOG  responses,  but only at much
higher concentrations (1-20
     Both ovulated and PGF2Q,- injected female goldfish release immuno re active
PGF to the water  (Sorensen, et al. 1988).  The rate of PGF release  increases
significantly at  ovulation and continues to  increase for at  least 6 hours but
rapidly declines  to basal rates if ovulated  eggs are removed by spawning or
stripping.  Because removal of eggs also abolishes female spawning behavior
(Stacey 1987), it appears that a single mechanism, activated by the presence
of ovulated eggs, produces the PGF2a,  which first acts  as  a hormone  to
stimulate female  sexual behavior and  later acts as a pheromone to stimulate
male sexual behavior.

     Although there is as yet no direct evidence that ovulated female goldfish
release 15K-PGF2a, there  is  strong  indirect evidence  that  this  is  the  case.
For example, the potency of the odor  of PGF2a- inj ected females  (as measured by
evoked EOG responses) is greater than would" be expected if the PGF2fl!  had been
                                     221

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 released without being metabolized, and similar to what would be predicted if-
 the PGF2a   were converted to 15K-PGF2o, prior to release (Sorensen et al. 1988).

      More recently, we (Sorensen  et al. 1988, Sorensen  et al. in press) have
 shown^that both water-borne PGF2a  and  15K-PGF2o! stimulate the courtship
 behaviors exhibited by males exposed to the odors of ovulated or PGF2Q,-
 injected females (Sorensen et al.  1986).  When PGFs were added to aquarium
 water, grouped males immediately decreased feeding activity and increased
 swimming activity and social contact with conspecifics.  These studies also
 have confirmed and extended the findings of Kyle et al. (1985) that GtH levels
 of male goldfish increase during sexual interaction with PGF2a-injected
 females.   In particular,  experiments comparing the effects of PGF exposure on
 grouped and isolated males provide strong evidence that the pheromone does not
 increase GtH directly,  but acts indirectly by stimulating sexual activity
 (Sorensen,  et al.  in press).  Thus,  the actions of water-borne PGFs are
 distinctly different from those of water-borne 17,20y9-P,  which induces
 equivalent reproductive responses  in grouped and isolated males (Stacey and
 Sorensen 1986).

     An unresolved and potentially important aspect of PGF pheromone function
 in goldfish is whether PGF2a and 15K-PGF2a,  perform the  same or  different
 functions.   Although the  presence  of separate olfactory receptors  for these
 PGFs suggests the  potential for distinct effects,  only qualitative differences
 in responses have  so far  been observed (Sorensen,  et al.  in press).   We
 believe that by responding to two  PGFs males are able  to  increase  the distance
 over which they can locate ovulated females.   For example,  were males to
 employ only a single class of PGF  olfactory receptor,  saturation would occur
 as the male approached the female, with the result that no further increase in
 concentration would be  detectable.   By employing two classes of PGF receptors,
 with different sensitivities but similar central effects,  males may extend the
 range  of PGF concentrations over which a gradient  can  be  detected.


 FURTHER ASPECTS OF  SEX  PHEROMONE FUNCTION  IN GOLDFISH

     We are only beginning to understand the  nature  of sex pheromone  function
 in goldfish;  new pheromones,  and new functions  for identified pheromones,
 certainly await discovery.   For  example, the  studies of Yamazaki and  Watanabe
 (1979)  suggest that sex-specific goldfish pheromones may be induced by
 estrogen and androgen treatment.  Also,  there are  suggestions that  the
 pheromonal  function of  17,20/3-P  is more  complex  than originally expected.  We
 recently have found that  low concentrations  of water borne  17,20/3-P increase
 the occurrence of ovulation in goldfish  (Sorensen  and  Stacey 1987 and un-
 published) ,  an effect consistent with  the fact that  17,20/3-P induces  equiva-
 lent EOG responses  in male  and female  goldfish  (Sorensen et al.  1987).  If
 this is a pheromonal effect, then the  concept of 17,20ft-P as a  female  sex
 pheromone will need  to be revised.    Indeed, because exposure to water-borne
 17,20/3-P increases  17,20/3-P blood levels (and presumably 17,20/3-P release) in
male goldfish (Dulka et al.  1987),  it  is entirely possible  that 17,20/3-P is a
bisexual pheromone, produced by, and acting on, both male and female.  Such a
pheromonal mechanism could synchronize the spawning of  local populations.
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                     HORMONAL PHEROMONES IN .OTHER TELEOSTS

     Our findings that goldfish sex pheromones are hormones provides strong
empirical support for theoretical arguments  (Doving 1976, Colombo et al. 1982)
that fish commonly use released hormones for intraspecific reproductive
synchrony.  Although the generality of hormonal pheromones in fish cannot be
assessed until more species are investigated, in all cases where the chemical
identity of a fish sex pheromone has been proposed, the active substance is a
sex hormone or metabolite  (Lambert et al. 1986, Stacey et al. 1987, Sorensen
and Stacey in press).

     Although pheromonal effects of progestational steroids (i.e., 17,20/3-P-
like compounds) appear to have been studied only in the goldfish, Van Den Hurk
et al.  (1987) propose that glucuronated androgens released by male zebrafish
(Brachydanio rerio) acts as a primer pheromone to trigger ovulation in the
female.  Another glucuronated androgen, etiocholanolone glucuronide, has been
suggested to act as a releaser, attracting the ovulated female Gobius joso to
the male's territory (Colombo et al. 1982).  In fathead minnows, Pimephales
promelas (Cole and Smith 1987), and milkfish, Chanos chanos (C.D. Kelley, C.S.
Tamaru, and C.-S. Lee, Oceanic Institute, personal communication), male
courtship responses are triggered by the odor of PGF2o!-injected females  and
water-borne PGF2a,  respectively.   Together  with the  finding  that PGFs  induce
EOG responses in arctic charr, Salvelinus alpinus (T. Sveinsson and T.J. Hara,
Freshwater Institute, personal communication), these recent studies suggest
that hormonal pheromones are widespread among teleosts.


                 PRACTICAL APPLICATIONS  OF  FISH SEX  PHEROMONES

     There are several reasons for considering the application of sex
pheromones to control certain reproductive processes that presently are
managed by hormone or drug injection.  First, because pheromone treatment does
not require the handling of fish, both labor costs and fish stress are
reduced.  Second, hormonal pheromone therapies will affect only fully mature
individuals because they simply mimic natural triggers of endogenous reproduc-
tive events (ovulation, spermiation).  It should be possible, therefore, to
eliminate a difficulty often associated with injection therapies:  determining
whether individual fish are in a reproductive state appropriate for treatment.
Third, present information suggests the level of technical expertise required
to carry out pheromone treatment will be less than that required for treatment
with exogenous hormones.  Fourth, fish marketability is not likely to be
affected by a treatment that applies a natural product exogenously, at
concentrations lower than those present in the fish.

     We believe that,  in each teleost species,  reproductive strategy and
reproductive endocrinology work in concert to determine what sex pheromone
functions will evolve (intersexual primer,  intersexual releaser, territorial
advertisement, etc.),  and what hormones will be selected to play these roles
(Sorensen and Stacey in press).   Because the reproductive biology of many
cultured cyprinids is broadly similar to that of the goldfish,  we also believe
that all these species use similar sex pheromone systems,  and that the
pheromonal effects we have demonstrated in goldfish,  therefore,  can be readily
                                    223

-------
applied to the reproductive management of 'cultured cyprinids.   There seems no
reason why 17,20y3-P could not be used to stimulate spermiation of cyprinid
broodstook.  On the other hand, because cyprinid ovulation often is
synchronized with daily photoperiod, further work on the ovulation inducing
effect of water-borne 17,20/3-P is required to establish the most effective
time of exposure, and minimum duration of exposure required.

     It also might be possible to advance gonadal development by elevating GtH
levels with longterm progestogen treatment (e.g., daily pheromone pulses).
Prostaglandin pheromones also may be useful in carp culture, one obvious
application being the use of PGF-treated females as pheromone sources to
segregate sexually mature males from mixed-sex schools or to attract and
collect wild males for broodstock.  Whether the inhibitory androgens have any
practical application is not yet clear,  although they may be useful agents for
delaying spermiation and ovulation to extend the period of egg production.

     The reproductive effects of hormonal sex pheromones in goldfish are
dramatic and can be reliably induced with minimal expense and experience.  If
the same effects can be demonstrated in farmed cyprinids, a novel approach to
the problem of controlled finfish reproduction may be at hand.


                                ACKNOWLEDGMENT

     This study was supported by grants from the Natural Sciences and En-
gineering Research Council of Canada (N.E.s.), the Alberta Heritage Foundation
for Medical Research (P.W.S.), and the Minnesota Agricultural Experimental
Station.
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Kyle, A.L., P.W. Sorensen, N.E. Stacey, andJ.G. Dulka.  1987.  Medial
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                                     225

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Sorensen, P.W. , N.E. Stacey, and P. Naidu.  1986.  Release of spawning
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Sorensen, P.W., T.J. Kara, and N.E. Stacey.  1987.  Extreme olfactory sen-
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Sorensen, P.W. , and N.E. Stacey.  1987.  17o:, 20/3-dihydroxy-4-pregnen-3-one
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Sorensen, P.W. , T.J. Kara, N.E. Stacey, and F.W. Goetz.  (1988).  F pros-
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Sorensen, P.W., and N.E. Stacey.  (in press).  Identified hormonal pheromones
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     tion.  Canadian Journal of Fisheries and Aquatic Sciences 39:92-98.

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Stacey, N.E., and P.W.  Sorensen.  1986.  17a,20-dihydroxy-4-pregnen-3-one:   a
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     goldfish.  Canadian Journal of Zoology 64:2412-2417.
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Stacey, N.E., P.W. Sorensen, J.G. Dulka, G.J. Van Der Kraak, and T.J. Kara.
     1987.  Teleost sex pheromones:  recent studies on identity and function.
     In:  Proceedings of the Third International Symposium on the Reproductive
     Physiology of Fish.  D.R. Idler, L.W. Grim, and J.M. Walsh (eds.).
     Memorial University Press, St. John's, Newfoundland,  pp. 150-154.

Stacey, N.E., P.W. Sorensen, G.J. Van Der Kraak, and J.G. Dulka.  (in press).
     Direct evidence that 17a,20j8-dihydroxy-4-pregnen-3-one functions as a
     goldfish primer pheromone:  preovulatory release is closely associated
     with male endocrine responses.  General and Comparative Endocrinology.

Ueda, H.,  A. Kambegawa, and Y. Nagahama.  1985.  Involvement of gonadotropin
     and steroid hormones in spermiation of the amago salmon, Oncorhynchus
     rhodurus.  and goldfish, Carassius auratus.  General and Comparative
     Endocrinology 59:24-30.

Van Den Hurk, R.,  W.G.E.J. Schoonen, G.A. Van Zoelan, and J.G.D. Lambert.
     1987.  Biosynthesis of steroid glucuronides in the testis of the
     zebrafish.  Brachydanio rerio. and their pheromonal function as ovulation
     inducers.   General and Endocrinology 68:179-188.

Van Der Kraak,  G.J., P.W. Sorensen, N.E. Stacey, and J.G. Dulka.  1989.
     Periovulatory female goldfish release three potential pheromones:
     17cc, 20/3-dihydroxyprogesterone, 17a, 20/3-dihydroxyprogesterone glucuronide,
     and 17o;-hydroxyprogesterone.  General and Comparative Endocrinology.
     73:452-457.

Yamazaki,  F., and K. Watanabe.  1979.  The role of steroid hormones in sex
     recognition during spawning behavior of the goldfish, Carassius auratus
     L.  Proceedings of the Indian National Science Academy, Part B 45:505-
     511.
                                    227

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                 EFFECT OF FEMALE-SPECIFIC PROTEIN COMPOUND
               TREATMENT ON HATCHING RATE OF FISH LARVAL AND
                 FISH GONAD MATURATION  (PROTEINS ENGINEER I")

                                     by

                         Wang Hao and  Liu Rongzhen
     The effects of partially purified fish gonad protein on increasing oocyte
maturation and hatching rate of fish larval were studied in adult freshwater
and seawater fishes (Hypophthalmichthys molitrix. Cyprinus carpios L,
Ctenopharyngodon idellus. Mugil soiuy. Aristichthys nobilis. Parahramis
perkinensis. Carassius auratus.  Monopterus albus).

     In our work, female-specific protein compounds from fish gonads were
isolated by using biochemical methods.  All experimental fishes received
intramuscular or intraperitoneal female-specific protein injections of 2 to 4
ml extractions for each fish in our experiment.  After injection, at 3 to 7
days the hatching rate of fish larval were 89% and 90% in female Aristichthys
nobilis.  and the hatching rate of Aristichthvs nobilis larval of control were
65% and 66%, respectively.  The results of observation of all experimental
female fish have increased the hatching rate by about 20%.  Gonad weights of
experimental fishes were higher than those of controls.   Controls received
injections of saline solution only.  Our extracted female-specific protein
from fish gonad was very effective.  When it was used, it induced oocyte
maturation and increased hatching rate of fish larval, as well as related
phospholipids.
Department of Biology, University of Nanjing, Nanjing, PRC
                                    228

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TABLE 1.    INJECTING  (INTRAPERITONEAL) FEMALE-SPECIFIC
            PROTEIN SOLUTIONS AND THE EFFECTS ON OOCYTE
                 GROWTH IN FEMALE Carassius auratus

No. of fish
2
3
15
16
17
18
19
20
21
Control
6
4
7
9
10
TABLE 2.

Body wt, g Gonad .
wt, g
167 ' 15.4
172 20
170 19
190 15.8
148 14
141 9
198 16.5
200 16 . 5
209 17.5

124 13
129 4.7
147 9
196 9.5
189 6.60
INJECTED FEMALE-SPECIFIC

Mean oocyte
diameter , /un
750
780
775
790
780
805
821.4
821.4
857.7

400
450
560
688.6
610.5
PROTEIN TREATMENT
ON FEMALE Mugll soiuy AND THE EFFECTS ON
HATCHING RATE OF FISH LARVAE
Experiment
A
B
C
D
Control
1
2
3
Dose, ml Fertilization Hatching
rate, % rate of fish
larval , %
2 62.9
2 72.2
2 71.6
2 76

0 70.6
0 58.5
0 40
24
23.4
X
22.5

7.4
6.07
X
                             229

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              ANTIFREEZE PROTEIN GENES:  PHYSIOLOGICAL REGULATION
                 AND POTENTIAL VALUE TO THE GENETIC  ENGINEERING
                            OF FREEZE RESISTANT  FISH

                                       by
           Garth L.  Fletcher1, Margaret A. Shears1,  Madonna J.  King1,
                 Ming H. Kao1, Peter L. Davies2 and Choy L.  Hew3

                                 INTRODUCTION

      The freezing  point of most aqueous solutions  depends upon the
concentration of dissolved  solute particles  (colligative properties).  The
higher the concentration of solute  particles  the  lower  the freezing point.
Thus distilled water freezes  at 0C, whereas the body fluids of most fishes
freeze at -0.5 to -0.8C depending on their salt content.   This means that
fishes inhabiting fresh water are in no  danger  of freezing, unless, of course
the water turns  solid.  Seawater has a salt concentration that is
approximately three  times that of fish body fluids.  Therefore, its freezing
temperature ranges  from -1.7  to -2C, depending on salinity.

      Since the  seawater temperatures in the polar  oceans and along the
Northeast Atlantic  coast of Canada  approach freezing for at least part of the
year, why do not fishes inhabiting  these waters freeze and die?  Scholander
and his colleagues  first attempted  to answer the  question by studying a number
of marine fish inhabiting the coast of Northern Labrador (Scholander et al.
1957, Gordon et al.  1962).  They made two important observations.   Some fish
do survive supercooled; that  is, at temperatures below their freezing point.
If they come into contact with ice, however, they immediately freeze and die.
Therefore,-it is the combination of supercooling and ice contact that is
lethal.

      The second observation made by Scholander's group was that the body
fluids of some fish had the same freezing point as seawater.   They termed the
responsible solute "antifreeze."  The early work of DeVries and Feeney
(Devries et al.  1970) established that the antifreezes were polypeptides.
Since that time,  four distinct classes of antifreeze polypeptides  differing in
            Sciences Centre, Memorial University of Newfoundland, St. Johns
NF, Canada

     Department of Biochemistry, Queens University, Kingston ON, Canada

     Department of Biochemistry, Research Institute Hospital for Sick
Children, Toronto ON, Canada

                                     230

-------
carbohydrate content, amino acid composition, protein sequence, and secondary
structure have been isolated from the blood sera of a diverse group of marine
teleosts.  These proteins are synthesized in the liver for export to the
blood, which distributes them throughout most of the extracellular space where
they effectively protect the fish from freezing at temperatures as low as the
freezing temperature of seawater (-1.8C)  (Hew and Fletcher 1985,  Scott et al.
1986, Davies et al. 1988).
                      WINTER FLOUNDER ANTIFREEZE PEPTIDES

      We have been studying the antifreeze polypeptides  (AFP) of the winter
flounder (Pseudepleuronectes americanus) (Figure 1) for  the past 15 years (Hew
and Fletcher 1985, Davies et al. 1988).  This marine flatfish inhabits shallow
inshore coastal waters of much of "the Northeast Atlantic coast of North
America and many populations are exposed to freezing temperatures (<-0.7C)
and ice every winter  (Fletcher et al. 1985).

      The AFP in winter flounder consist of a family of  at least seven
independently acting polypeptides ranging in size from 3300 to 4500 daltons.
The two major AFP (3300 daltons) found in the blood plasma have been sequenced
completely and one of them has been  crystallized to reveal that they are
amphiphilic a helices.  The majority of the hydrophilic  amino acid side chains
project along the length of one side of the helix and the opposite side is
predominantly hydrophobic (Figure 2)  (Davies et al. 1982; Pickett et al. 1984;
Fourney et al. 1984a; Hew et al. 1986 a,b; Yang et al. 1988).  It is believed
that the hydrophilic residues bind (hydrogen bond) to embryonic ice crystals,
thus preventing their growth by blocking the further addition of water
molecules (DeVries and Lin 1977).  Yang et al.  (1988) recently refined this
model and elaborated on the mechanism by which the antifreeze peptides complex
with ice.
           Figure 1.  Gyotaku  print  of  a 35-cm winter  flounder
             (Pseudopleuronectes  americanus)(artist  Ron  Fourney)
                                     231

-------
              Th'r2   Asp5  fhr.
13   Asn|6  Thr24  Asn27  Thr35
                          WINTER  FLOUNDER
                                AFP

         Figure 2.  Structure of winter  flounder  AFP.  [The alpha-helical
           secondary structure of the AFP is indicated by the coil.  Residues ()
           with hydrophilic side chains are indicated and those that are postulated
           to interact with the ice lattice project downwards. The broken line
           between lysine and glutamate indicates an intramolecular salt bridge. The
           molecule is about 50 nra long.]
       In Newfoundland populations of winter  flounder,  mature AFP appear in the
blood plasma in November when the water temperature  approximates 4 to 6C,
reach peak values by January, and disappear  during May when the temperature
begins to rise above 0C (Figure 3)  (Fletcher 1977).  These  seasonal .changes
in plasma AFP levels are accompanied by concomitant  changes in the winter
flounder's resistance to freezing.  Moreover, the winter increase in freezing
resistance over that observed during the summer  is identical to the winter
increase in plasma antifreeze activity (Figure 4) (Fletcher et al.  1986).

       The environmental and physiological factors regulating the annual AFP
cycle in winter flounder have been reviewed  in detail by Davies et al.  (1988)
and Fletcher et al.  (1988a).   All of the available data suggest that the
annual cycle is endogenous, with photoperiod acting  via the pituitary gland to
regulate the precise time of onset of AFP production in the Fall (Fletcher
1977;  Fourney et al. 1984 b,c).   Water temperature does not appear to play a
major role in controlling the time of AFP production, but it does affect the
rate  of AFP disappearance from the blood.  At the water temperatures  that
prevail in Newfoundland in the Spring (March and April)  (0  to -1C) plasma AFP
levels remain at winter values (February) despite the fact  that AFP synthesis
has stopped.   Only when the water temperatures increase above 0C will AFP
levels decline (Fletcher 1977, Hew et al. 1986b).  This means that  the  fish
are protected from freezing even when there  is an unusually prolonged winter.

                ANTIFREEZE PEPTIDES AND ATLANTIC  SALMON CULTURE

       Approximately  7 years ago,  Dr.  Arnie Sutterlin brought to our attention
the economic  desirability of culturing Atlantic salmon  in sea cages along  the
Atlantic  coastline of Canada.  No evidence for the presence  of AFP  has been
found in any  salmonid;  therefore, the problems associated with this endeavour
were  obvious  to all  of us:   the  icy marine environment was  lethal  to  salmon
(Fletcher et  al.  1988b).   Because the only fish capable  of  surviving  in  such
an environment were  those possessing antifreeze polypeptides,  we decided to
                                     232

-------
-1.8
O
o
I -1.6
LU
H -1.4
CD
N |,
LU ''
LU
o:
LL.
-1.0
2
CO
5 -0.8
_j
CL
-0.6




_ , i , i , , i i i i i i _
-,. J  Jv WATER
xX \J TEMP-/ _
" ^ / \ 
\ I \ '
L X / 1 / "
^J I /  
r- \'
1 \ A
/ X -' \
/ - 	 -' \
/FREEZING \
I TEMP . \
/ T \
/ IK-
,.-/ A v
/ r ;
/ \ -
/ \ :

14
o
o
10 -'
QL
LU
6 H
CC
LU
2 t
0 *
-2
la" "=

^
01
14 ^
LU
N
LU
10 g
LL
h-
6 <
2 1
<
                           ASONDJFMAMJJA
                                     MONTH
        Figure  3.   Annual cycles of plasma freezing temperatures and
          plasma  antifreeze peptide (AFP)  levels in Newfoundland winter
          flounder.  [All values plotted as means  one standard error (N=5 to 10).]
attempt to make Atlantic salmon more freeze resistant by giving them a set of
antifreeze genes.

          DO ANTIFREEZE PEPTIDES CONFER FREEZING RESISTANCE TO FISH?

      Because we were embarking on a project to improve the freezing
resistance of Atlantic salmon by winter flounder gene transfer, we needed
direct evidence that the AFP was not species specific and that it would
increase the freezing resistance of salmon.

      The lethal freezing temperature of both seawater-acclimated Atlantic
salmon and rainbow trout was approximately -0.75C (Figure 4) (Fletcher et al.
1988b).  When AFP purified from the blood plasma of winter flounder were
injected into the rainbow trout, their freezing resistance increased in direct
proportion to blood AFP levels (Figure 4) (Fletcher et al. 1986).  These
results indicate that Atlantic Salmon can be engineered to tolerate subzero
water temperatures and ice if winter flounder AFP genes can be inserted into
the salmon genome and expressed in physiologically significant quantities.

                       WINTER FLOUNDER ANTIFREEZE GENES

      During the course of our studies on the antifreeze peptides and their
regulation in winter flounder, the genes coding for these peptides were

                                     233

-------
                          -1.6

                       o
                       -  -1.4
                       Q.
                       UJ
                         -1.2
                       CS

                       N -1.0
                       UJ
                       UJ
                       CO
                         -0.8
                                        WINTER
                                        FLOUNDER
                                               TROUT
                                               PLUS ANTIFREEZE
                                /+ ATLANTIC SALMON
                                *- RAINBOW TROUT
                               -0.8  -1.0   -1.2  -1.4  -1.6

                                 PLASMA  FREEZING POINT (C)
                                 0 2.5    5.0    12.0
                                 PLASMA  ANTIFREEZE (mg/ml)
          Figure 4.   Relationship between  lethal freezing temperatures
            and plasma freezing  temperatures.  [Lethal freezing temperatures
           were determined on seawater-adapted fish by gradually lowering the temper-
           ature of the aquaria with the use of crushed seawater ice. "Winter" floun-
           der are winter flounder acclimatized to winter conditions. "Summer" floun-
           der are winter flounder acclimated to  summer conditions.  Experiments on
           salmon and rainbow trout were carried  out during late winter.  Crosses
           represent means  one standard error.  Solid dots represent individual
           samples from rainbow trout injected intraperitoneally with winter flounder
           AFP.  The diagonal line indicates the  water temperature at which the fish
           should die as predicted from the plasma freezing points.  Data summerized
           from Fletcher et al.  1986, 1988.]



isolated (Davies  et al.  1984).   The  functional AFP gene  is  1  kilobase  (kb)
long and contains a 600  base pair  (bp)  intron between two  exons.  Genomic
Southern blots show that there are approximately  30 to 40  genes per haploid
genome.  The  majority of these AFP genes (>20) are present  in 7 to 8 kb
elements of DNA,  which codes for the major AFP found in  flounder blood plasma
(Scott et al.  1985).


       In preparation for our gene  transfer experiments,  one of the genomic
tandem (Bam HI) repeats  (7.8kb) (subclone 2A-7) was cloned  into the Bam HI
site of plasmid pUC-9 using E.  coli as  the host to produce  large quantities of
the  gene (Figure  5).  The plasmid  containing the  antifreeze protein gene  was
harvested from the  E. coli.  purified, and linearized for injection by
digestion with Eco  Rl (Fletcher et al.  1988c).  Because  the gene used for
injection was  an  entire  tandem  repeat of the winter flounder antifreeze gene,
it should contain all of the DNA sequences responsible for controlling its
expression (Scott et al. 1985).


                           TECHNIQUES FOR GENE TRANSFER


       From the outset of our studies, we  have concentrated on  microinjection
techniques to  transfer the  antifreeze genes  to salmon eggs.  The most
                                        234

-------
effective place  to inject any foreign  gene  is directly into the nucleus.   For
a number of  reasons,  however, this  is  not easy to do with salmonid eggs.
Although salmon  eggs  are very large (5mm diameter) they are also  opaque,
making it impossible  to see the nuclear area.  In addition to this,  once the
eggs are fertilized in water, the chorion separates from the perivitelline
membrane and hardens, making it very difficult to penetrate with  fine glass
needles.  This is unfortunate, for  once the perivitelline space has formed,
the germinal disc is  always on top  of  the yolky egg where it can  be readily
located for  microinjection.

       Salmon eggs are  fertilized by the entry of  a  sperm through the
micropyle,  which  is  located close to the dividing egg nucleus  (Figure 6)
 (Ginsburg 1968,  Riehl  1980).   Thus it was evident that the  best means of
 locating the female nucleus was  to find the  micropyle.   Although it took us  a
number of spawning seasons to  perfect this technique, we can now do it with
 relative ease under  a  dissecting microscope  using reflected light.  Once the
micropyle is located,  a 3 to 5 /am  (outside diameter)  glass  needle can slide
without resistance into the germinal vesicle area of the egg.

       One potential  problem with using  the micropyle as an injection route is
 that it may become altered in such a manner  as  to interfere with the normal
 fertilization process.  Thus we decided to carry out the microinjections after
 fertilization.
         Figure  5.   Schematic diagram of tandemly repeated antifreeze
           gene  locus  in winter flounder.  [B=Bam HI site, E=EcoRl.  The dis-
           continuity indicates the varied number  of repeats in a gene cluster.  The
           AFP gene (1 kb long) is indicated in the enlargement showing its single
           intron and two exons.  One of the tandem repeats was cloned into the Bam
           HI site of the plasmid Puc 9.  The plasmid containing the AFP gene (clone
           2A-7) was harvested from E. coli and linearized for injection using re-
           striction endonuclease EcoRl.]'
                                        235

-------
                                               B
                                                            >  '  - - fc .   v	
                                                          -'".: 7.' -' ".' .'. 'JQu,
      Figure 6.   Schematic of fertilized salmon egg  (A)  and micropyle (B).
         [ABL=blastodisc, CH=chorion, M=micropyle, PV=perivitelline space, VM=vitelline
         membrane, and Y=yolk.   B--CA=cortical alveoli, CH=chorion, CY=cytoplasm,  FS=fer-
         tilizing spermatozoa,  MII=metaphase of second maturation division, MC=micropilar
         canal, and PBI=first polar body.   Some of the ideas for these drawings are from
         Ginsburg (1963, 1968).]
       Once fertilized,  the chorion immediately begins  the water hardening
 process, thus the  time  available to use the micropyle  for injection is
 relatively short.  To extend this time, we followed up on Ginsburg's (1963)
 technique of fertilizing the eggs in a salmon ringer solution.   When this
 procedure is used, the  eggs are fertilized but not activated and the water
 hardening process  is delayed until the eggs are placed in.fresh water.

       The injection apparatus consists of a 3 to 5 //m  (O.D)  glass needle
 driven by a micromanipulator.   The volume of DNA solution injected (2 to 3 nl)
 was regulated using short bursts (100 to 1000 ms) of N2 at 200 kPa.   The
 timing of the bursts was  controlled using a Grass Stimulator.   The'eggs were
 injected with approximately 1 X 106 copies  of the linearized antifreeze gene
 plus plasmid (pUC-9) through the micropyle within 2 hours  of  fertilization
 and incubated in freshwater at 8C.                                         '

                             ANTIFREEZE GENE TRANSFER

       Details of the antifreeze gene transfer experiments have been published
 (Fletcher et al. 1988c).   Of the 1800 Atlantic salmon eggs injected with the
 antifreeze genes, approximately 80% survived to hatching.  This  survival rate
 was  the same as  that of uninjected eggs.

       Eight months after  injection 30 fingerlings (1  to 2 g each) were
 collected for DNA analysis.  Genomic  DNA was  isolated from individual
 fingerlings as described by  Scott  et  al.  (1985).   These DNAs were screened for
 the presence of  the winter flounder antifreeze gene using genomic Southern
blotting procedures (Southern  1975).   In this  technique, the high molecular
weight DNA was cut into smaller  fragments using restriction enzymes  and the
resulting solution was  electrophoresed in 0.7% agarose  gels.   The DNA
fragments,  now separated according  to  size, then were  transferred to
                                      236

-------
nitrocellulose and probed for the presence of antifreeze genes using a short
32P  labelled  segment  (2.7 kb)  of  DNA containing  the complete antifreeze gene
(Figure 7).   If winter flounder antifreeze gene sequences are present in the
salmon DNA,  the 32P labelled probe will bind  to  them and  they  can be  localized
using autoradiography.

           EVIDENCE FOR ANTIFREEZE GENE TRANSFER TO ATLANTIC SALMON

      A preliminary screening demonstrated that the DNA from 2 of the 30
salmon fingerlings tested contained the antifreeze genes.  A more detailed
analysis of these two positive DNAs (No.  26 and No. 36) along with the DNA of
one negative control (No. 45) was carried out in order to be certain that the
complete antifreeze gene was integrated into the salmon genome (Fletcher et
al. 1988c).

      When the salmon DNA was not cut with restriction enzymes, the 32P-
labelled probe only hybridized to large molecular weight DNA,  indicating that
the injected genes were present within the salmon genome rather than free
within the cell.  When the DNA was cut with restriction endonucleases Sst 1
and Bam HI,  the probe hybridized to 2.7 and 7.8 kb DNA segments, respectively.
Segments of this size are what would be predicted if the injected DNA gene
sequence was cut with the same enzymes.  When the salmon DNA was cut with
restriction enzyme Hind III, the probe hybridized to DNA fragments of 9.4 kb
and longer in one of the two positive salmon and 13.5 kb and longer in the
other.  Because the gene used for injection had only one site at which this
enzyme could cut, the other cut had to be within the salmon DNA itself.
Because we cannot predict where this cut would be, the size of the fragment
would be at least 7.8 kb (the length of the injected winter flounder gene) and
have a variable upper length  (Figure 7) (Fletcher et al. 1988c).

      The minor bands in the Bam HI and Hind III lanes of 26 and 36 possibly
represent cleavage products of 2A-7 that have been independently incorporated
into the salmon genome.  In this situation, one would expect to see fewer of
these accessory bands .in the Sst I lanes than in the other two digests, simply
because there is less chance of the breaks in 2A-7 occurring within the
central 2.7-kb Sst I fragment than within the 7.8-kb Bam HI fragment.  When
the overall hybridization signal derived from the 30 to 40 genes in the Sst I
digested winter flounder standard (Figure 7) is considered, it is clear that
more than one copy of the AFP gene had been incorporated into the transgenic
salmon.  The exact number of copies per cell must await further research.

      The integration frequency of the injected genes in the present study (2
out of 30; 6%) appears to be  similar to that observed by other investigators
using microinjection procedures.  Hammer et al. (1985) found an integration
frequency of 1 to 13% for several mammals.  Maclean et al. (1987) reported a
5%  integration frequency of rat growth hormone-mouse metallothionein fusion
gene in the  genome of rainbow trout.  Similarly, Dunham et al.  (1987)
demonstrated, using restriction analysis, the successful integration of a
mouse metallothionein-human growth hormone fusion gene in 2 out of 10  (20%)
channel catfish.  Ozato et  al. (1986) found evidence to suggest a 30%  (10 of
30) integration frequency of  a chick S-crystalline gene construct.  McEvoy et
al. (1988) using dot blot hybridization procedures suggest that 2 out of 15
                                      237

-------
                               26
                                                  H   S   '
               23,1 -. %,


                 9.4-

                 6.7-


                4.4-
                                                                     * ^-
                2.3-
                2,0-
ESB
T
j
s s
j | 3^^
Lo^i
probe
2.7 Kb
,S BH
J  	 1


E
	 J


Figure  7.   Genomic Southern blots  of  salmon DNAs.  [26,  36,  and  45  represent
   individual  salmon. WF=DNA from winter flounder. U=undigested DNA.  B,H,  and S repre-
   sent DNAs digested with restriction enzymes Bam HI, Hind III,  and  Sst I,  respectively.
   The origin  of  the gel  (0) is indicated by the arrow, and the length  (kb)  of  DNA stan-
   dards are indicated below it.  On the right-hand side, the arrow  indicates the point
   of migration of  the AFP gene containing Sst I fragment of flounder DNA  and Sst I
   fragment of 2A-7.  The schematic diagram represents a restriction  map of  the integra-
   ted, linearized  plasma 2A-7.  Salmon genomic DNA is indicated by  the  broken  line.  The
   pUC-9 section  of 2A-7  is indicated by the double lines.  The 1-kb  gene  is represented
   by the small rectangular box.  The open areas of the box are the exons  (AFP  coding
 ,  sequences)  and the hatched area is the intervening sequence (intron).   The cleavage
   sites of restriction enzymes Bam HI (B), Sst I (S), and Hind III  (H)  are  marked.  The
   2.7-kb Sst  I fragment  used as a radioactive probe is underlined.   This  figure is pro-
   duced from  a combination of Figure 1A and 2 of Fletcher et al.  (1988c).]
                                          238

-------
(13%) Atlantic salmon embryos (14 weeks old) contained detectable levels of
the E. coli ft galactosidase-mouse metallothionein promoter fusion gene.  Zhu
et al. (1985) reported that 50% (3 out of 6) of their goldfish injected with a
human growth hormone gene construct were transgenic; Chourrout et al. (1986)
claimed an integration frequency of 75% by injecting a human growth hormone
cDNA sequence into rainbow trout eggs.

      An excellent study carried out by Stuart et al. (1984) examined 547
adult zebrafish that had been injected as embryos with a linearized bacterial
plasmid (pSV-hygro) and found that only 5% of them retained the foreign DNA.
The amount of foreign DNA present averaged less than one copy per cell.  When
20 of their positive (transgenic) fish were crossed to uninjected control
fish, only one consistently transmitted the foreign sequences to its
offspring.  One of their Fj progeny was crossed with a control fish to yield
an F2 generation of which 50% contained the foreign DNA sequence.   Their
experiments demonstrate unequivocally that injected DNA can be integrated into
the fish genome to give rise to a stable breed of transgenic fish.

                          ANTIFREEZE GENE EXPRESSION

      We are screening the blood of potentially transgenic salmon for the
presence of antifreeze peptides using immunoblotting procedures (Burnette
1981).  Samples of blood plasma, approximately 10 /il, are analyzed on a 15%
acrylamide sodium do'decyl sulfate, polyacrylamide gel electrophoresis (SDS-
PAGE) and electrophoretically transferred to nitrocellulose paper.
Immediately after the proteins are transferred, the nitrocellulose sheet is
incubated first in a solution containing bovine serum albumin and then in a
solution containing rabbit anti-flounder AFP antibodies.  The antibody-AFP
complex is located on the nitrocellulose sheets by hybridization with 125I-
protein A followed by autoradiography (Hew et al. 1986).

      The results obtained from screening blood plasma collected from 46
salmon fingerlings during February 1986 indicated that three of them contained
low but detectable levels of winter flounder AFP.  The immunoblotting results
from one of the three AFP-positive salmon are illustrated along with several
AFP-negative fish in Figure 8.

      The molecular mass of the presumptive AFP found in the salmon plasma was
approximately 6 kd, twice that of mature AFP found in winter flounder (3.3 kd)
(Figure 8).  An explanation for this observation comes from our knowledge of
AFP gene expression in winter flounder.  The winter flounder AFP gene used in
this study codes for an 82 amino acid pre-proAFP (Figure 9) (Pickett et al.
1984).  The 23 amino acid pre-sequence is cleaved off intracellularly and the
proAFP is secreted into the blood where the pro-sequence is removed within 24
hours to yield a 37 amino acid mature AFP.  The molecular size of the AFP-
immunoreactive protein found in salmon plasma is essentially the same as that
of the pro-AFP (6kd) isolated from flounder.  This suggests that, although the
Atlantic salmon can properly express the inserted winter flounder gene and
secrete the proAFP into the blood, it lacks the necessary (enzyme)- systems to
process the pro-AFP to mature AFP.  A similar result has been obtained in
transgenetic Drosophila melanogaster for which only proAFP was found in the
hemolymph (Rancourt et al. 1987).
                                     239

-------
      Studies by Hew et al.  (1986b)  have demonstrated that  proAFP isolated
from winter flounder liver possess  significant antifreeze activity (Figure
10).  Thus  although the proAFP  are  less effective than mature AFP at
depressing  the freezing temperature,  they nevertheless would be capable of
conferring  increased freezing resistance to salmon.

      We  currently are attempting to repeat our observations of AFP gene
expression  by screening all  (>700)  of our individually tagged,  potentially
transgenic  salmon.  In this  set of  observations, DNA will be extracted from
the red blood cells of fish  with detectable plasma levels of AFP and analyzed
for AFP genes using genomic  Southern blotting procedures.   All salmon showing
good evidence for the presence  of AFP genes and their expression will be
selected  for breeding experiments to determine their inheritability.

               HOW CAN AFP GENE  EXPRESSION IN SALMON BE IMPROVED?

      It  is evident from our preliminary screening that  transformed salmon
express the winter flounder  AFP genes and secrete the proprotein into the
blood.  If  these results are substantiated by further analysis, it appears
that the  level of expression and secretion of the proAFP will be low and not
sufficient  to confer a significant  increase in the salmon's ability to resist
freezing.   Because blood plasma AFP concentrations of 10 mg/ml will be
required  for winter survival of sea-cage-farmed salmon in most locations along
                        2   3456789  10
                                         AFP
                                         STD
            Figure 8.  Immunoblots of Atlantic salmon  plasma.  [Blood
              plasma from Atlantic salmon  that developed from fertilized eggs in-
              jected with winter flounder  antifreeze genes  (clone 2A-7) were ana-
              lised on a 15% acrylamide SDS-PAGE and transferred to nitrocellulose
              paper.  The paper was incubated with anti-AFP antibodies followed by
              1251 lableled protein A. Lane 7 AFP standard.  All other lanes contain
              salmon plasma.  The arrow under lane 3 indicates an AFP-immunoreactive
              protein of approximately 6 kd.  Numbers on the right side of the gel
              indicate the location of molecular weight markers.]
                                      240

-------
         MET ALA LEU  SER LEU PHE THR VAL GLY GLN LEU  ILE PHE LEU
                                            23
         PHE TRP THR  MET ARC ILE THR GLU ALA SER PRO  ASP PRO ALA

         ALA LYS ALA  ALA PRO ALA ALA ALA ALA ALA PRO  ALA ALA ALA
              44-
         ALA PRO ASP  THR ALA SER ASP ALA ALA ALA ALA  ALA ALA LEU

         THR ALA ALA  ASN ALA ALA ALA ALA ALA LYS LEU  THR ALA ASP
                                                        82
         ASN ALA ALA  ALA ALA ALA ALA ALA THR ALA ARG  GLY
                   PRE PRO AFP  =    82 AA

                   PRO AFP      =    59 AA

                   MATURE AFP   =    37 AA
Figure  9.   Amino acid sequence of the antifreeze precursor  from winter
  flounder  (HPLC component  6) .  [The underlined region indicates the sequence
  of the mature protein (Hew et al.  1986).]
              O
              o
              (S)
              (f)
              LU
              FLOUNDER
                                        PRO-AFP
                                2     3
                                 AFP (mM)
          Figure 10.  Antifreeze activity of winter flounder
            pro-AFP, winter flounder mature AFP,  and ocean
            pout AFP.  [Thermal hypteresis is a measure of. antifreeze
            activity.   Data summarized from Hew et al.  (1986) and Kao
            et al. (1986).]
                                    241

-------
 the East coast of Canada, the expression of these exogenous genes will have to
 be improved.

       Studies of the antifreeze genes and their expression in natural
 populations provides us with a number of clues as to which way to proceed.
 Our research on the antifreeze peptides in a broad variety of taxanomic groups
 has led us to suggest that they evolved relatively recently in response to the
 appearance of seawater ice (Scott et al. 1986).  In many species, the genes
 coding for these antifreezes are amplified,  suggesting intense selective
 pressure to produce large amounts of protein.   Recently we have found evidence
 that the degree of gene amplification (gene  dosage) can be correlated directly
 with the level of AFP produced (Fletcher et  al. 1985,  Scott et al.  1988a  Hew
 et al. 1988).

       Because gene amplification appears to  be a common mechanism by which
 fish have increased their circulating AFP levels,  it seems reasonable to look
 for ways to increase AFP gene copy number in salmon.

       The winter flounder DNA sequence used  in the present study represents
 one of the tandemly repeated genes;  therefore,  it  may  well contain elements
 responsible for gene amplification.   If such sequences are present,  they could
 lead to a similar expansion of AFP gene number in  transformed salmon.   Our
 breeding experiments with the transformed salmon should determine whether this
 will occur.   These experiments,  however,  may take  some time for Stuart et al.
 (1988) found,  using zebrafish,  that  it took  until  the  F2 generation to produce
 a  stable line of transgenic fish.

       It does not seem wise to  rely  entirely on the winter flounder AFP  genes
 to transfer freezing resistance to salmon.   For one thing,  the  proAFP  produced
 by the transformed salmon is  a  less  active antifreeze  than the  mature  protein.
 Furthermore,  we recently have found  that the winter flounder AFP  genes are
 regulated by growth hormone (Vaisius et  al.  1988).  Growth hormone must be
 absent,  or  at low levels,  in  the plasma  before  AFP  genes can be transcribed.
 If this  regulatory system is  passed  on to the  salmon,  there may be conditions
 under  which  growth hormone levels  may not be low enough during winter  for
 transferred  AFP genes  to  be transcribed.

       Recently, we have been  considering the potential value of wolffish and
 ocean  pout AFP  genes as candidates for gene  transfer to salmon.  Wolffish
 (Atiarhichas  lupus) and Ocean  pout  (Macrozoarces  americanus") are members of
 different families from the suborder Zoarcoidea.  Both species produce high
 concentrations  (20 to  30  mg/ml) of AFP (6 kd), which differ markedly from that
 of the winter flounder in that they  are not  a helical and  do not possess an
 abundance of alanine (Hew et  al. 1984, 1988;  Scott et al.  1988b).

       There  are four reasons  why the AFP from wolffish and ocean pout may be
 useful for gene transfer.   First, both species  appear to be expressing their
AFP genes constitutively  for  they maintain significant plasma concentrations
 all year round  (Fletcher  et al. 1985  and unpublished data).  Furthermore,
plasma AFP levels, at  least for the  ocean pout,  are controlled to some degree
by water temperature.  This is not the case with winter flounder where AFP
 genes  are controlled by the pituitary.  Second,  the fact that they are not
                                     242

-------
biased, in the way winter flounders  are, -to  the  use  of alanine might make it
easier for the salmon to synthesize  them in  large  quantities.   Third, neither
the wolffish nor the ocean pout produce pro  AFP.   Therefore,  no special
mechanisms would he required by the  salmon to  produce fully efficient mature
antifreeze.  Fourth, on a molar basis, the ocean pout AFP,  and most likely the
wolffish AFP, are approximately 40%  more active  than the mature winter
flounder AFP (Figure 10).

      The AFP genes from the wolffish and  the  ocean  pout are encoded in large
multigene families of approximately  80 and 150 members,  respectively (Hew et
al. 1988, Scott et al. 1988b).. Detailed studies of  AFP gene organization in
the wolffish indicate that two-thirds of the 80  to 85 genes are organized in 8
kb tandem repeats, each of which  contains  two  genes  in inverted orientation.
These repeats are clustered in groups of at  least  six (Scott et al. 1988b).   A
restriction map of one genomic clone (Wr4A)  containing two-and-one-half
repeats (five AFP genes) is illustrated in Figure  11.  We are now injecting
this 16 kb stretch of wolffish DNA into the  salmon eggs.  By using this
relatively long sequence, we are  not only  attempting to directly increase the
AFP gene copy number incorporated into the salmon  genome but are also
increasing the potential for gene expansion  or amplification by unequal
crossing over during meiosis  (Hew et al. 1986a).

      In summing up, it is apparent  that,  as we  learn more  about the
regulation of antifreeze genes and their expression  in natural populations,  we
will devise more appropriate gene constructs for engineering freeze-resistant
fish.  Although we look forward to the ideal result  of finding that all
transgenic fish produce enough antifreeze  protein  to survive down to the
freezing point of seawater, it is more realistic,  however,  to anticipate that
there will be a more limited expression of the transferred genes and that the
production of freeze resistant salmon will have  to rely on appropriate
selection and mating techniques.
              SHSa   SH  SaHS
                                    XWr4A
      BH SHSa  SH  SaH
                                                        BH SHSa
B=BamHI  H=HindIE
                                               = SstI  Sa = SalI
                                  WOLFFISH
        Figure 11.  Restriction map of a wolffish  genomic  clone containing
          five antifreeze protein  genes.   [Arrows indicate positions of the' genes
          and the directions of transcription.

                                     243
             Redrawn from Scott et al. (1988).]

-------
                                ACKNOWLEDGMENTS

      We would like  to dedicate anything of merit in this work to Dr. Wu Shan-
Chin formally of  the Institute of Oceanology Academica, Sinica, Tsingtao
China.  Dr. Wu spent 6 weeks in Newfoundland where she taught us a little egg
surgery and a great  deal about patience and dedication to science.  This
research was supported by strategic and operating grants from the Natural
Sciences and Engineering Research Council of Canada and by the Medical
Research Council  of  Canada.  (Ocean Sciences Centre Contribution No. 19)


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 Hew,  C. L. , N. C.  Wang, S.  Joshi,  G.  L.  Fletcher,  G.  K.  Scott,  P.  H.  Hayes,  B
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       antifreeze protein diversity and  dosage  in the ocean pout,  Macrozoarces
       americanus .   J.  Biol.  Chem.   263:12049-12055.

 Hew,  C. L., N. C.  Wang, S.  Yan,  H. Cai,  A. Sclater,  and G. L.  Fletcher.
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 Kao,  M. H., G. L.  Fletcher,  N.  C.  Wang,  and C.  L.  Hew.   1986.   The
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 MacLean,  N. , D.  Penman, and Z. Zhu.  1987.  Introduction of novel genes  into
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 McEvoy,  T., M. Stack,  B. Keane,  T.  Barry,  J. Sreenan and F. Gannon.   1988.
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 Ozato,  K. ,  H.  Kondoh, H. Inohara,  T.  Iwamatsu, Y. Wakamatsu, and T. S. Okado
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 Pickett,  M. , G.  Scott,  P. Davies, N. Wang,  S. Joshi,  and C. L. Hew.   1984.
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 Scott,  G. K., P. L. Davies,  M.  H.  Kao,  and G. L.  Fletcher.   1988a.
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 Scott,  G. K., G. L. Fletcher, and  P.  L. Davies.   1986.   Fish antifreeze
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 Scott,  G. K., P. H. Hayes, G. L. Fletcher,  and P. L. Davies.   1988b.  Wolffish
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 Scott,  G. K., C. L. Hew,  and P.  L.  Davies.  1985.  Antifreeze protein genes
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 Southern, E. M.  1975.  Detection  of  specific sequences  among DNA fragments
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 Stuart, G. W., J. V. McMurray, and M.  Westerfield.  1988.  Replication,
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Vaisius, A., G.  L. Fletcher, and D. R.  Idler.  1988.  Growth hormone represses
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Zhu, Z., G.  Li,  L. He, and S. Chen.  1985.   Novel gene transfer into
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      Ichthyol.   1:31-33.
                                    247

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                     BIOENERGETICS MODELING OF FISH GROWTH

                                      by
                          Cui Yibo1 and R. J. Wootton2

                                 INTRODUCTION

      Modeling the growth of fish is of great interest to fish biologists.
Adequate growth models are needed for predicting the production of fish
populations, which is important in the management of fisheries and for an
understanding of the role of fish populations in aquatic ecosystems.

      A popular approach to  modeling fish growth is to regard the growth rate
as a function of body size,  such as the von Bertalanffy model.  This approach
does not explicitly consider the effects of causal factors.  The model only
reflects the growth of the fish under the average conditions of the water body
concerned; it is descriptive rather than predictive.

      Bioenergetics modeling has provided a promising alternative for
predicting fish growth in varying environments.  This approach considers the
effects of causal factors--usually rate of food consumption, water temperature
and body size--on each component of the energy budget of fish and integrates
these effects to predict a growth rate.  Bioenergetics models have been
applied to a number of fish  species (Kitchell et al. 1974, 1977; Kitchell and
Breck 1980; Rice et al. 1983; Stewart et al. 1983; Diana 1983; Bevelhimer et
al. 1985; Cuenco et al. 1985a,b,c; Stewart and Binkowski 1986) and have been
applied to natural systems (Cochran and Rice 1982, Hurley 1986, Kitchell and
Crowder 1987, Carline 1987).  There has not been much improvement in the
structure of the model, however, since it was first developed over a decade
ago.  Before we can be sure  of extensive applications of this approach, the
assumptions adopted in the model need to be justified, and the model has to be
validated against data from  independent, rigorous experiments.

      The purpose of this study was to evaluate the use -of bioenergetics
models for predicting fish growth.  A bioenergetics model was developed for
the European minnow, Phoxinus phoxinus (L.).  Using this model, the impacts of
some assumptions commpnly adopted in other bioenergetics models were assessed.
Data from an independent experiment was used to validate the model.
     Institute of Hydrobiology,  Academia Sinica,  Wuhan,  PRO

     Department of Zoology,  University College of Wales,  Aberystwyth,  UK

                                     248

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                              METHODS AND RESULTS

MODEL DEVELOPMENT

      According to the energy budget of  fish, growth can be expressed as:
                              G = C-F-U-SDA-Rs-Ra
where G is growth, C  is food consumption, F  is fecal production, U  is
excretion, SDA is specific dynamic action, Rs is standard metabolism, and Ra
is activity metabolism (Stewart  et al. 1983).  In the model, each of the
components on the right side of  the equation is regarded as a function of a
series of predictor variables--i.e., rate of food consumption, temperature and
body weight.  Sub-models  also were developed for the dry matter content and
energy content of fish, so that  growth in energy can be converted into wet
weight.

      Most parameters in  the model were  derived from a series of controlled
experiments, referred as  "original experiments" in this paper.  Procedures for
these experiments were described elsewhere (Cui 1987; Cui and Wootton
1988a,b,c; in press a,b), and only a summary is given below.  In each
experiment, 25 minnows were equally divided  into five ration groups--0, 1, 2
and 4% of initial body weight per day and ad libitum.  Each experiment lasted
21 days, during which each fish  was kept in  an individual aquarium  and fed
daily the prescribed  quantity of Enchytraeus worms.  Food consumption, fecal
production, excretion, dry matter content and energy content were determined
directly, and metabolism was estimated indirectly as the difference between
consumption and other components.

      The metabolic rate of the  starved  fish was assumed to be the  standard
metabolism.  SDA was assumed to be 15% of food energy, an assumption adopted
in most other bioenergetics models for fish.   Equations relating' each of the
model components were calculated from the experimental data by multiple
regression.  They are summarized in Table 1.

      The relative dry matter content (% wet weight) and energy content (cal
mg"1 dry matter) of  the fish at  the end of the original experiment were termed
equilibrium dry matter content (DRYE) and equilibrium energy content
(ENERGYE), respectively.   The dry matter content on day t (DRYt)  was
calculated as:
                        DRYt  = DRY,..! + (DRYE-DRYt.^/21
      The energy content on day t (ENERGYt) was  calculated in a  similar way.
      The total energy content on day t  (Et,  cal) was:
                                  Et  = Et^+G
      E was converted to wet weight (W)  by
                            W =  (E/DRY/ENERGY)xlOO

      A computer program,  written in FORTRAN 77,  was used to calculate the
growth of fish based on a time-step of 1 day.

EVALUATION OF ASSUMPTIONS

      The assumptions commonly adopted in other bioenergetics models for  fish
include:   (1)  fecal production (F)  is a constant fraction of food consumption
(C),  (2)  excretion (U) is a constant  fraction of C,  (3)  activity metabolism

                                     249

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 (Ra) is a fixed multiple  of standard metabolism (Rs),  (4)  dry matter content
 and energy content of'the fish  are  constant.  These  assumptions were not  used
 in the model for the minnow.  To  assess  their implications,  the minnow model
was modified to adopt these assumptions.

      In the original experiments,  the average  fractions of  food energy lost
 in feces and excreta were 0.0652  and 0.0511, respectively; the average dry
matter content and energy content for all the fish used were  25.77%  and 5 02
cal mg  , respectively.  These values were used  in the modified models.

      Thus,  to adopt the  assumption  that F is a constant fraction of C,,the
sub-model for fecal production  (Equation 3 in Table 1) was substituted'by
                                  F - 0.0652C.


      To adopt the assumption that  U is  a constant fraction  of C,  Equations 4
 and 5 in Table 1 were substituted by
                                  U  - 0.0511C.
 TABLE 1.   EQUATIONS  USED IN THE BIOENERGETICS  MODEL FOR THE MINNOWa
No.
1
2
3
4
5
6
7
8
Equation
Cmax = o.017W-806T1.22
F = 0.032C0.863260.1093T
U = -2. 0518+0. 0.528C+0.2338T, when C>0
U = 9xlO-5wO-703e0.549T-0.018T2, when C=0
SDA = 0.15C
Rs = 0.0162wO-8108e0.1001T
Ra - -24. 3989+0. 465C+0.017W+0.2739T2-0.00258WT
DRYE = exp(3.5117-0.05711n(FL=l)+0.03731n(FL+lUnT
r2
0.922
0.824
0.911
0.874
_
0.557
0.753
n ATI
             -0.01921nWlnT)

      ENERGYE = exp(5.6722-0.48161nW-l.75671nT-0.0071Ln(FL-KL)lnW'
                +0.20331nWlnT+0.00491n(FL+l)InWlnT)
0.489
 aAlso shown are the coefficients of determination (r2) of the equations
  fitted to the original data. Cmax: maximum food consumption (cal d"1);
  W: body weight (mg); T: temperature (C); F: fecal production (cal d"1) ;
  C: food consumption (cal d"1); U: excretion (cal d-1); SDA: specific
  dynamic action (cal d"1); Rs: standard metabolism (cal d"1) ; Ra: activity
  metabolism (cal d l);  DRYE: equilibrium dry matter content (% body weight);
  ENERGYE: equilibrium energy content (cal mg"1 dry matter): FL = feeding
  level - 100xCmax(%).
                                     250

-------
      To adopt the assumption of constant dry matter and energy  content,  the
dry matter content and energy content of fish were assumed  to be constant
throughout the simulation at 25.77% and 5.02 cal mg"1,  respectively.

      To adopt the assumption that activity metabolism  is a fixed multiple  of
standard metabolism, Equation 8 in Table 1 was substituted  by
                                   Ra = AxRs
where A = 0,1,2 or 3 and was constant in each simulation.

      These modified versions of the model were used to predict  the 21-day
growth by a fish with an initial weight of 2500 mg at 10C.   Three  ration
levels were used:  starvation, 3% of initial weight per day, and ad libitum.
The growth predicted from each modified model was compared  with  that from the
original model (Figure 1).  The assumptions that fecal production and
                                                    0%
                   -500
                   2000-

                   1600-

                   1200-

                    800-

                    400
MAX
                                     3456
                                      Assumption
          Figure 1.   Effect of different assumptions on the 21-day
            growth of a 2500 mg minnow at 10 C at three rations
            (% initial body weight day~l)  predicted by the bioener-
            getics model.   Assumptions:  1. nominal model, 2.  F=0.0652C,
            3. U=0.0511C,  4. dry matter and energy constents  are con-
            stant, 5.  Ra=0, 6.  Ra=Rs,  7.  Ra=2Rs,  8.  Ra=3Rs.
                                     251

-------
excretion are constant fractions of food energy caused only small deviations
in the predicted growth; the other assumptions caused large deviations.

VALIDATION OF THE MODEL

      Data from an independent experiment, referred to as "test experiment,"
was used to validate the bioenergetics model for the minnow.  The experiment
was carried out at 10C (Cui 1987).   Thirty-six minnows were equally divided
into three ration groups:  starvation, 3% of initial weight per day, and ad
libitum.  The experiment lasted 21 days.  Growth, food consumption, fecal
production, excretion and dry matter content of each fish were determined.
The energy content was not determined but calculated from an empirical
relationship between dry matter content and energy content (Cui and Wootton
1988c).  Temperature, -initial body weight, ration, and initial dry matter and
energy content (estimated from a group of control fish sacrificed at the start
of the experiment) were input into the model.  The predicted growth from the
model was plotted against the observed, growth (Figure 2).   The model gave
reasonable predictions for the starved fish but over-estimated growth by the
feeding fish, particularly fish fed ad libitum.

REASONS FOR MODEL FAILURE

      Validation of the bioenergetics model for the minnow showed that the
model was not successful in predicting the growth of fish in the "test
experiment."  As the model gave reasonable predictions for the starved fish,
failure to predict growth at higher rations may be caused by an inadequate
simulation of the growth-ration relationship.  A simulated relationship
between specific growth rate (SGR),  conversion efficiency (K),  and ration is
shown in Figure 3.  SGR was calculated as:  100x(lnWfc-lnW0)/t where Wt is the
predicted final, W0 is the initial weight of fish (mg),  and t is the period of
growth (day).  K is calculated as:  100x(Wt-W0)/CT where Ct is the total amount
of food consumed (mg).  In the simulations, the initial weight of the fish was
assumed to be 2500 mg; the initial dry matter content and energy content were
25.77% and 5.02 cal mg"1,  respectively;  the  growth period was 21 days; ration
ranged from starvation to ad libitum: four temperatures were used:   5,  9, 12
and 15C.

      At all temperatures, the predicted SGR increased with increased ration.
There was no approach to an asympote at high rations.  The predicted
conversion efficiency also increased with increased ration without a decrease
at high rations.  These predictions did not agree with results from most
empirical studies, which showed that the relationship between SGR and ration
in fish is a decelerating curve and the relationship between conversion
efficiency and ration is an inverted "U"-shaped curve (Brett and Groves 1979,
Cui and Wootton, in press b).

      To find out why the model failed to predict the growth of fish in the
test experiment, observed values for some of the energy budget components in
the experimental fish were used in the model instead of the predictive sub-
models.  The resulting "models" were used to predict the growth of the fish
and the predictions were compared with those from the original model.  If use
of observed values for a component yields a great improvement in the
                                     252

-------
predictions, then errors due to  the  sub-model  for  this  component may be an
important source for the errors  in the growth  predictions.

      Data for the 12 fish fed ad libitum  in the test experiment were used for
this analysis.  The total summed growth by the 12  fish  was  compared with that
predicted by each of the modified "models"  as  well as the original model
(Table 2).   Use of observed values for fecal production and excretion resulted
in little improvement in the predictions,  whereas  use of observed values for
food consumption and dry matter  and  energy content yielded  great improvements.
This indicated that errors in growth predictions may be largely caused by the
errors in the sub-models for consumption and dry matter and energy content.
                -200
             0>
             ~  -400
                  -400
             O
             73
             
-------
                 2 -i
            tt    1
            a
            w
                 o -
                -1
                                4         8
                                  Ration
                                                    12
                20-
                10-
                                    5
                                  Ration
                                                     10
    Figure 3.   Relationship between specific growth rate (SGR,  % day"1),
      conversion efficiency (K,  %), and ration (% body weight day1)
      predicted by the bioenergetics model for the minnow at four
      temperatures 5 C, A9  C, X i2 C,H15 C.
      As  food consumption is strongly related to body weight in the model,  the
 over-estimation of consumption may be a result of the over-estimation of
 growth  due to the poor estimates of other components.   This was proved to be
 true as the average predicted ration, when expressed as a percentage of mean
 body weight,  was within the range observed for the 12 fish in the test
 experiment.   (The average predicted ration was 6.2%,  while the observed values
 ranged  from 4-7%.)  When the observed values for all these components were
 used in the model, there was still a substantial deviation in the predicted
 growth, indicating that the sub-models for metabolism, which was not directly
 determined in the experiment,  also are important in causing the prediction
.errors.  As the model gave reasonable predictions for the starved fish, the
 sub-model for standard metabolism should be suitable;  the errors lie in the
 sub-models for SDA and activity metabolism.
                                      254

-------
    TABLE 2. ERRORS IN THE PREDICTIONS OF  THE GROWTH OF THE 12 MINNOWS FED
         ad libitum IN THE TEST EXPERIMENT FOR "MODELS" USING OBSERVED
                         VALUES FOR1 DIFFERENT COMPONENTS
                                               Predicted growth

                                           "Model" in which observed values
                                           were used for the following
                      Observed   Original  component(s)a	
                       growth     model      C     F+U      DE   C+F+U+DE
Growth (mg)
Error (mg)
Relative error (%)
6465
0
0
16485
+10020
154.99
13607
+7142
110.47
16792
+10327
159.74
11232
+4767
73.74
8814
+2349
36.33
    energy content.
                                  DISCUSSION
      The results of this study indicated that, in the bioenergetics modeling
of fish growth, it is relatively safe to assume that fecal production and
excretion are constant fractions of food energy; however, major errors may
arise from the assumptions that activity metabolism is a fixed multiple of
standard metabolism and that energy content of fish is constant.

      Although most parameters in the bioenergetics model for the minnow were
derived from controlled experiments and the model adopted fewer assumptions
that most other models of its sort, the model failed to accurately predict the
growth of fish under laboratory conditions.  The model's prediction of the
relationship between growth and ration did not agree with that observed in
empirical studies.  This may be an important cause for the failure of the
model.

      In most other bioenergetics models, the predicted relationship between
growth and ration was not explicitly examined.  Kitchell et al. (1977) stated
that their model for Perca flavescens predicted maximum conversion
efficiencies at maximum rations.  This was in agreement with the prediction
from the minnow model.

      Inaccurate estimates of dry matter and energy content, SDA and activity
metabolism were the major sources of errors in the growth prediction from the
minnow model.  In most other bioenergetics models for fish, simplifying
assumptions were made for these components.

      This study suggested that, at the present stage,  bioenergetics modeling
may have limited use for predicting fish growth.   Future studies should focus
on the development of more accurate sub-models for the dry matter and energy
content of fish,  SDA and activity metabolism to improve the performance of the
model.
                                     255

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Kitchell, J. F. and J. E. Breck.  1980.  Bioenergetics model and foraging
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                                    257

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   ROLE OF SCIENCE AND TECHNOLOGY  IN CONSERVATION OF THE STURGEON RESOURCE

                                      by
                         Liu Jiankang1 and Yu Zhitang2

                                 INTRODUCTION

      The Zhonghua sturgeon  (Acipenser slnensis Gray) is an anadroraous fish of
enormous size, reaching a maximum  body weight of 550 kg.  It inhabits the
continental shelf of the east coast of Asia.  Every year in July and August,
individuals approaching maturity ascend the Chang Jiang (the Yangtse River),
where their gonads undergo further development, and spawn in the fall of the
following year.  Its spawning ground is distributed mainly in the lower
reaches of the Jinsha River and the upper reaches of the Chang Jiang.  The
annual catch of the sturgeon in the provinces along the Chang Jiang averaged
400 to 500 fish in the 1970s (Yu et al. 1986).

      The construction of the Gezhouba Hydro-electric Project blocked the
passage for the spawning migration of this fish.  The need to construct a dam
by-pass for the fish was hotly debated from 1970 to 1982.  Finally, on
December 28, 1982, six leading officials sent a report to our central
government pointing out that "construction of a fish by-pass may not be
considered among the measures for  the salvation of the Zhonghua sturgeon in
order to avoid serious economic loss and waste," that "more impetus should be
given to the experimentation on the induced spawning of the sturgeon below the
dam," and that "strict prohibition of commercial sturgeon fishing must be
enforced" (Academia Sinica 1984).  These decisions from the central government
are well grounded.

      It might be helpful here to  summarize the discrepancy in opinions in
this matter.  Those who advocated  the construction of a fish by-pass
maintained that the natural spawning ground of the sturgeon lies in the upper
reaches of the Chang Jiang some 1000 km from Yichang.  The gonads of those
sturgeon detained at the Gezhouba  dam could not develop to maturity, hence it
would be impossible for them to spawn below the dam.  For the same reason,
qualified brood sturgeon for induced spawning (whose gonads must attain Stage
IV development) would not be available.  Therefore, the sturgeon is faced with
the crisis of extinction.  In their opinion, a fish by-pass is a sort of
advanced installation that is indispensable.  Even if the effectiveness of
     ^Institute of Hydrobiology,  Academia Sinica,  Wuhan,  PRC

     2Institute of Reservoir Fisheries,  Ministry of Water Conservancy and
Academia Sinica, Wuhan, PRC

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such an installation may not be satisfactory for sturgeon, it is nevertheless
better to construct one than none.

      Those who objected to the construction of a fish by-pass held that when
the sturgeon ascend to below the dam, there was some evidence that the gonads
could develop to Stage IV, at least in a part of the spawning shoal.
Moreover, there are river sections downstream of the dam that offer bed
conditions and hydrological features similar to those in the natural spawning
ground of the Jinsha River.  These sections possibly could form a new spawning
ground.  Experiments on the induced spawning of sturgeon in the upper reaches
proved successful early in 1972.  Induced spawning of the sturgeon below the
Gezhouba Dam should not be "beyond the capability of our generation" as
someone pessimistically predicted.  What is more important is the fact that,
although fish by-passes intended for sturgeons built at hydro-power stations
abroad are generally unsuccessful (Doroshov 1977),  the Zhonghua sturgeon has
even larger body-size and sparser population, and,  coupled with the vast
breadth and great depth below the dam, the effectiveness of such construction
seems very doubtful.  Suppose we built a fish by-pass at the cost of scores of
millions of yuan (RMB) that required millions of kWh of electric power to
operate but achieved nothing but a "monument of stupidity" (LaBounty, J. F.,
personal communication).

      Besides, the construction of a fish by-pass at the Gezhouba Dam is a
matter that must be considered in the light of the proposed Three-Gorge High
Dam.  To have the sturgeon pass the high dam over 100 m above the water level
is certainly much more difficult than to have it pass the low dam of Gezhouba.
And if it is decided in the end that no fish by-pass should be built at the
high dam, then what is the sense of constructing a fish by-pass at the dam of
Gezhouba.

                SCIENTIFIC AND TECHNOLOGICAL EFFORTS  AFTER THE
                   INTERCEPTION OF FLOW AT THE GEZHOUBA DAM

      Interception of flow at Gezhouba was effected in January 1981.
Beginning from the fall of that year, inspection of individuals with mature
gonads was made each year at the sturgeon shoal at the Yichang River section
below the dam.  The results were 1981, 13.5%; 1982, 12.5%; 1983, 25.4%; 1984,
51.1%; (Zhao et al. 1986) 1985, 44.0%; 1986, 49.6% (Deng et al. 1987).  The
sturgeon aggregated below the dam normally includes individuals that will not
spawn until next fall; therefore, a percentage of nearly 50% is to be regarded
as normal (Zhao et al. 1986).  In October-November 1982, members of the
Institute of Hydrobiology disclosed for the first time a new spawning ground
of the sturgeon downstream of the dam and recovered a large number of sturgeon
eggs from the intestines of benthic fishes.  Thirty newly hatched, live
sturgeon fry also were collected 20 km below the Gezhouba Dam (Deng et al.
1987).

      A joint investigation group dispatched by the National Economy
Committee; Ministry of Agriculture, Animal Husbandry and Fisheries; and the
Ministry of Water Conservancy and Electric Power verified this finding and
wrote in their report of investigation, "The spawning and normal hatching of
the sturgeon below the dam is a fact beyond any doubt and should be affirmed"
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 (Academia Sinica).   Subsequent surveys  for 3  years  (from 1982  to  1984) not
 only confirmed the  fact that the gonads of sturgeon below the  dam can develop
 to  maturity,  but also  delineated the  site  of  the new  spawning  ground as  the
 river section between  the Yichang Shipbuilding Yard of  the Bureau of
 Changjiang Navigation  and the vicinity  of  Aijiahe and Jiangjunmao--a section
 of  about  14 km in length.   Besides, a brood sturgeon  with running eggs was
 caught near Huyatan (Deng et al.  1987).

       The earliest  success of induced spawning of the sturgeon at the site of
 its natural spawning ground dated back  to  1972.  After  the interception  of
 flow at Gezhouba, the  Collaboration Group  for Induced Spawning (Liu  1987)
 embarked  on a test  for the sturgeon caught from below the dam  in  November
 1983,  adopting the  method of chaining up the  brood  fish at the river bank.
 Fertilized eggs  were kept  in a circular  "race-course,"  where fry  were hatched.
 This  implies  the first success of induced  spawning  of sturgeon below the dam
 (Liu 1987,  Fu et al. 1985).   During the  course of the experimentation,
 technical improvements were  made  in several ways, such  as  the  use of the
 commercially  available estrogen LRH-A to replace the  pituitaries  of  sturgeon
 and carp  completely (Yi et al.  1986), the  rearing of  immature  female sturgeon
 (with ovaries  of the developmental stage III) to sexually  ripe parent fish in
 the  course of 1  year (Liu  1988),  and  the use  of a new "Sieve net"  type of
 incubator.  Newly hatched  fry were kept  temporarily in  a  loop-shaped pond, and
 after 7 days,  they  were introduced into  the rearing pond  at the density of 500
 fry per square meter.   After 137  days of rearing, the fingerlings  grew to a
 body weight of 200  g.   This  result demonstrates clearly that sturgeon caught
 below the dam can be used  for induced spawning (Liu 1987).

       The Sturgeon  Release Station was organized by the Fishery Division of
 the Gezhouba Bureau of Engineering in April 1982, and by 1984,  the base was
 already in a workable  condition.   From 1982 to 1984,  the station  engaged in
 transporting  the would-be  spawners over  the dam from  below, in the hope that
 they might ascend further  to  reach the natural spawning ground.   In  the three
 fishing seasons, the station captured and  released  82 fish to the newly formed
 reservoir above  the dam (Fishery  Division  1984).  As  to the induced spawning
 of sturgeon below the  dam, the  station obtained more  than 40,000  fry from its
new spawning pond and  released 6000 fingerlings 4 to  5 cm  in length downstream
 as the first batch.   From  1984  to  1986,   467,000 7-day baby sturgeon 1.8 to 2.3
 cm in  length  (69 -78 mg in weight) were  released downstream (Liu  1987) .
Tagging of various  sorts were  done on fingerlings and on a limited number of
 large  fish (average weight 715  g), but the number was too small,  and the time
 interval  too short,   to  get any  returns as yet.  A summary on the cultivation
 techniques  of  fry and  fingerlings has been published  recently  (Xiao et al.
 1988).

      No matter whether natural or induced spawning is concerned,  the
prerequisite at present  is an abundance  of brood sturgeon  (with ripe gonads)
 in the river.   Regretfully,  in  the first 2 years after flow interception,
batches and batches  of brood sturgeon were captured and killed when the
migrating  fish were  impeded by  the dam.   In the fall and winter of 1981,  more
than 800  sturgeon were  caught in  the Hubei Province,  i.e., 5.5  times the
multiyear  average of 145 before dam construction.   Again, a total of nearly
400 fish were caught in 1982 and  in the  spring of 1983  (Yu 1986).   Since the
                                    260

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fall season of 1983, a number of Fishing Administration Stations have been set
up and strict protection of the brood fish has been brought into effect.  The
action began to obtain good results in the second year.  The number of brood
sturgeon caught since 1984 has decreased to a large extent, and, in 1984,  the
season of natural spawning occurred earlier than in 1982 and 1983.   The
duration of two batches of spawning was prolonged and the range of spawning
ground was extended in comparison with the previous years (Academia Sinica
1984).

      Having affirmed the effects of brood fish protection, research workers
now turn to the protection for the juvenile sturgeon (Zhao 1986).  Their
investigation in the estuary indicated that the number of specimens of
juvenile sturgeon they could collect (477 in 1984 and 571 in 1985)  was
substantially increasing.  However, juveniles descending a long way from
Gezhouba were captured in large numbers by the set-net fishery at the estuary.
The authors stress that the protection of juvenile sturgeon is an important
link in the chain of sturgeon resource management and should arouse the
attention of fishery management departments.   The proposal is made to the
effect that during the peak period of juvenile "emigration" (mid-June to mid-
July) ,  set-net fishing should be banned for a month, so as to give the
juveniles more chance to descend to the sea for growth.

      To sum up, from the refutation of the idea of building a fish by-pass
for the sturgeon to the confirmation of the possibility that the sturgeon
below the Gezhouba Dam can develop ripe gonads, and to the actual finding of
the anticipated new spawning ground below the dam and from the improvements in
the techniques of induced spawning to the protection of brood sturgeon and the
installation of release stations for the sturgeon, along with the protection
of juvenile sturgeon, all these point to the guiding role of science and
technology in the conservation of the sturgeon resource.
                                   REFERENCES

 Academia Sinica.   1984.   Circumstance Report on the Work of Fish Salvation at
       Gezhouba.   Institute of Hydrobiology,  Academia Sinica.
 Deng et al.  1987.
 Doroshov,  S.  I.   1977.   Passage of sturgeon through the  fishlocks in the USSR.
 Fisheries  Institute.   1981.   Summary for the Biotechnical Research on the
       Artificial  Breeding of Zhonghua Sturgeon by the Method of Chaining.
       Fisheries  Institute of Chang Shou Lake,  Chongqing.
 Fishery Division  of the  Gezhouba Bureau of Engineering.   1984.   Manuscript.
 Fu,  C-j.   1985.   Freshwater Fisheries,  No.  1,  1-5.
 Liu,  Y.   1987.  Preliminary studies on the induced spawning and fingerling
       rearing of  the  Zhonghua sturgeon (Manuscript).
 Liu,  Y.   1988.  Technical study of the artificial proliferation of the  Chinese
       Zhonghua sturgeon--The Status Quo and Prospects.   Shuiliyuye Water
       Conservancy Fisheries No.  4,  20-23.
 Xiao,  H.  et  al.   1988.   Preliminary studies  on the techniques of cultivation
       for  the fry and fingerling of the Zhonghua sturgeon.   Shuiliyuye  (Water
       Conservancy Fisheries) No.  4,  24-29.
 Yi,  J-f.  et  al.   1986.   Shuiliyuye (Water  Conservancy Fisheries)  No.  2, 44-46.
 Yu,  Z-t.   1986.   Transactions of the Chinese Ichthyological Society.  No.  5,
       1-16.
 Zhao,  Y.   1986.   Shuiliyuye (Water Conservancy Fisheries)  NO. 6,  38-41.

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               PROTECTION OF WATER QUALITY IN THE UNITED STATES

                                      by

                              Rosemarie C. Russo1
     Since 1970 the United States Environmental Protection Agency (EPA) has
exercised primary responsibility within the Federal government to address the
environmental problems confronting our nation.  The Agency administers nine
comprehensive environmental protection laws:  the Clean Air Act; the Clean
Water Act; the Safe Drinking Water Act; the Comprehensive Environmental
Response, Compensation, and Liability Act (better known as "Superfund"); the
Resource Conservation and Recovery Act; the Federal Insecticide, Fungicide,
and Rodenticide Act; the Toxic Substances Control Act; the Marine Protection,
Research, and Sanctuaries Act; and the Uranium Mill Tailings Radiation Control
Act.

     Some of these Acts were created since the EPA was first established;
others are amended versions of legislation first enacted as far back as 1899.
The 1899 legislation was the Rivers and Harbors Act, which was the first U.S.
Federal law in environmental protection.  Subsequent legislation in water
pollution control produced the Clean Water Act, which was recently amended and
reauthorized in 1987.

     Using the environmental laws as the foundation, the Agency develops
regulations and sets standards for pollutant levels in the individual environ-
mental media and then monitors compliance by, pollutant generators within the
individual states.  To ensure that its activities are based on the best
available scientific and technical knowledge,  the Agency conducts an extensive
research and development program.

     EPA is organized into four major regulatory offices to carry out its
responsibilities to protect the environment and human health.   These are
(Figure 1) Air and Radiation, Water, Pesticides and Toxic Substances, and
Solid Waste and Emergency Response.  In addition,  there is an Office of
Research and Development with twelve research laboratories whose function is
to conduct both fundamental and applied research in support of the regulatory
offices.  There are ten regional offices whose function is to work with the
^Environmental Research Laboratory,  U.S.  Environmental Protection Agency,
 Athens, GA, USA
                                     262

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     Office of
  Administration and
Resources Management
    Office of
 Enforcement and
Compliance Monitoring
                                  Criminal
                                Enforcement
                                  Senior
                                Enforcement
                                  Counsel
Office of
Water



Water
Enforcement
and Permits

Water
Regulations
and Standards

Municipal
Pollution
Control

Drinking
Water
Marine and
Estuarlnas
Protection
Ground Water
Protection

Wetlands
Protection
Office of
Solid Waste and
Emergency Response
-

Solid Wasto

Emergency
and Remedial
Response

Waste
Programs
Enforcement

Underground


                                        Storage Tanks
                                                                     Office of
                                                                    Air & Radiation
                                                                      Air Quality
                                                                      Planning
                                                                    and Standards
                                                                       Mobile
                                                                      Sources
                                                                     Radiation
                                                                     Programs
Office of
Inspector General


Audit

Investigations

Management
                                                                                      Modeling,
                                                                                    Environmental
                                                                                    Monitoring and
                                                                                   Quality Assurance
                                                                                    Environmental
                                                                                      Engineering
                                                                                      Technology
                                                                                     Demonstration
                                                                                     Environmental
                                                                                     Processes and
                                                                                        Effects
                                                                                        Research
                                                                                                                Health Research
                                                                                                                Health and
                                                                                                               Environmental
                                                                                                                Assessment
              Figure  1.    U.S.   Environmental  Protection  Agency  Organization

                                                              263

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states to implement EPA's environmental regulations throughout the country.
Finally, there are several administrative offices, including Administration
and Resources Management; Enforcement and Compliance Monitoring; Policy,
Planning, and Evaluation; International Activities; General Counsel; and
Inspector General.

     EPA also is empowered to enforce compliance with its regulations through
administrative, civil or criminal actions in court.  By seeking and winning
large financial and criminal penalties against significant violators, EPA can
perhaps remove any incentive to non-compliance.  EPA had a strong record in
enforcement in 1986 and 1987.  There were 373 civil referrals in 1986, highest
in history; and 274 civil referrals in 1987, second highest in history.  There
were 3,200 administrative orders issued by EPA in 1987, the highest in
history.  A record $24 million in civil penalties was assessed in 1987.  Sixty
percent of the total of all EPA civil penalties have been collected over the
last three years.

     Since 1972, EPA has awarded $44.6 billion for sewage treatment construc-
tion grants.  The 1987 Clean Water Act Amendments authorize an additional $18
billion, total, for construction grants through 1990.  Of this amount, at.
least $8.4 billion and as much as $13.2 billion is authorized for capitaliza-
tion grants to establish state revolving funds.  Federal money in the state
revolving funds must be matched by at least 20% state funding.  The State
Revolving Funds will be used for construction of sewage treatment plants and
other water pollution control activities.  They are part of the transition
from the Federal construction-grants program to a state-operated loan program.
EPA is increasingly helping states develop their own criminal enforcement
capabilities to detect and prosecute environmental crimes.  In 1988 program
enforcement priorities include an emphasis on municipal compliance for water
quality.  State enforcement is reaching increasingly high levels, with State
agencies prosecuting a total of 723 environmental cases in 1987--twice that of
the previous year.  States also initiated 3200 administrative orders.

     Clearly, water quality protection is a high environmental priority.  The
keystone for water pollution control is the National Pollutant Discharge
Elimination System (NPDES).  Under the NPDES, community sewage facilities must
secure permits that specify the types and amounts of pollutants that may be
discharged.  Industries discharging pollutants into waterways also are subject
to control requirements.  These effluent limitations are designed to reach an
ultimate goal of completely eliminating the discharge of pollutants into the
nation's waters.

     The water pollution control program seems to be succeeding.  Since 1970,
municipal sewage treatment has been provided for more than 80 million people.
Most industrial plants have installed water pollution control technology.  As
a result, organic wastes discharged from industrial sources have been reduced
by 38 percent.  When all the mandated effluent guidelines are in place,
discharges of toxic pollutants will have been reduced 96 percent from 1972
levels.

     To deal with problems of contamination of surface waters from toxic
organic pollutants such as pesticides and inorganic pollutants such as lead

                                     264

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and arsenic, the Agency established National Water Quality Criteria for
Protection of Aquatic Organisms and Their Uses.  The process of deriving
national criteria is shown schematically in Figure 2 (U.S. EPA 1985).  The
goal of these criteria is to prevent unacceptable long-term and short-term
effects on commercially and recreationally important aquatic species; other
important species; fish and benthic invertebrate assemblages in rivers and
streams; and fish, benthic invertebrate, and zooplankton assemblages in lakes,
reservoirs, estuaries, and oceans.  The national criteria for each chemical
are given as two numbers:  (a) criterion maximum concentration is intended to
be protective against acute adverse effects due to high, short-term concentra-
tions ;  (b) criterion continuous concentration is intended to be protective
against unacceptable chronic effects of lower, long-term continuous concentra-
tions .

     National criteria for a specific chemical may be applied to a particular
body of water directly, or may be modified to take into account local condi-
tions of water quality and aquatic species present ("Site-Specific Water
Quality Criteria").  Chemical characteristics of the local water may be such
as to increase or decrease the toxicity of the chemical of concern.  Some
species in the local water may be very sensitive or insensitive to the
chemical of concern, or the resident species may be stressed by other factors
such as parasites, disease, predators, other pollutants, etc.

     The states are responsible for establishing and enforcing standards for a
particular surface water body.  The standards are set taking into account the
national criteria, any site-specific modifications of the criteria based on
data about field conditions, plus social and economic considerations and
environmental and analytical chemistry capabilities.  EPA assists the states
in setting and enforcing state water quality standards.

     To regulate chemicals in surface waters, we need to know:  what chemical
is being discharged, the quantity being discharged, the fate of that chemical
in the aquatic environment, the short- and long-term hazard it poses to
aquatic life, and what the national criteria are, if available.

     To derive National Water Quality Criteria, certain specific information
is required:  the individual chemical, data on acute and chronic toxicity to
representative species of aquatic animals, toxicity to freshwater algae or
vascular plants, and bioaccumulation by aquatic organisms (if a maximum
permissible residue concentration in tissue is available).  Other pertinent
data that may be available are also taken into consideration.

     Data on acute toxicity to animals consist of 96-hour LC50 (concentration
causing mortality in 50% of tested animals) or EC50 (concentration causing
immobility or other appropriately defined effect) values.  These LC50 or EC50
values are generated by continuous exposure in laboratory toxicity tests on
fishes and macroinvertebrates.

     Data on chronic toxicity to aquatic animals consist of results of
continuous exposure laboratory tests on fish and macroinvertebrates.  These
tests may use any of several endpoints to measure unacceptable effects, such
as survival, growth, reproduction, or histological or other effect.  Data on
                                     265

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                 266

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bioaccumulation are used for chemicals where residues in aquatic organisms
may be harmful to the animal; or residue concentrations may adversely affect
wildlife, including fishes and birds, that consume these organisms; or resi-
due concentrations may adversely impact human health or marketability due to
flavor impairment.

      For derivation  of national water quality  criteria  (U.S.  EPA 1985),  the
minimum  set  of data  required for acute  toxicity to aquatic  animals is  results
of acceptable  acute  tests  with at  least one  species  of  freshwater animals  in
at least eight different families  such  that  all of the  following are  included.

      a.   the family  Salmonidae in  the class  Osteichthyes

      b.   a second family in the class Osteichthyes,  preferably a commer-
          cially or recreationally  important  warmwater species (e.g., blue-
          gill, Lepomis macrochirus:  channel  catfish,  Ictalurus punctatus.
          etc.)

      c.   a third family  in the phylum Chordata (may  be  in the class
          Osteichthyes or may be an amphibian,  etc.)

      d.   a planktonic crustacean  (e.g., cladoceran,  copepod,  etc.)

      e.   a benthic crustacean (e.g., ostracod, isopod,  amphipod, crayfish,
          etc.)

      f.   an  insect (e.g.,  mayfly,  dragonfly, damselfly, stonefly, caddisfly,
          mosquito, midge,  etc.)

      g.   a family in a phylum other than Arthropoda  or  Chordata (e.g.
          Rotifera, Annelida, Mollusca,  etc.)

      h.   a family in any order of insect or any phylum not already
          represented.

       The minimum set of data for chronic toxicity to animals is information
 from tests with species of aquatic animals for which acute toxicity data are
 available, and the tested species must be in at least three different famili-
 es.  Of these three species, at least one must be a  fish, at least one must be
 an invertebrate, and at least one must be an acutely sensitive freshwater
 species (the other two may be saltwater species).

      Results are required from at least one acceptable test with a freshwater
 alga or vascular plant.   Furthermore, if plants are  among the aquatic organ-
 isms that are most sensitive to the pollutant, results of a test with a plant
 in another phylum (division) also should be available.   Finally, at least one
 acceptable bioconcentration factor determined with an appropriate freshwater
 species is required, if a maximum permissible tissue concentration is avail-
 able.

      These data requirements were developed based on the assumption that data
 from toxicity tests with representative species provide a useful indication of
                                      267

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 the sensitivities of appropriate untested species.  It also must be recognized
 that there is a great deal of variability in toxicity testing.  Species tested
 are often selected on the basis of availability alone; tests are conducted in
 laboratory waters that are free of pollutants and typically low in particulate
 matter and organic matter.  Duration of tests can vary considerably   Also
 tests on single species are used to represent biologically complex systems;
 i.e., to reflect the response of ecosystems to pollutants.  Furthermore, these
 data requirements are quite expensive and time-consuming to meet.   Therefore
 it is worthwhile to compare the toxicity of given chemicals to a range of
 aquatic species.  This would help to determine whether a single species might
 be used as a surrogate for others, and to determine what minimum amount of
 toxicity data could adequately, reflect the susceptibilities of the aquatic
 animals that we are trying to protect.

      Only one experimental study,  by Thurston and coworkers (1985),  has been
 conducted to determine,  under standardized test conditions and procedures,  the
 toxicity of a selected set of chemicals to a selected group of aquatic
 animals.   The chemicals  tested were selected based on their exhibiting (from
 literature values)  a wide range of toxicity (from 26 grams/liter to  1  micro-
 gram per liter)  to  the fathead minnow,  Pimephales promelas.  and also on their
 having different physiological mechanisms of toxic action.   The animals tested
 were selected based on their frequent use for laboratory toxicity  tests  their
 ease of culture  in  the laboratory,  and  at the same time  to provide a reason-
 able diversity of species.   Six fishes,  two  crustaceans,  a chironomid,  and an
 amphibian were selected.   Acute toxicity tests  were then conducted for ten
 chemicals on ten aquatic  species (Table 1).

      Two  general conclusions  were  drawn from this  study.   First, there was no
 consistent relative  susceptibility,  or  orders of  sensitivity,  among  the test
 species for this group of chemicals.  Second, if  one averaged  the  individual
 LC50 values  to obtain a mean  toxicity,  positive deviations  from the  mean tox-
 icity  tended to  be much greater  than negative deviations.   This suggests that
 the  observed variation in toxicity between any  two  species  for  any one  chemi-
 cal  is likely attributable to  one of the  species being relatively  insensitive
 to the action of the  chemical  rather than one of the species being much more
 sensitive  than the mean sensitivity  for all  other species.

     The  study also found a relationship between the toxicity data for  rain-
bow  trout  (Salmo gairdneri) and  fathead minnows (P.  promelas'), whereby
 toxicity data for either organism could be relied on to classify properly ary
of the chemicals tested.  In addition, equations were developed to estimate
the  lethal concentrations of chemicals with each species from the toxicity
data for fathead minnows.

     Finally,  the largest existing compilation of aquatic toxicity data  in
terms of both chemicals and species, is EPA's AQUIRE (Aquatic Information
Retrieval) toxicity data base (Russo and Pilli 1984), which is a computerized
compilation of literature toxicity data from acute and chronic tests  for
freshwater and marine organisms.  AQUIRE contains information from 100  000
toxicity tests for 5000 chemicals on 2300 aquatic species.  Data included in
                                    268

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this data base are rated for quality, based on the test procedures used.
Access to AQUIRE is available through the Scientific Outreach Program, EPA
Environmental Research Laboratory, 6201 Congdon Boulevard, Duluth, MN  55804.


TABLE 1.  COMPARATIVE TOXICITY OF TEN ORGANIC CHEMICALS TO TEN COMMON
          AQUATIC  SPECIES  (Thurston  et al. 1985)
                  Chemical
      Species
          2-(2-Ethoxyethoxy)-ethanol

          2-Methyl-2,4-pentanediol

          2-Methyl-l-propanol

          2,2,2-Trichloroethanol

          2,4-Pentanedione

          2-Choroethanol

          Hexachloroethane

          Pentachlorophenol

          Permethrin

          Endrin
Daphnia magna

Tanytarsus dissimilis

Orconectes immunis

Rana catesbiana

Salmo gairdneri

Lepomis macrochirus

Gambusia affinis

Ictalurus punctatus

Carassius auratus

Pimephales promelas
                                  REFERENCES
Russo, R.C. and A. Pilli.  1984.  AQUIRE:  Aquatic Information Retrieval
     Toxicity Data Base.  EPA-600/8-84-021.  U.S. Environmental Protection
     Agency, Duluth, MN.

Thurston, R.V., T.A. Gllfoil, E.L. Meyn, R.K. Zajdel, T.I. Aoki, and G.D.
     Veith.  1985.  Comparative toxicity of ten organic chemicals to ten
     common aquatic species.  Water Research 19(9):1145-1155.

U.S. EPA  (Environmental Protection Agency).  1985.  Guidelines for deriving
     numerical national water quality criteria for the protection of aquatic
     organisms and their uses.  National Technical Information Service
     Accession Number PB85-227049.  U.S. Environmental Protection Agency,
     Washington, D.C.
                                     269

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                        THE FISH EMBRYO-LARVAL PROCEDURE!
                           PREDICTING CHRONIC TOXICITY
                             AND ECOLOGICAL EFFECTS

                     by Jeffrey A.  Black and Wesley J.  Birge


                                  INTRODUCTION

      Numerous studies relating to fish toxicology generally have indicated
 that piscine reproduction is one of the most sensitive target sites of aquatic
 contaminants (Birge et al. 1974).   Reproductive processes can be severely
 impaired by toxicants at concentrations that apparently have no overt effects
 on adult organisms.  As a result,  test procedures for establishing water
 quality criteria have included determinations of chemical concentrations that
 produce chronic-level effects on aquatic life.

      Methodologies historically used for such purposes have involved periods
 of exposure incorporating all or most life-cycle stages (Mount and Stephan
 1967, Pickering 1974,  McKim et al.  1976).   Such tests generally require 8
 months to 2 years for completion,  are labor-intensive,  and have impeded the
 generation of chronic data.   To obviate such problems,  more economical early-
 life-stage procedures  were developed (U.S.  EPA 1978).   These tests span 30 to
 90 days and chronic-effect endpoints appear to  be in reasonable agreement with
 those observed in the  longer life-cycle and partial-life-cycle tests  (McKim
 **/// 

      Because of the thousands  of chemical substances requiring toxicological
 evaluations,  however,  the need persisted for  still shorter and more economical
 tests for reliably estimating  chronic  toxicity  to fish  species.  Two  such
 "short-term"  methodologies have been developed  for these purposes.  The  first
 involves  a 7-day test  performed with larvae of  the fathead minnow  (Pimephales
 promelas),  in which organisms  are fed  throughout  the exposure  period.  Testing
 is  initiated with 1-day-old  larvae,  and endpoints  are quantified on the basis
 of  survival frequencies and  growth differentials between control and
 experimental  animals.  Specific procedures for  the applications of this  test
 have  been described elsewhere  (Horning and Weber  1985, Mount et al. 1985
 Norberg and Mount 1985).                                                 '

     A second procedure,  the short-term embryo-larval test, may be performed
with  a variety of fish and amphibian species.  This  test is initiated shortly
 after egg  fertilization and continues  through 4 days posthatching.   The
     1.  Graduate Center for Toxicology and School of Biological Sciences
University of Kentucky, Lexington, KY, USA.
                                    270

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exposure period is 5 to 8 days (25C) and typically is conducted with species
such as the largemouth bass (Micropterus salmoides), bluegill sunfish (Lepomis
macrochirus), and the fathead minnow.  No feeding is required.  Test responses
include embryonic mortality, egg hatchability, larval mortality, and
teratogenesis.

     Log problt analysis (Finney 1971) can be used to calculate median lethal
(LC50) and toxicant threshold (LCI) concentrations.  These values are
generally calculated by combining frequencies of dead organisms and grossly
teratic larvae observed at the end of the test.  Conventional statistical
analyses such as Dunnett's multiple comparison procedure (Horning and Weber
1985) and the chi-square Fisher's exact test (Armitage 1971) can be used to
calculate no-observed-effect concentrations (NOEC).  Rainbow trout (Salmo
gairdneri) also have been used in this system, in which the total exposure
period is about 28 days (13C).

     These'latter two tests can be performed using either static-renewal or
continuous-flow procedures and have produced chronic values similar to those
determined in traditional life-cycle experiments (Birge et al. 1981,  Birge and
Cassidy 1983,  Birge et al. 1985,  Norberg and Mount 1985).

     This paper reviews the reliability and usefulness of the short-term fish
embryo-larval test for estimating chronic-level effects of contaminant stress
on aquatic organisms.  Primary attention given to specific applications of the
procedure, including assessments of (1) the toxicity of single compounds and
chemical mixtures, (2) differential species sensitivity,  (3) teratogencity as
a toxicological endpoint, (4)  structure-toxicity relationships, (5) effluent
and receiving water toxicity,  and (6) effects produced by sediment-associated
chemicals.

                      GENERAL ASPECTS  OF THE  SHORT-TERM
                            EMBRYO-LARVAL PROCEDURE

     Procedures for performing the short-term embryo-larval toxicity test were
reviewed briefly in the above section and have been further detailed in
previous publications (Black et al. 1983,  Birge et al. 1985, Horning and Weber
1985).  An overview of test characteristics is given in Table 1.  Selection of
the particular exposure system to be used (static-renewal,  flow-through) is
dependent on several considerations.  The static-renewal test, in which
solutions are changed at regular intervals (e.g., 12 or 24 hours),  is
economical and can be used with toxicants that are relatively stable in
solution.  Flow-through procedures are recommended for testing rapidly
degradable materials.  One such system developed in our laboratory has been
used successfully in regulating exposure concentrations of volatile or
insoluble organic compounds (Birge et al.  1979a, Black et al. 1982).   We
presently are developing new toxicity test procedures that will require
solution volumes of only 50 ml or less.

     Biological endpoints subject to evaluation in embryo-larval tests are
summarized in Table 2.  Endpoints not discussed in this paper (e.g.,
avoidance/attraction behavior, tumor formation) have been described elsewhere
(Black and Birge 1980, Hendricks et al. 1984).  Growth, not typically used as
an embryo-larval test endpoint,  is currently being evaluated for reliability

                                     271

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Table 1. GENERAL CHARACTERISTICS OF THE SHORT-TERM EMBRYO- LARVAL TEST
 1.  Five or more exposure concentrations plus control
 2.  Two or four replicates/exposure
 3.  20-50 organisms per 50-400 mL chamber
 4.  Dilution water: natural or reconstituted
 5.  Exposure through hatching plus 4 days posthatching
 6.  Static-renewal or flow-through system
 7.  No feeding requirement
 8.  Laboratory or field applications
 9.  Multiple animal species capable of being tested in one chamber
10.  Multiple test endpoints
11.  Regression analysis (LC50, LCI)
12.  Analysis of variance, Dunnett's test, Duncan's test (NOEC, LOEC)
 and reproducibility.   The  short-term embryo-larval procedure has a number  of
 applications  and includes  among  others  the evaluation of  single toxicants,
 effluents  and other  complex mixtures, and sediment-associated chemicals.
 Several types of environmental media evaluated using early-life stages  are
 given in Table 3.  The following sections .summarize various aspects  of  the
 test,  including the  reliability  and applicability of the  data generated.

                       REPRODUCIBILITY AND RELIABILITY OF
                            EMBRYO-LARVAL PROCEDURES

      The reproducibility of the  short-term embryo-larval  test has been
 investigated  using various compounds, and an  example of the precision of test
 endpoints  is  presented in  Table  4 (Birge et al.  1985).  Six continuous  flow
 tests (two replicates each) were performed with  cadmium on the fathead  minnow
 over an 8-month period.  The  LC50 values varied  only from 0.067 to 0.084 mg/L
 and LCI values were  between 0.010 and 0.014 mg/L.  The 95% confidence limits
 were relatively small and  probit-derived LCls (threshold  toxicity values)  were
 in good agreement with NOECs  determined by independent statistical procedures.
Table 2.  EMBRYO-LARVAL TEST ENDPOINTS
Acute toxicity - LC50
Chronic Toxicity - Estimated chronic values or toxicity threshold values (LCI)
Teratogenesis - defective or retarded development (embryos, larvae)
Growth - length, weight
Behavior - avoidance/attraction responses, locomotor impairment, feeding
Bioconcentration - toxicant uptake
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Table  3.  ENVIRONMENTAL MEDIA SUBJECT TO EMBRYO-LARVAL TOXICOLOGICAL
          EVALUATIONS
 Single  chemicals/substances  (soluble,  insoluble, volatile)
 Chemical  combinations  -  toxic  interactions
 Complex mixtures
     Whole  effluents/dilutions
     Effluent  fractions
     Chemical  leachates
     Persistence  of toxicity
 Receiving systems
 Sediment-associated chemicals
     Natural bulk sediments
     Chemically enriched sediments
     Sediment  elutriates
Similar precision was observed in static-renewal tests,  using the same
compound and fish species.

     The short-term procedure also has been used to perform multiple species
tests, in which embryos of different species have been maintained in the same
exposure chamber.  In one such experiment, eggs of the carp (Gyprinus carpio),
the fathead minnow, and the largemouth bass were exposed to cadmium, and the
LC50 values were 138.9, 107.0, and 244.1 jig/L, respectively.  The
corresponding LCls were 4.1, 11.5, and 13.9 /zg/L.  To achieve a broader
toxicity base on cadmium with warmwater species, additional experiments were
performed on the channel catfish  (Ictalurus punctatus) and goldfish (Garassius
auratus), and the LCI values (Mg/L) obtained with these species were 8.6 and
3.0, respectively.

     The range of LCI values calculated for these 5 species was 3.0 to 13.9 pg
Cd/L.  This range compared favorably to chronic values of 5.8 to 15.0 /ig/L
achieved in longer-term chronic tests reported in the EPA criterion document
for six warmwater fish species (U.S. EPA 1980).  Although data for precise
species to species comparisons were not always available, these results lend
support to the premise that the toxicity threshold values (LCls) obtained in
short-term embryo.-larval tests can be used reliably to estimate chronic
toxicity.

      Further evidence  of this nature has been shown in tests with the rainbow
trout.  Embryo-larval  stages exposed in static-renewal tests to seven
different metals  gave  LCI values  that compared favorably to maximum acceptable
toxicant concentrations  (MATC) calculated from chronic tests with rainbow
trout and other  salmonid species  (Table 5, Birge et al. 1985).

                     TOXICOLOGICAL  SCREENING  OF CHEMICALS

      On the basis of data indicating that the embryo-larval test can be used
for predicting long-term effects, this procedure may  afford a practical means

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 by which to screen a great number of contaminants for toxic properties,
 identify those of greatest concern to aquatic ecosystems, and estimate
 concentrations that produce chronic toxicity.  As an example, the short-term
 procedure with the rainbow trout was tested.on 33 elements found in fossil
 fuels (Birge et al. 1980).  In this study, the LC50 values-were less than 1
 mg/L for 19 of 33 elements and LCI values were 10 //g/L or less for 12.
 Results indicated the high sensitivity of trout developmental stages and the
 potential application of these procedures for use in testing programs designed
 to prioritize compounds for further toxicological evaluations.

      The test also has been used to establish a toxicological response range
 for a number of different aquatic organisms.   The heterogeneity of species
 response is important in understanding the degree to which aquatic biota vary
 in their sensitivity to environmental contaminants.   The toxicity data on
 cadmium given above demonstrate the differential susceptibility among certain
 warmwater fish species.   Another example of this concept was reflected by
 comparative tests in which inorganic mercury was administered to embryo-larval
 stages of 14 amphibian species (Birge et al.  1979b).   The LC50  values spanned
 nearly 2 orders of magnitude,  ranging from 1.3 /ig/L with the narrow-mouth toad
 (Gastrophryne carolinensis")  to 107.5 fig/L with the marbled salamander
 (Ambvstoma opacum).
Table  4.  REPRODUCIBILITY OF 8-d EMBRYO-LARVAL TESTS  WITH CADMIUM USING THE
          FATHEAD MINNOW IN A CONTINUOUS-FLOW SYSTEM3
Test
no.
1
2
3
4
5
6
LC50
(mg/L)
0.067
0.084
0.073
0.079
0.084
0.070
95% confidence
limits
0.056-0.078
0.073-0.097
0.062-0.085
0.068-0.091
0.072-0.098
0.060-0.081
LCI
(mg/L)
0.010
0.013
0.012
0.012
0.014
0.010
95% confidence
limits
0.006-0.014
0.008-0.017
0.007-0.017
0.008-0.017
0.009-0.020
0.006-0.015
NOEC
(mg/L)
0.012
0.011
0.013
0.011
0.010
0.014
"Reprinted with permission from Environmental Toxicoloty and Chemistry,  vol.
4, W.J. Birge, J.A. Black, and A.G. Westeman, Short-term fish and amphibian
embryo-larval tests for determining the effects of toxicant stress on early
life stages and estimating chronic values for single compounds and complex
effluents, 1985, Pergamon Press.
                                    274

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                                  TERATOGENICITY

       Use of the short-term embryo-larval test provides the opportunity to
  quantify frequencies of teratogenesis present in hatched populations   For
  most toxicants,  the number of teratic larvae generally has been found to
  increase with exposure level,  but the dose-response relationship usually is
  not as  precise as that observed for mortality.   In most experiments, the
  incidence of teratogenesis has ranged from 0 to 5% at or near the toxicity
  threshold and frequencies  of 10% or greater usually have not been observed
  except  at or above median  lethal concentrations.   There are exceptions to this
  trend for certain metals and organic contaminants,  and these occurrences have
  been discussed previously  (Birge and Black 1977,  Birge et al  1983)    Gross
  anomalies most often encountered have included  defects of the head and
 Table 5.
                                                         METALS
              LClb
 Element
 Cadmium
8.0
         95% confidence    MATC
             limits
                                                       Species
                                                        Test0
5.4-10.9
1.7-3.4
3.8-11.7
Brook trout
Brown trout
clc
els
Chromium
Copper


Lead


Mercury
Silver
Zinc
21.5
3.4


10.3


0.2
0.1
216
10.3-35.2
1.6-5.9


6.9-14.6


0.1-0.3
0.1-0.2
157-275
51-105
3.0-5.0
5.0-8.0
9.4-17.4
4.1-7.6
7.2-14.6
71-146
0.29-0.93
0.09-0.17
532-1368
Rainbow trout
Brook trout
Brook trout
Brook trout
Rainbow trout
Rainbow trout
Rainbow trout
Brook trout
Rainbow trout
Brook trout
els
els
els
clc
pic
pic
els
clc
pic
pic
aLCl values determined in 28-d static-renewal tests with rainbow trout
 terminated 4 d after hatching.
bMATCs taken from published data for 60- to 90-d early-life-stage (els)
partial life-cycle (pic) or complete life-cycle (clc) tests
 Reprinted with permission from Environmental Toxicolnt-y and Chenngt-r-y  Vol
embr'/^'/^' J1*^' and A'G- Westeman, Short-term fish and amphibian
embryo-larval tests for determining the effects of toxicant stress on early
eff?u!^SeS,^ Timatlng chronlc values for single compounds and complex
ettluents, 1985, Pergamon Press.
                                   275

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vertebral column, dwarfed bodies, partial twinning, microcephaly, absent or
reduced eyes and fins, and amphiarthrodic jaws.  Approximately 80% to 90% of
the gross anomalies observed have 'been defects of the skeletal system.  Most
have involved the vertebral column and included lordosis, scoliosis, kyphosis,
and rigid coiling.

     Based on these observations, it was proposed that fish and amphibian
embryo-larval stages may constitute simple and effective models with which to
investigate teratogenesis and that such test systems possess high potential
for (1) determining the mechanisms of teratogenesis, (2) evaluating the impact
of environmental toxicants on aquatic biota, and (3) preliminary screening and
identification of environmental teratogens that may be of concern to human
health (Birge et al. 1983).

                       STRUCTURE-ACTIVITY RELATIONSHIPS

     Because aquatic ecosystems will continuously be affected by a broad
spectrum of pollutants, environmental hazard assessments must rely on an
orderly and systematic process for screening toxicity of diversified
contaminants.  The vast number of chemicals introduced to the environment
places constraints on the extent of toxicity testing that can be accomplished
within a practical time frame.  For this reason, considerable attention has
been given to developing procedures to allow for the estimation of toxicity
based on certain structural characteristics of such compounds (Konemann 1981,
McCarty et al. 1985, Hodson et al. 1988).  Given an adequate toxicological
baseline for a representative number of organic chemicals or chemical classes,
the toxicity of many untested compounds could potentially be predicted by
examination of their structures or other physical-chemical characteristics.

     The usefulness of the short-term embryo-larval procedure for predicting
toxic effects of structurally related chemicals has been evaluated for certain
classes of organic compounds  (Birge and Cassidy 1983, Black et al. 1983,
Millemann et al. 1984).  In embryo-larval studies  testing compounds within
each of three chemical classes  (i.e., hydroxylated aromatic hydrocarbons,
azaarenes, polycyclic aromatic hydrocarbons),  toxicity to both the largemouth
bass and rainbow trout was found to increase with  increasing ring number
 (Black et al. 1983).  Similarly,  toxicity of azaarene compounds  to the leopard
frog  (Rana pipens)  increased with ring number  and  increased octanol/water
partition coefficients  (Birge and Cassidy 1983).

     Fish and amphibian  short-term tests also  were used  to assess the
relationship between toxicity and degree of chlorination of certain
polychlorinated biphenyl compounds and chlorinated alkanes.  Toxicity was
 observed to be  greater  as  the chlorination  of  these compounds  increased.   For
 example, LC50 values  with the leopard frog  were  48,  4.16, and  1.64  mg/L  for
methylene chloride  (2 chlorine  atoms), chloroform  (3 chlorine  atoms), and
 carbon tetrachloride (4  chlorine atoms), respectively.   Corresponding octanol
water  partition coefficients  (log P)  were  1.51,  2.02,  and 2.79  (Birge and
 Cassidy 1983).   Based on these  and other data,  the sensitive  and economical
 short-term embryo-larval procedure may be  extremely advantageous for use in
 broad-based testing programs  designed to establish structure-toxicity
 relationships  for a wide variety of  compounds.
                                      276

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         EMBRYO-LARVAL BIOMONITORING OF  EFFLUENTS AND RECEIVING WATERS

     The above descriptions of the short-term embryo-larval test primarily
have been concerned with assessments of single compounds.  This procedure,
however, has been used successfully in a number of field investigations
involving direct toxicological monitoring of effluents and receiving waters.
In biomonitoring studies on effluents from a tannery-sewage treatment plant,  a
synthetic rubber plant, a metal plating plant, and a chemical manufacturing
plant, both acute and embryo-larval tests using fathead minnows were performed
concurrently on each of these NPDES (National Pollutant Discharge Elimination
System) discharges (Birge and Black 1981).

     The embryo-larval tests gave greater detection of effluent toxicity.  The
LC50 and LCI (percent effluent by volume) values for the four effluents, as
determined in flow-through tests, ranged from 0.3% to  29.4% and 0.001% to
2.8%, respectively.  In the acute tests, it was not possible to calculate LC50
values for three of the four effluents.   The acute LC50 for the most toxic
effluent was 8%, a value about 27 times greater than the embryo-larval LC50
(0.3%) and different from the embryo-larval LCI (0.001%) by more than three
orders of magnitude.

      In  three of the  on-site studies, tests also were conducted to determine
the utility of embryo-larval biomonitoring for identifying toxic effluent
fractions and for evaluating the effectiveness of waste treatment processes.
Results  indicated that more accurate evaluations could be achieved with
embryo-larval data that reflect the "net toxicity" of complex mixtures than
could be predicted with chemical analyses of  the effluents or effluent
fractions.

     In  another major field investigation, chemical monitoring, ecological
surveys, hydrological measurements, and fathead minnow embryo-larval tests
were conducted on an effluent from a secondary sewage treatment plant (STP)
and the  affected'receiving stream (Birge et al. in press).  A good correlation
existed between embryo-larval toxicity and the degree of impact determined
using  faunistic survey data evaluated at the  different sampling stations.  In
addition, an independent flow-through toxicity test performed on the STP
effluent and 4 effluent dilutions gave an effluent LCI value of 30.6% of
volume.  The LCI compared closely with the instream effluent concentration
(33%) observed for the last downstream monitoring station, which was the only
station  at which ecological survey data revealed no significant impact on fish
and macroinvertebrate populations.

        EVALUATION OF EFFECTS  PRODUCED BY SEDIMENT-ASSOCIATED CHEMICALS

     It  is evident that short-term embryo-larval tests have broad uses for
evaluating the toxicity of waterborne contaminants.  More recently, this
procedure has been adapted for determining chronic effects of sediment
contamination.  The importance of sediment toxicity and the development of
criteria for sediment-associated chemicals are receiving considerable
attention by the U.S. EPA and other interested parties.  An in-depth treatment
of this  subject has been presented in a recent volume edited by Dickson et al.
(1987).
                                     277

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     A number of different types of sediments, both naturally contaminated and
chemically enriched  (spiked), have been evaluated using fish and amphibian
embryo-larval stages.  Test procedures and findings from these studies have
been presented in earlier publications (Francis et al. 1984, Birge et al.
1987) .  In one such  investigation, experiments were performed to determine the
effects of mercury-enriched sediment on trout eggs.  Samples of sandy loam
sediments were taken from a local stream with biotic conditions characteristic
of a healthy aquatic ecosystem and were enriched with inorganic mercury at
concentrations ranging from 0.1 to 100 Mg/g.  Sediments were layered to a
depth of 2 cm in 500 mL-Pyrex test chambers and covered with 400 mL of
mercury-free reconstituted water.  Eyed eggs of the rainbow trout were placed
on the sediment layer and exposed from 10 days before hatching through 10 days
after hatching.

     A distinct inverse correlation existed between sediment mercury
concentrations and percent survival (Table 6).  Trout embryos and alevins
accumulated significant quantities of mercury during the 20-day exposure, and
tissue concentrations rose proportionally with increasing sediment levels of
mercury.  It is likely that contaminated sediments, when used as spawning
substrates, could present a formidable hazard to fish reproduction.

     With respect to these results and those reported by others (Chu-fa et al.
1979, Swartz et al.  1982, Malueg et al. 1984, and Nebeker et al. 1984), there
is a considerable evidence that toxicological biomonitoring is a practical and
reliable procedure by which to evaluate sediment contamination.  Taking into
account results based on early-life-stage toxicity tests, it is apparent that
bulk sediment chemistry is important for evaluating the hazard of sediment-
associated chemicals and that such tests should significantly augment the
development of regulatory criteria.

                                    SUMMARY

     The embryo-.larval toxicity tests described above have important
implications in the  assessment of chronic toxicity produced by environmental
contaminants (Table  7).  This procedure may be performed both in the
laboratory and in the field and can be used for development of aquatic and
sediment criteria, the establishment of structure-toxicity relationships, and
effluent and receiving water biomonitoring.  Test endpoints observed in field
studies have been found to correlate closely with independent ecological
parameters used to quantify effects of point-source discharges on receiving
water systems.  In addition, tests conducted with an effluent dilution series
are useful in estimating the likelihood or degree of instream impact (Birge et
al. in press).  Not only is the test routinely employed for assessing chronic-
level effects under the NPDES biomonitoring program,  but it is also  applicable
for evaluating toxicity associated with hazardous waste sites in which liquid
discharges, leachates, or surface water contamination are involved.   Moreover,
this methodology may prove to be an economical and predictive means  by which
to prescreen chemicals for potential effects on human health.
                                     278

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279

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 Table 7.  APPLICATIONS OF EMBRYO-LARVAL TOXICOLOGICAL EVALUATIONS
 Chemical criteria development
 Ranking toxicity and animal sensitivity
    Animal test species (species response range)
    Chemicals (toxic effects range)
 Structure-toxicity relationships
 Field biomonitoring (effluents, sediments,  hazardous  waste sites)
 Predicting ecological impact (receiving streams,  sediments)
 Surrogate testing for prescreening  health effects (teratogenesis)
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                                   283

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                    TOXICITY OF FENVALERATE TO SIX SPECIES
                 OF FISH AND  TWO  SPECIES  OF FISHFOOD  ORGANISMS

                                      by

                  Ding  Shurong1,  Zhou Fengfen, and Zhang Min
      Fenvalerate is one of the pyrethroid insecticides with broad spectrum
and high activity.  It is highly toxic to fish and crustaceans when it enters
rivers and aquaculture ponds.   Some information is needed about its toxicity
to aquatic animals,  especially fish that have economic value and the organisms
on which they feed.   In the past decade, some researchers studied the toxicity
of fenvalerate to many species of aquatic animals (Mulla et al. 1978, Coats
and O'Donnell-Jeffery 1979, Mcleese et al. 1980, Anderson 1982, Clark et al.
1985, Mckee and Knowles 1986,  Bradbury et al. 1987).  Few common fish with
economic value in China and fishfood organisms were included in these studies.
Only a few subacute toxicity studies of the toxicity of fenvalerate to aquatic
animals have been done.  The present study evaluates both the acute toxicity
of fenvalerate to six species of fish with economic value in China and two
species of fishfood organisms (Daphnia and algae) and the subacute toxicity on
the aspects of behavioral toxicity (avoidance reaction),  genetic toxicity and
accumulation toxicity of fenvalerate to fish.  Acute toxicity of fenvalerate
to goldfish (Carassius auratus) and silver carp (Hypophthalmichths molitrix)
was evaluated to provide some ecotoxicological data for the safety assessment
of the pesticide.
                             MATERIALS AND METHODS
CHEMICALS
      Fenvalerate (Technical, 90%) was supplied by the Research Institute of
Hormone, Jintan, Jiangsu Province.  It was dissolved in ethanol, heated
slightly and then diluted to various concentrations for testing.
     ^Department of Environmental Sciences,  Nanjing University,  Nanjing,  PRC.
                                     284

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STATIC ACUTE TOXICITY TEST

Test animals

      FIngerlings of silver carp (Hypophthalmichths molitrix), average length
3.10  0.15 cm, average weight 0.49  0.15 g.

      Fry of silver carp, obtained from the Aqualculture Farm of Nanjing City,
10 to 15 days old.

      Fingerlings of the common carp (Gyprinus carpio), average length 3.78 
0.50 cm, average weight 1.06  0.09 g.

      Fry of the common carp, 20 days old.  The common carp were obtained from
the Aquaculture Farm of Nanjing.

      Finless eels (Monopterus albus). average length 24.4  0.9 cm, average
weight 13.9 g, were obtained from the market.

      Goldfish CCarassius auratus) , 20 to 30 days after hatching, obtained
from the market, with average length of 1.0 to 1.5 cm and average weight of
0.3 to 0.5 g.

      Grass carp (Ctenopharyngodon idellus). average length 3.96 cm, average
weight 1.1 g, obtained from the Aquaculture Farm of Nanjing.

      The loach (Misgurnus anguillicaudatus). average length 11.9 cm, average
weight 13.6 g, obtained from the market.

      All the fish were laboratory-acclimated for 7 to 10 days before testing.
They were not fed 24 hours preceding or during the 96-hour exposures.

      Pregnant daphnia (Daphnia carinata) were cultured in 1000-ml beakers 12
hours before testing.  Each beaker contained 30 animals.  After culturing for
12 hours,  the animals were filtered and screened with webs to obtain the
newborn daphnia with instar of 12 hours.

      Algae (Scenedesmus obliquus and Chlorella pyrenoidosa1) were cultured
with Bold Basal solution (Stein 1973) and inoculated to 1000-ml flasks
containing culture media under the condition of 24 to 26C, 2000 lux.  The
cultures were transferred at each 120-hour interval.  After three transfers,
the algae culture reached a stage of synchronus growth and they were ready for
testing.

      Water quality for fish test:  pH 6.0 to 6.5, conductivity 220 to 310 x
102 ^mho/cm,  dissolved oxygen 7 mg I"1, COD^ 1.03  to 1.19  mg I"1, chloride 7.0
to 8.5 mg I"1, hardness 5.38  to  6.05  (Deutsche degree).

      Water quality for daphnia test:  Tap water filtered by activated
charcoal and aerated with micropumps, dissolved oxygen >7.6 mg I"1,
conductivity 1.25 To 1.60 X 102 ^mho/cm.
                                     285

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      Bioassay procedures:  Static exposure tests using fingerling were run
using 40-L aquariums, each containing 10 randomly assigned fish.  A series of
five exposure concentrations and a control were used.  Mortality counts were
recorded once every day during the 96-hour exposure.  LC50 values were
calculated (APHA, AWWA, WPCF, 1980).  The static exposure tests using fry were
run using 1000-ml and 5000-ml beakers.

      A series of five exposure concentrations and a control were used for
static acute toxicity tests for daphnia.  Ten parallel groups were set up for
each concentration.  Each ten animals in one 150-ml beaker containing 100 ml
of test solution were tested under the condition of 21  1C and 12 to 14-hour
light periods with the intensity of 4500 to 5000 lux.  Mortality counts were
recorded once every day during the 48-hour exposures.  The 48 hour-LC50s were
calculated according to the data obtained (APHA, AWWA, WPCF, 1980).

      To perform the static acute toxicity test for algae, the prepared test
solution of fenvalerate was added to each 70-ml test tube containing fresh
culture media for algae to make up test solutions having different
concentrations, i.e., 1000 ppm, 100 ppm, 10 ppm, 1 ppm, 100 ppb, 10 ppb, and 1
ppb (6 tubes for each concentration).  An ethanol control and a blank control
were used.

      After making cell suspensions of the algae, with cell cultures attaining
the stage of synchronous growth, the cell densities were counted.  The cell
densities of the cultures of chlorella pyrenoidosa and Scenedesmus obliquus
were 5.31 x 107 cells/ml and 7.15 x 106  cell ml,  respectively.   Cell
suspensions of 1.13 ml and 2.09 ml were inoculated to 27 tubes containing
culture medium with 3 tubes for each concentration.  The initial
concentrations of chlorella pyrenoidosa and Scenedesmus obliquus were 2 x 106
cells/ml and 5 x 10s cells/ml,  respectively.

      Culture conditions:  25 to 29C, 2500 lux, light/dark = 14:10,  shaking
speed 110 rpm.  The growth amounts during the culturing periods of 24, 48, 72,
96 and 120 hours were determined using a cell counting method.
ACCUMULATION TOXICITY TEST

      Accumulation toxicity tests using loach (average length 11 to 13 cm,
average weight 10 to 12 g) were run using 401 aquariums each containing 20
randomly assigned fish, divided into treatment groups and control.  The tests
were done by exposure with toxicant dose increasing progressively.  The amount
of toxicant in each step was 1 to 1.5 times the amount of the preceding
period.  The increase of doses continued until half of each group of fish were
dead.  The accumulation coefficients, K, were calculated by the WHO formula
(WHO 1978)
                              K
LD50(n)

LD50(1)
                                     286

-------
      where:  LD50(n) = accumulative total dose for 50% dead animals
                        exposed repeatedly   ,       .  . .   .

              LD50(1) = dose for 50% dead animals exposed once
TEST FOR INDUCTION OF MICRONUCLEI IN THE PERIPHERAL BLOOD ERYTHROCYTE OF THE
LOACH

      The loaches (average length 11.9 cm, average weight 13.6 g) were
laboratory acclimated for 7 to 10 days before testing.  Feeding ceased before
and during the exposure.  The loaches were exposed both by contact with
fenvalerate solution and by intraperitoneal injection.  Three to five dose
groups were set up according to the 96-hour LC50 of the loach.  A positive
control with mitomycin and two solvent controls with ethanol and dimethyl
sulfoxide were included in the experiment.  Peripheral blood was taken with a
heparinized capillary tube after severance of the caudal peduncle.  Blood
smears were made and slides were fixed in methanol and stained with Feulgen.
The erythrocytes were examined with a microscope.


AVOIDANCE TEST

      Test animals:  silver carp (average length 3.6 Cm, average weight 0.64
g), the common carp (average length 3.1 cm, average weight 0.9 g), and grass
carp (average length 3.96 cm, average weight 1.1 g).  The fish were
laboratory-acclimated for 7 to 8 days before testing.  Feeding ceased before
exposure.  Water for dilution was aerated and dechlorinated tap water.  Four
to six treatment groups between 1/13 to 1/2 96-hour-LC50  of  the carp were
chosen and a "clean" control and solvent (ethanol) control were set up.
Avoidance reaction tests were run in straight-type avoidance equipment
provided with constant water flow.  Experiments were repeated four times for
each concentration.  Each experiment was done with ten fish, which were put
previously into the avoidance test equipment for 15 minutes to acclimate.
Each experiment lasted for 20 minutes.  The frequencies of appearance by fish
both in "clean" region and in polluted region were recorded separately each
minute.  Calculated data were judged by X2 test.
TEST FOR TOXICITY OF SOAKED-OUT LIQUID THROUGH ADSORPTION BY SOIL

      Test animals:  fry of silver carp (10 to 15 days after hatching) were
obtained from Aquaculture Farm of Nanjing City and goldfish were obtained from
the market with average length 1.0 to 1.5 cm and average weight of 0.3 to 0.5
g.  Soil from a rice field was collected from the suburbs of Nanjing City.
The air-dried soil was put into 13 beakers (5000 ml in volume) with each 400
g.  Four groups were divided according to the duration of adsorption, i.e.,
24, 48, 72 and 96 hours.  The concentrations for each group were 100 //g I"1,
500 ng I"1  and  1000 /ig  I"1.   A control group was included.  The amount of 3500
ml of fenvalerate solution with different concentrations was poured into each
beaker separately.  After stirring with a glass rod, it was allowed to stand

                                     287

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for precipitation.   The amount of 2500 ml of soaked-out liquid were drawn from
3 beakers  each at the interval of 24 hours to conduct the acute toxicity test
for goldfish.   Ten fish were included in each experimental group.   The method
of toxicity test for fry of silver carp was just the  same as that for goldfish
but with smaller containers.   The experimental temperature was 23  1C.
                             RESULTS AND DISCUSSION
 THE ACUTE TOXICITY OF FENVALERATE TO FISH,  DAPHNIA AND ALGAE


      Fenvalerate is highly toxic to silver carp, common carp,  goldfish  and
loach (Table 1) .  According to the classification on the degree of  grade of
toxicity proposed by an international group (Joint  IMCO/FAO/UNES CO/WHO Group
of Experts 1969), it is "very toxic" to these fish, but "toxic" to  finless
eel.  Mcleese et al. (1980) reported that fenvalerate was highly  toxic to
Atlantic salmon juvenile  (Salmon salar) : the 96-hour LC50  was 1.2 ng I"1  only.
Coats and O'Donnell-Jeffery (1979) found the 24-hour LC50  for fingerling of
rainbow trout (Salmo gairdneri) tested with fenvalerate was  76.0  ppb.  'The
acute toxicity  (96-hour LC50)  of fenvalerate to six estuarine fish was
determined in flow- through laboratory tests by  Clark et al.  (1985)  and 96-hour
LC50's  for cyprinodon variegatus.  Menidia menidia.  Menidia penissulae. Menidia
beryllina. Leuresther  tenuis . and Opsanus beta  were 5.0 /jg I"1, 0.3 pg I"1, 1.0
fig I"1,  1.0 pg I'1, 0.3 /*g I"1  and 2.4 /jg I"1, respectively.  The values of 96-
hour LC50's for five species  of fish with economic value exposed to
fenvalerate were coincident with these previous studies.  Therefore,  there is
seemingly a potential  menace to fishery and it  is worthwhile  to understand
much about the  toxicity of fenvalerate to aquatic animals.
               TABLE  1.  ACUTE TOXICITY OF  FENVALERATE  TO  FIVE
                         SPECIES  OF  FISH AND  DAPHNIA

Test organism

Silver carp (fingerling)
Silver carp (fry
Common carp (fingerling)
Common carp (fry)
Goldfish (fry)
Finless eel
Loach
Daphnia
Temp . ,
C
91 4- 1
jLL IE J.
O o 4-1
/.:>  i
O1 +1
on 4- i
i\J 21 L
23  1
OR 4- 1
OR 4-  1
OT -4-1
ZJ_ IE L
LC50(ppb)
24 h 48 h 72 h 96 h
	 	 } 1 S 2 35
	 QC;
	 	 38
	 70
12.10 11.55 11.40 10.2
1100 1000 81 0
	 	 169
11 /. 	

                                     288

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       As one kind of fishfood organism, Daphnia do occupy an important
 situation in the food chain and food web in aquatic ecosystems.  It is very
 sensitive to fenvalerate.  Meyer and Ellesieck (1986) found that the acute
 toxicity of fenvalerate to Daphnia magna was 2.1 /ig I"1.  During the 21-day
 test period, survival of Daphnia magna was not significantly (p = 0.05)
 affected by the 21-day exposure; however, reproduction was reduced at
 fenvalerate concentration of 0.25 and 0.5 ^g I"1  (McKee and Knowles 1986).
 The results obtained by Anderson (1982) indicated that fenvalerate can affect
 behavior and cause death for certain stream invertebrates at constant exposure
 concentration of 0.030 //g I"1.   The most  sensitive animals in fenvalerate
 exposure were the amphipods.   Within 4 to 7 days, over 50% of the gammarids at
 the low concentration of 0.022 fig I"1 were dead.  The results of the present
 study indicated that 48-hour LC50 for Daphnia carinata exposed  to fenvalerate
 was 1.14 /zg I"1.  We think that  datum showing high sensitivity  as this may be
 useful in the process of comprehensive analysis for the potential hazards of
 fenvalerate to aquatic ecosystems.

       With the toxicity test for Chlorella pvrenoidosa and Scenedesmus
 obliquus,  it was found that under low concentrations,  fenvalerate showed a
 promotive action to these two species of algae (Tables 2 and 3).   Within a
 certain range of fenvalerate concentrations,  this action increased with
 increasing concentrations.   The stimulating actions appeared at the
 concentration range of 100 ppb to 10  ppm.  It is  analogical  to  the data
 obtained by Chen and Zeng (1982) in their study on the toxicity of some
 pyrenthroids to algae.   The inhibition effects of fenvalerate appeared at the
 higher concentration.   The EC50  for Scenedesmus obliquus and Chlorella
 pyrenoidosa ranged from 100 ppm to  1000 ppm.   In general,  during degradation
 pesticides containing nitrogen and  phosphorus  can liberate plant nutrients.
 The CN"  group  in fenvalerate molecule may play  a  role in stimulation.


RESULTS  OF ACCUMULATION TEST


      According  to the assessment criteria for  accumulation (WHO 1978), when
K>5, it belongs  to "slightly accumulated."  As  the K value obtained by us in
the present study equals 6.4, which is more than  5, there is only slight
accumulation of  fenvalerate in the loach.  The  experimental method for
accumulation toxicity adopted by us is a method denoting functional
accumulation.  There is a relationship between  the intensity of functional
accumulation and the strength of metabolism as well as the degree of
functional change in fish body.  But information  about the accumulation
toxicity of fenvalerate to fish has not yet been reported.  The data would be
useful to evaluate the toxicity  of fenvalerate.


RESULTS OF MICRONUCLEUS TEST

      Results obtained are listed in Tables 5 and 6.

      Results indicated that the basal value for the rate of micronucleus in
the loach was rather low.  It basically coincides with the data obtained by
                                     289

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-------
                     TABLE 4.   ACCUMULATION OF FENVALERATE
Group

Number
of
Fish

Dose
1/20x0 . 02ml
2/20x0 . 02ml
Number
of
Fish
0
0
Accumulation
Mortality, coefficient
% (K)

Control
(ethanol) 20
Treatment
20


4/20x0 . 02ml
8/20x0 . 02ml
16/20x0. 02ml
32/20x0. 02ml
1/20 LC50
2/20 LC50
4/20 LC50
8/20 LC50
16/20 LC50
32/20 LC50
0
0
0
0
0
2
2
3
2
1
0 No accumulation

50 6.4


 Liu (1981).  After exposure, micronuclei do appear, but the difference of the
 rates of micronucleus between treatment group and control group was not
 significant.  It denotes that fenvalerate may not be a cytogenetic agent to
 fish.
 RESULTS OF AVOIDANCE REACTION TEST

       Results were expressed as the number of observations in clean water as a
 percentage of the total number of observations.  Significant avoidance was
 judged to occur when the number of counts in the clean arm of the trough was
 statically higher than in the polluted arm (X2>3.84, p<0.05)  (Table 7).
 According to the data shown in Table 7, the concentration in which silver carp
 and common carp could avoid the fenvalerate were the 1/10 to 1/2 values of 96-
 hour LC50  for common carp,  and that  for grass  carp  were  the 1/8  to  1/2 values
 of 96-hour LC50  (10.3 > X2  3.84, 0.05 > P > 0.01) .

       The capacity of silver carp, grass carp and common carp to avoid
 fenvalerate  solution related to the concentration of fenvalerate.  The
 concentration threshold of fenvalerate fell in the  range of 0.36 mg I"1  to
 0.40 mg I"1  for  silver  carp and common carp  and that for grass carp was  0.40
                                      292

-------
            TABLE 5.  INCIDENCE OF FENVALERATE TO MICRONUCLEUS OF
                       THE PERIPHERAL NUCLEATED ERYTHROCYTES IN THE
                       LOACH (By Contact Exposure)

Dose,
ppm
Number of
erythrocytes
scored

Number of
micronucleus
Rate of
micronucleus ,
%


P
0.05

0.02

0.01

0.005

0.002

Mitomycin
(1.8 mg/kg)

ethanol
(0.01 ml)

Control
50000

30000

35000

25000

25000


10000


50000

60000
1

1

1

1

1
1

1
0.02  0.02       >0.05

0.03  0.03       >0.05

0.028  0.02      >0.05

0.04  0.04       >0.05

0.04  0.04       >0.05


0.6  0.24        <0.01


0.02  0.02       >0.05

0.017
Control:  tap water

mg I"1 to  0.50  mg I"1.  The avoidance capacity of silver carp and common carp
seems stronger.  The avoidance capacity would be an important factor to
evaluate the actual hazard of fenvalerate to fish.


RESULTS FROM TEXT FOR TOXICITY OF THE SOAKED-OUT LIQUID THROUGH ADSORPTION BY
SOIL

      The acute toxicity of soaked-out liquid from soil adsorption decreased
rapidly along with the prolongation of time intervals for soil adsorption
(Figure 1, Figure 2).

      The decreasing degree was greater for those groups with higher initial
concentrations.  All the soaked-out liquid adsorbed by soil for 4 days did not
show any acute toxicity.  The decrease of acute toxicity may be due to both
soil adsorption and aerobic degradation of fenvalerate (Sumito Chemical Co.
1982, Ohkawa 1980).   Can and Chen reported that through an isotope tracer
experiment using "C-labeled fenvalerate they found that the mud  in water
system showed a strong action of adsorption and it is responsible mainly for
the water purification.  Fish could not only take up but also liberate the
fenvalerate residues very quickly from and to waters.
                                     293

-------
      To sum up, although the acute toxicity of fenvalerate to aquatic animals
was very high in laboratory experiments, the actual toxicity to aquatic
ecosystems in the real world would be less than predicted.


          TABLE 6.   INCIDENCE  OF FENVALERATE  TO MICRONUCLEUS  OF
                    THE PERIPHERAL NUCLEATED ERYTHROCYTES IN THE
                    LOACH (By Intraperitoneal Injection)
Dose,
ppm
0.02
0.01
0.005
Mitomycin
(1.8 mgAg)
Dimethyl
Sulfoxide
(0.01 ml)
Distilled water
Number of
erythrocytes
scored
20000
20000
25000
10000

20000
30000
Number of
micronucleus
1
1
1
6

1
1
Rate of
micronucleus ,
% P*
0.05  0.05 >0.05
0.05  0.05 >0.05
0.04  0.04 >0.05
0.6  0.24 <0.01

0.05  0.05 >0.05
0.03
 *P>0.05, not significant
  P<0.01, significant
                                   REFERENCES
 Anderson, R.L.  1982.  Toxicity of fenvalerate and permethrin to several
       nontarget aquatic invertebrates.  Environ. Entomol.  11:1251-1257.

 APHA, AWWA and WPCF.  1980.  Standard methods for the examination of water
       and wastewater, 15th edition.  American Public Health Association.

 Bradbury, S.P., D.M. Symonik, J.R. Coats, andG.J. Atchison.  1987.  Toxicity
       of fenvalerate and its constituent isomers to the fathead minnow,
       Pimephales Promelas and blue, Lepomis macrochirus.   Bull. Environ.
       Contain. Toxicol.  38:727-735.

 Chen Zhitao and Zeng Zhaoqu.  1982.  Toxicity of 26 kinds of pesticides  to
       bacteria and algae in water.  China Environmental Science.  2:24-28.
                                      294

-------
    TABLE  7.   CAPACITY OF THREE SPECIES OF FISH TO  SEEK
                WATER FREE OF  FENVALERATE
Species
Silver
carp






Grass
carp




Common
carp





Concentration ,
Pg/1
2.00 (1/2 96hLC50)
1.00 (1/4 96hLC50)
0.50 (1.8 96hLC50)
0.40 (1/10 96hLC50)
0.36 (1/11 96hLC50)
0.30 (1/13 96hLC50)
ethanol 0.02 ml
(ml/1000 ml)
tap water
2.00 (1/2 96hLCso)
1.00 (1/4 96hLC50)
0.50 (1/8 96hLC50)
0.40 (1/10 96hLC50)
ethanol 0.02 ml
(ml/1000 ml)
tap water
2.00 (1/2 96hLC50)
1.00 (1/4 96hLC50)
0.50 (1/8 96hLC50)
0.40 (1/10 96hLC50)
0.36 (1/11 96hLC50)
ethanol 0.02 ml
(ml/1000 ml)
tap water
Number of fish**
In In
water fenvalerate
22 3
17 6
15 5
16 7
13 9
9 7
11 9
11 10
18 6
17 6
18 7
14 9
9 8
12 10
26 7
22 8
21 10
22 10
10 9
16 15
14 14
Percentage
in water
87.4
73.0
74.3
69.5
59.0
54.6
5 2'. 3
50.0
76.8
75.2
71.6
59.0
52.8
53.8
78.3
72.4
71.0
68.2
53.3
51.0
50.0
X2*
value
13.90
4.66
4.58
3.89
0.850
0.101
0.045
0.046
6.84
5.72
4.76
0.76
0.056
0.13
10.56
5.61
4.34
4.22
0.848
0.019
0.0005
*X2 - P (3.84 - 0.05; 6.63 - 0.01; 10.83 = 0.001), P > 0.05 not significant;
     0.05 >. P > 0.01 significant; P  0.01 very significant.

**Does not include fish in the holding area.
                               295

-------
          J?
           (0
          *-
           o
              100
               80 -
               6O-
               40-
               20-
       e	e   1000ugL
       o  -e   500 ugL "1
       G~o   100ugL"1
                          24
48        72
 Time, hours
96
             Figure 1.   The acute toxicity of fenvalerate to silver
               carp after adsorption by soil at different intervals.
Cheng Haihong, Li Shurong, and Yin Guihua.  1986.  Study on the toxicity of
      fenvalerate.  Pesticide.  No. 4.

Clark, J.R., J.M. Patrick, Jr., D.P. Middaugh, and J.C. Moore.  1985,
      Relative sensitivity of six estuarine fish to carbophenothion,
      chlorphyrifos, and fenvalerate.  Ecotoxicology and Environ. Safety.
      10:382-390.

Coats, J.R. and N.L. O'Donnell-Jeffery.  1979.  Toxicity of four synthetic
      pyrethroid insecticides to rainbow trout.  Bull. Environ. Contain.
      Toxicol.  23:250-255.

Can Jianying and Chen Ziyuan.  1986.  Dynamics of fenvalerate in rice-water-
      fish system.  Acta Scientiae Circumstantiae.  6:263-271.

Joint IMCO/FAO/UNESCO/WHO Group of Experts on the Scientific Aspects of
      Marine Pollution.  1969.  Abstract of the report of the first session,
      Water Research.  3:995-1005.

Lewis III, F.G. and R.J. Livingston.  1977.  Avoidance of bleached kraft
      pulpmill effluent by pinfish (Lagodon rhomboides) and gulf killifish
      (Fundulus grandisl.  J. Fish Res. Board Can.  34:568-570.

Liu Changjie.  1981.  Study on the monitoring for environmental pollution
      using nucleate erythrocytes micronucleus of vertebrates.  Acta of
      Huazhong Normal College (Natural science edition).   No.  2.
                                    296

-------
Mayer, F.L. and M.R. Ellersieck.  1986.  Manual of Acute Toxicity:
      Interpretation and Data Base for 410 Chemicals and 66 Species of
      Freshwater Animals.  U.S. Fish and Wildlife Service, Washington DC.

Mckee, M.J. and C.O. Knowles.   1986.  Effects of fenvalerate on biochemical
      parameters, survival, and reproduction of Daphnia magna.  Ecotoxicology
      and Environ. Safety.  12:70-84.

Mcleese, D.W., C.D. Metcalfe and V. Zitko.  1980.  Lethality to permethrin,
      cypermethrin and fenvalerate to salmon, lobster and shrimp.  Bull.
      Environ. Contamin. Toxicology.  25:950-955.

Mulla, M.S., H.A. Nawab-Gojrati and H.A. Darwazeh.  1978.  Toxity of
      mosquito larvidical pyrethroids to  four species of freshwater fish.
      Environ. Entomol.  7:428-438.

Ohkawa, Hideo, Ryoichi Kikuchi  and Junshi Miyamoto.  1980.  Bioaccumulation
      and biodegradation of the  (s)-acid  isomer of fenvalerate  (sumicidinR) in
      an aquatic model ecosystem.  J. Pesticide Sci.  5:11-22.

Stein, J.R.  (ed.).  1973.  Handbook of Phycological Methods, Culture Methods
      and Growth Measurement, Cambridge,  the University Press.

Sumito Chemical Co. Ltd.   1982.  Selected Papers on Pesticides--Synthetic
      pyrethroides, Sumicidin.
           (0
           *J
           o
               100 -
               80 -
               60 -
               4O -
               20 -
   o   1OOOugL'
 -   500 ugL M
       100ugL"1
                            24        48        72
                                     Time, hours
          96
           Figure 2.   The acute toxicity of fenvalerate  to  gold-
             fish after adsorption by soil  at different  intervals,
                                     297

-------
WHO.  1978.  Principles and Methods for Evaluating the Toxicity of Chemicals,
      Part 1. Geneva.

Zhu Qinghua, Liang Xinglan, and Liu Fengming.  1987.  Study on the toxicity of
      fenvalerate, pesticide.  No. 2.
                                   298

-------
                       MODELING THE EFFECTS  OF TOXICANTS
                              ON FISH POPULATIONS

                                      by

      Thomas G.  Hallam1,  Ray R.  Lassiter2, Jia Li1,  and William McKinney1
                                 INTRODUCTION

      The relationships between toxic chemicals and their effects on
populations are intricate and generally poorly understood.  One reason for
this lack of understanding is that the biology of the stressed organisms
frequently is not considered in the determination of the risk associated with
an exposure to a toxicant.  The first steps in current chemical assessment
procedures are generally based upon quantitative structure-activity relations
(QSARs).  QSARs are mathematical expressions that relate biological activity
(molar concentrations causing quantal effects) to descriptors of molecular
properties of a sequence of chemical compounds.  These approaches are based on
properties of the chemicals and do not include biological properties of the
exposed organisms.

      A theme of this article is that the present theoretical basis for
determining effects of chemicals on populations is inadequate primarily
because past developments do not account for biological detail.  This
inadequacy is magnified considerably when it is noted that an improper
investigative focus at the population level is usually employed.  These
deficiencies have contributed to a lack of substantial developments in
ecotoxicology.  Such hindrances are especially restrictive for the
foundational work relating to the determination of effects of toxicants on a
biological system.

      Another theme of this article is that a proper focal level for
ecotoxicology must account for the individual organism.  Indeed, in the
classical ecological organizational scheme, the individual is special and
unique.  In the hierarchy from the cell to tissue to individual organism to
population, the levels below the individual are sets of genetically identical
elements, whereas the population is structured by genetic variation (Lomnicki
1988).   It is individual variation that often is missing or suppressed in
     "Department of Mathematics,  University of Tennessee,  Knoxville TN,  USA

     Environmental Research Laboratory,  U.S.  Environmental Protection Agency,
Athens GA, USA
                                      299

-------
studies of the effects of chemicals on populations.  This variability'is
needed to properly investigate and develop the appropriate theoretical basis
for ecotoxicology.  Variation in the genetical, physiological and physical
distributions of individuals in a population together with the biogeochemical
environment of the population determines the characteristics of the effects
resulting from chemical exposure.  In this article, we will generate some of
the genetic and physiological distributions associated with a fish population
that are relevant to the chemical effects problem.

      Chemical impact occurs at the level of the individual not at the
population level.  Even though the target site of a chemical may be specific
tissues, the exposed affected individual is the appropriate reference point
for extrapplation to the population level.  A basic concept of toxicology,
susceptibility, intrinsically implies the existence of variance-variation- that
is viewed here as structure in the population.  Susceptibility to chemical
exposure, an .individual property, is not static but is a variable related to
the dynamics of the individual.  Dynamic susceptibility of individuals must be
reflected in the distribution of susceptibility for the population.  A basic
premise of this article is that effects of chemical stress at the population
level are the cumulation of effects of the chemical on the individuals that
compose the population.

      For investigations of effects, it is essential to note distinctions
between individual properties and population properties.   Individual
properties include physiological variables such as body size and composition
as well as tolerance or susceptibility to a toxicant.  Population properties
include distributions of individual properties such as distribution of
tolerances or susceptibility and moments of these distributions.

      For modeling purposes, techniques exploring the role of the individual
in determining population dynamics are a relatively recent phenomenon but are
now evolving at a rapid rate (Metz and Diekmann 1986).   Most of the original
works in mathematical ecology unfortunately do not reflect this individual
perspective, primarily because aggregation in many early studies  was done at
the population level.  Developments at the population or higher organizational
levels have proved to be generally unsuccessful in ecotoxicological studies
because individual variation is lost in these representations.   Recent
progress in the analysis of individual-based population models is encouraging
because there have been significant developments in the area of accessible
computing power.  This allows one to track large numbers  of individuals in a
reasonable computational time period.   We shall utilize an approach that first
develops a physiologically structured population model and then performs the
analysis by numerical techniques.

      This article presents a theoretical study of the effects of a lipophilic
narcotic on a dynamic fish population.   The work is theoretical because data,
facilities and techniques are not presently available to  generate the
information needed for corroboration of the basic hypotheses or outcomes.   The
conclusions obtained are conjectures that must be tested;  however,  because the
foundations of our model formulations  are solidly grounded in the biological
and toxicological literature,  we believe they merit the efforts necessary to
check their consistency.   The effects  literature is sparse because theoretical
efforts in ecotoxicology have virtually ignored biology.   Our efforts

                                    300

-------
 represent an initial  attempt to  include  relevant biology  in an assessment
 procedure.

       Effects are  limited for the  present  discussion to mortality  in the
 population.   This  restriction is not  necessary but  it is  sufficient  to
 illustrate  the procedure  that we suggest.   The rudiments  of the underlying
 theory for  mortality  in a static population are given in  Lassiter  and Hallam
 (1989a).  This static theory,  intuitively  nicknamed "survival  of the fattest,"
 is  developed for acute chemical  exposures  and a static population.   It
..assesses  the effects  of toxic exposure by  relating  the n-octanol-water
 partition coefficient to  the partition coefficient  of the lipid and  aqueous
 phases of the animal,  by  hypothesizing equilibration within the body, and by
 employing quantitative structure-activity  relationships as a component  of the
 biological  response assessment.  Because the chemical is  lipophilic,  a  known
 distribution of lipid in  the population  is necessary to apply  this static
 theory.'  We are aware of  only two  distributions of  lipid  in an aquatic
 population  [Brockway  (1979)  and  Clark (1988,  personal communication)].  These
 distributions,  for static fish populations,  indicate that there can  be  much
 variation in lipid content of fish of the  same relative size in both
 laboratory  and field  populations.

       The dynamic  behavior of individuals  coupled with the possibility  of
 chronic or  multiple toxicant exposures requires a new perspective.   To  our
 knowledge,  the dynamic distribution of lipid in any aquatic population  is
 nonexistent at the present time.   Nondestructive sampling methods  currently
 are being developed and will ultimately  lead to progress  in this area.  For
 our purposes,  however,  it is currently necessary to obtain dynamic lipid
 distributions by methods  other than experimental ones.  The approach espoused
 here--to  focus at  the biological-chemical  interface of the individual--
 necessitates development  of  a dynamic representation of an individual
 organism.   The specific individual representation employed is  one  developed
 for the purpose of determining the effects of a chemical  on an individual
 fish.   The  individual model,  based upon  energetics  and described in  detail in
 Lassiter  and Hallam (1989b) ,  is  an important part of the  population  model.  A
 brief  summary of the  individual  model is presented  below  so that its  role in
 the dynamics of the population can be understood.

       Exposure and uptake of chemical also must be  modeled to  determine the
 effects of  a toxicant on  a population.   We utilize  an analogue of  the uptake
 model,  FGETS,  developed by Barber  et  al. (1988).  GETS and FGETS were both
 formulated  for fish and need little modification for our  purposes.

       Population dynamics are  a  compilation of all  individual  dynamics.  We
 model  population dynamics by employing a partial differential  equation  that
 incorporates individual dynamics explicitly in the  population  representation
 and describes the  behavior of the  population in terms of  a density function
 that depends on individual physiological model variables  and time.   The
 population  model,'  its  behavior in  the absence of the chemical,  and the
 behavior  in the presence  of  the  toxicant will be discussed here.

       The primary  objectives of  this  paper are to indicate theoretical
 developments formulated to explore the effects of a chemical on a fish
 population  when toxic  exposure is  allowed  through both the environmental and

                                      301

-------
the food chain pathways.  In a recent paper  (Hallam et al. 1989), we have
studied the effects of different chemicals on a Daphnia population.  In this
article, we study  the effects of exposure duration on a fish population.


                      A MODEL OF INDIVIDUAL FISH DYNAMICS

      The dynamics of an individual fish are well documented in the
literature; see books in the series edited by Hoar and Randall (1969).
Shul'man (1974) explores the role of lipids and their dynamics in fish
populations.  Other papers that explore energetics and model growth in fish
population include Stewart et al. (1983) and Kitchell et al. (1974, 1977).  We
know of no application of energetically based models to the assessment of
effects of a chemical on a fish population other than those presented here.
Kooijman and Metz  (1984), Kooijman (1986), and Hallam et al. (1989) have
developed model formulations for Daphnia that are analogous to the ones
presented here.  Philosophically, our model is closely related to these
efforts.  Technically, this work is similar to our previous work on Daphnia;
however, the specifics of the individual (and subsequently, the population
model) are different than the Daphnia population model.

      Any modeling project must be compatible with its objectives; hence, our
individual model must allow for relevant interaction with the chemical and
must be able to account for its toxicity.  Appropriate model components must
be chosen with the specific chemical and type of exposure in mind.

      Most industrial chemicals and many chemicals of environmental concern
are non-ionic, are nonreactive, induce baseline narcosis and are, to some
degree, lipophilic (Veith et al. 1983).  The chemicals employed in our
illustrations are assumed to have these characteristics although the procedure
does not explicitly require any of these characteristics except the
lipophilicity and the nonreactivity.  A method of determining the toxicity
effects threshold such as application of a QSAR also is needed.  The lipid in
an individual buffers the action of a lipophilic chemical, allowing larger
body burdens in fatter individuals than in less fat organisms to elicit
biological response.  Thus, additional lipid in an individual results in an
extension of the toxicity effect thresholds for an acute exposure.  Lipid
storage provides protection against toxic stress from transient exposures only
if the organism is not forced to rapidly mobilize quantities of stored lipids.
When an organism rapidly utilizes stores of lipid in a situation where high
body burdens have been obtained, internal release of the chemical can lead to
toxicity effects under conditions where there is no change in the external
environmental concentration of a chemical.   These considerations indicate
that,  for the class of chemicals under consideration,  a lipid compartment is
important and appears necessary in any individual model that is utilized to
represent the biological-chemical interaction.


                        A MATHEMATICAL MODEL OF A FISH

      We now summarize the model of the life history of a fish.  More detail
and background information can be found in Lassiter and Hallam (1989b).
Figure 1 is the conceptual model listing associated compartments and the flow

                                     302

-------

1L

Resource

1P




Food Lipid
\
2L ;

i
Fecal
i
3L
Energy Integrator;
Mass to Energy
i i
Food Protein
t
3P
2P

5LR IT
4M
I W
4W
4R
*"
3PR _

Egg Lipid

Energy Losses
Maintenance
Work (activity)
Reproduction

Egg Protein
Figure 1.  Compartment and flow'diagram for the individual fish model.
 chart  for  an adult  female  fish.   The model  assumes  that  the  only inputs  to  the
 lipid  and  protein compartments  are  obtained from a  decoupling of the  food
 lipid  and  structure;  that  is, no  synthesis  of fat can occur  from the
 carbohydrates and proteins of the resource.   This is  not a valid hypothesis in
 most situations  (Vel'tishcheva  1961, Amlacher 1961, Menzel 1960)  as Shul'man
 (1974)  indicates.   On the  other hand,  the diets  of  many  fishes contain small
 amounts of carbohydrates that can be converted into lipids,  so in these  cases
 this model might  be useful.

       Let  mL  and ms denote  the mass  of the lipid and mass of the structure,
 respectively,  in  an individual  organism.  Structure is regarded as primarily
 protein and  carbohydrates.   Each  of these components  are assumed to have both
 labile and nonlabile  portions.  The nonlabile portion of the structure is
 viewed as  protein and carbohydrates bound in soma and is designated in the
 model by mps, the mass of the protected structure.  The labile portion of the
 structure  component is represented  by  ms - mps.   The nonlabile lipid is
 assumed to be proportional to mps and in the model is  represented by efflps;
 hence, the labile lipid is mL - emps.

      The  density of  the resource is denoted by  x and we assume that  x - XL +
 xs where XL and xs are the  lipid and structural portions  of the resource
 density, respectively.  The resource is assumed  to  be utilized for growth
 according  to  a hyperbolic  uptake  law (Lassiter 1986).  For details on the
 particulars  of the  representation used here  refer to  Lassiter and Hallam
 (1989b).   The losses  of energy  for  the maintenance  and the activities
 compartments  are  assumed to operate on a continuous time scale.   Hence,  on
 intervals  where there is no reproductive loss  the fish is  assumed modeled by
 the differential  equations:
                                     303

-------
dmL
-    -
XT
TED > AE

TED <: AE
                                                                           (1)
dms
 dt

                                        TED > AE

                                        TED < AE
     The quantity ad is the encounter rate coefficient  (liters per day)
obtained from Gerritsen and Strickler  (1977)  and is  given by
                             8.64.10Wd(v2+3v2h)/1033vh
                                                 (2)
with sd defined as the reactive distance (cm) of the fish  (Breck and Gitter
1983) and given by Sd - 4.775Lf1/2, where Lf denotes the length of the fish; vp
is the velocity of the prey  (cm/s)  and is  given by vp = folsp  . Lp where Lp
denotes the length of the prey and  blsp denotes the body  lengths per  second of
the prey; and vh is the velocity of the fish  (cm/s) while hunting for prey
defined by (blsh) (Lf) with blsh  denoting  the body lengths  per second of the
fish while hunting.  The symbol Wp represents the mass of the  prey  (g dry  wt) ;
v is the difference in the swimming velocities of the fish and its prey:  Sy -
(vc - vp)8.64.10A; k  is the gut emptying rate  (1/cf) and Ms  is  the mass  capacity
of the fish's stomach (g).

     A generic derivation for the feeding  rate  of  a pursuit feeder  may be
found in Lassiter (1986, Equation 66).  The formulation is  a hyperbolic
function F  x/(A1 + Azx)  in g/d where x is the density of the prey
population.  When formulated in terms  of mass consumed, the above form may be
written as a hyperbolic function  of numbers of  prey Np:
                      F =
     The terms in the denominator relate  to  characteristic  times  (d/g)  for
pursuit, encountering prey, and digestion, respectively:
                       T0=Sd/(5vWp)  (d/g captured) ,
                                    (d/g captured) ,
                       rd=l/(ktfs/)   (d/g eliminated).
                                     304

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      The feeding rate is thus F = (Tc + Te  + T^y1.   In our computations,  we
 assume that the number of prey is fixed.  The reproductive losses are assessed
 at discrete times of reproduction.  Reproduction is allowed in a time window
 to represent seasonality aspects of reproduction.  This reproduction interval
 is utilized to set, .the birthing times for the specific species and is utilized
 because we do not indicate hormone triggers such as temperature-related
 phenomena.  The losses associated with the individual fish model include
 energetic needs associated with maintenance, activity and reproduction as well
 as losses of lipid and structure for egg production.  Maintenance energy
 requirements are assumed to be proportional to the compartment.  Activities
 are modeled according to Gerritsen (1984) who states a relationship for power
 consumed by swimming fish as


                      P = 0.275Av2-5ir-5q-1     (ergsec'y1)


 where A is wetted surface area (0.4L2),  v is swimming  velocity (part  of the
 time hunting,  part of the time chasing and  is given as the appropriate product
 of bis and L),  and q is swimming efficiency (0.2 according to Gerritsen).
 Thus,  after converting to appropriate power units


                              P - 4.75.10~351s5/2L4


 or when cruising and chasing are included


                     P - 4.75.10-3L4Mf(&lsh5/2TeF+&lscs/2TcF)


      The  reproductive  losses are assessed at the discrete times of  reproduc-
 tion.   It is clear  that the  process of allocation of bulk mass to eggs  occurs
 over^a continuous  time  frame but, because there  is  little information on  the
 specific  time  scales of this process  and because it is small  relative to
 population time  scales, we treat them as  discrete events.   The reproductive
 losses  include bulk  allocation to eggs  and  the energy  required to deposit this
 mass  in the eggs.  These operations,  as well as  the mechanism employed  to
 determine  the number of eggs produced,  are  described in detail in Lassiter and
 Hallam  (1989b) and Hallam et al.  (1989).

     Examples of the numerical  solution of  the individual  model are given in
 Figure  2.                                                           6


                           UPTAKE IN AQUATIC ANIMALS

     The uptake model that is employed in connection with  the  above individual
model is a modification of the FGETS model  (Barber  et  al.  1988).  The model,
based upon thermodynamic potential, represents the  chemical exchange between a
fish^and the aqueous environment that occurs across gill membranes and the
chemical exchange that occurs across gut walls and  the contaminated ingested

                                      305

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                                                                       9.0
       Figure 2.  Lipid (MI) and structure (MS) cycles in the individual
         model.   The dynamics illustrate the decrease of size while in the
         egg sack, exponential growth as a juvenile, and the reproductive
         cycles.  The nonlabile structure, mps, is a nondecreasing function
         of age.

food.  The model  in its simplest  form without  food  uptake  is  of  the classical
bioaccumulation form; however,  in this  approach the parameters of  the uptake
model are represented in considerable detail and  include representations  of
many factors that can influence internal  concentrations such  as  the fractions
of the organism that are lipid  (PL) ,  aqueous (PA)  and structure (Ps) ;  the
partition coefficients that  indicate the  affinity of  the chemical  towards
lipid (KL)  and structure (Ks) ;  the conductance  of  the exposed membrane (Kg);
the total weight  of the organism  (WT) and the active  (effective)  exposure8 area
(Sg)  of the gill.   The general form of the model is thus that of a classical
bioaccumulation model plus a term for exchange across the  gut:
                     dt
                           s
                                                                          (3)
     In Equation 3, BT represents the total toxicant in the organism; Cw and
CF represent the concentrations of toxicant in the environment and in the

                                     306

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food, respectively; F and E are the mass fluxes of food and feces, respective-
ly; BCF is the bioconcentration factor (total concentration in the organ -
ism/Cw) ;  kE is  the  partition coefficient  of chemical  to  excrement;  CE = kECA,CA
is the concentration of toxicant in the aqueous portion of the organism; and
>A is the aqueous distribution coefficient (D^CL+PgP^Ks+P^^K^'1 .  The unit
conductance kg may be calculated from the molecular weight of the chemical and
the n-octanol- water partition coefficient.  Barber et al. (1988) show that kg
is approximately N^D^h^'1 where Nsh is. the Sherwood number, Dw is the toxi-
cant's diffusion coefficient, which is a function of the molecular weight of
the chemical, and hw is the characteristic dimension of interlamellar chan-
nels .
     A seemingly natural approach to model the uptake of chemical from the
food chain pathway would be to proceed by employing hypotheses similar to
those imposed for the environmental pathway.  The complications of this
hypothesis and other options for modeling the uptake from contaminated food
are discussed in Barber et al. (1988).

     The basic assumption of this model representation, that of equilibration
of chemical between the organism's body and the gut contents, is, of course,
not necessarily true.  It has been demonstrated that this requirement is a
worst case assumption during increasing body concentration when exposed to
contaminated food; that is, no additional chemical could be taken up under any
thermodynamically consistent assumption than would be taken up when food and
body equilibrate.  During depuration, however, this assumption leads to
predicted minimum depuration times; that is, any other thermodynamically
consistent assumption would lead to longer depuration times.  For toxicity
evaluations, this would usually not be considered the worst case scenario.


                      EFFECTS  OF  TOXICANTS ON  INDIVIDUALS

     The basic ideas employed to assess the effects of chemicals on an
individual and on a static population are given in Lassiter and Hallam
(1989a) .   We review these ideas to set the stage for this study on the effects
of a chemical on a population.  Again the particular effect focus is on
mortality of the individual but sublethal effects could be considered by the
same methods .

     The assessment of mortality due to chemical action is implemented by
utilizing formulations obtained from QSARs.   The processes relating to mode of
action and concentration- response relationships must be coupled to determine
the effects.  The procedure then consists of combining the following into an
assessment protocol:

     (a)   The differential equations for development of the individ-
          ual dynamics .

     (b)   The differential equation for the total concentration of
          toxicant (or,  equivalently, the total body burden) in the
          organism.

     (c)   The determination of mortality from QSARs.
                                     307

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     The first two of these steps couple the dynamics of the individual with
the dynamics of the chemical in the individual.  The chemical uptake is
coupled to the individual through the weight terms in each of the Equations 1
and 2.  Step c, the assessment of mortality, utilizes results of Veith et al.
(1983) and Konemann (1983) (see Figure 3).  These bioassays are developed for
baseline narcotic chemicals and relate a chemical property, KOW,  to  mortality
of the individual.

     There are numerous QSARs in the literature for modes of action other than
baseline narcosis.  For example, the modes of polar narcosis (Veith et al.
1985) and uncoupling of oxidative phosphorylation (Schultz et al. 1986) have
been documented.  A common feature of each of these modes of action is that
lipophilicity of the chemical as measured by KQW is  important.  We do not
present modes of action other than narcosis.  Because the analogous QSARs are
based at least in part upon KOW, however,  it appears  that  assessment of risk
due to exposure to a chemical with these other modes also must utilize lipid
as an individual model component.
u -
Ttll * _



 p 


o
10-3-
o
_j
2-4-
_J
-5-
-6-

-7-
Q n .-I
D ^

















D
R] O
0 D
Q3
DD, ft,
o t?1 ff
a
O D
a a a1^ gg apa
a C? D nD P
 ^ DaD
D ri^ci
a  j3
a  cP
D DO
O .Q

1 1 1 I "1 	 "
-2 ' 0 2 4 6
                                     Log Kow
    Figure 3.  Relationships between log LC5Q and log KQJJ.  Fitted result
      is log (LC5o)=-0.8 - log (KQW)  Data from Veith (1983) and Kone-
      man (1981).
                                    308

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                  AN ACUTE EXPOSURE-STATIC POPULATION THEORY:
                            SURVIVAL OF THE FATTEST

      In a recent article (Lassiter and Hallam 1989a), we have developed an
 approach that utilizes individual variation  to structure a population and to
 explain the effects  of an acute  toxicant  exposure from  a lipophilic narcotic
 on that population.   The basic idea  is that  lipid provides a buffer against
 toxic stress and this factor  must be utilized in any  consideration of effects.
 According to this theory,  in  an  assessment of mortality,  an individual with a
 smaller lipid fraction body content will  die  before another individual with a
 larger lipid fraction given equal exposure (Lassiter  and Hallam 1989a).   The
 hypotheses for this  theory are related directly to the  static state of the
 population.   This static state allows  only acute toxicant exposures.   The
 pathway of exposure  is not important for  effects on static populations.


            A DYNAMICS THEORY:  EFFECTS OF TOXICANTS  ON POPULATIONS

      Our prototype dynamic population  model is  based  on individual response so
 that  effects can be  determined directly and the cumulative effect  at  the
 population level ascertained.  The toxicant-population  model  is  formulated so
 that  a toxicant  may be released  at numerous times  and for arbitrary exposure
 lengths  during the study period  so that chronic as well as acute exposures may
 be  investigated.

      First,  we will  sketch the approach used  to model the population.  Then,
 we will  discuss  the effects of the toxicant on  the population due  to  duration
 of exposure.

     An  approach that  allows  incorporation of individual  dynamics  into a
 dynamic  population formulation is the McKendrick-von Foerster equation (Metz
 and Diekmann 1986).  This partial differential  equation allows explicit
 representation of physiological variables as  they determine the  dynamics  of
 individuals.   It  also  keeps track of the total  population through  the  popula-
 tion  density function.   The assessment of effects of a  chemical  that  is a
 lipophilic narcotic mandates  that the individual model  should minimally
 include  some measures  of the  physiological variables age,  lipid, and struc-
 ture.  Age is  necessary  to reflect the life history of  an individual.  Lipid,
 a bioconcentration site  for the toxicant,  is necessary  to  assess the effects'
 of the chemical  on the individual.  Structure is necessary to account  for  size
 related measurements such as weight or length of the organism.

     If p=p(t,a,mL^,ms)  is the  population density function--which  depends upon
 time and the physiological variables a, representing age; mL,  representing the
mass of the  lipid, and ms,  representing the mass of the  structure compartment-
 -and gL and gs are the  growth  rate  of the  lipid  and structural components  of
an individual as given by the Equations 1  and 2 respectively, then an equation
that incorporates these physiological variables into  a population scheme is
                                     309

-------
The subscripts denote partial derivatives with respect to that subscript.


     The birth process for the population is represented by a boundary condi-
tion, and the mortality rate is given explicitly in the differential equation
as /L*.  For physiologically structured models, the particular form of the birth
process may be written in several equivalent representations.  One of these
forms is
                      ,, - III
/3 (t, a, mLo, mS(), 2
where mLo is the mass of the lipid and mso is the mass of the structure at
age-0 and 0  is  the birth function which represents the number of eggs with
lipid content mLo,  structure content mso born to an individual of age a with
lipid content m^ and structure content ms  at time t.   One can use  this  form
because  there is a one-to-one correspondence between lipid or structure mass
and age  for  fixed mLo.

     Several different  types of mortality are represented in our numerical
model formulation.  We  include formulations for age dependent mortality, size
dependent mortality, and density dependent mortality.  The age dependent
mortality is assessed uniformly along  cohorts, whereas the density dependent
mortality is assessed uniformly across the population.   The size dependent
mortality is viewed  as  possibly caused by predation and  is determined by
weight of the individual.   The population model is, in general, nonlinear but
it would be  linear if the density dependent mortality term were omitted.  Some
restriction  on  mortality generally  is  needed to prevent  the population from
growing  beyond  reasonable size.  The specific forms of these mortalities are
generic  and  have a form that can be structured to test processes.  Formula-
tions of the mortalities are given  in  Figure 4.

     This population model  is a first  order hyperbolic partial differential
equation that may be represented in an alternate manner  by the method of
characteristics.  In this method, the  partial differential equation is reduced
to a set of  ordinary differential equations that are valid along certain
special  curves  called characteristics.  Specifically, in this model an equiva-
lent representation  for the partial differential equation is the following
system of five  ordinary differential equations.
                        d\
                        dX
                                     310

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         Individual Mortality:
           Weight-Dependent Mortality : fj,w = jw . fiiw = jw . fjilw(W),   W = weight (ing)
                     Vc,
                    continuous and linear,   elsewhere
           Age-Dependent Mortality:
{
                   Ka,    0 < a < 50
                   oo
                                       a>50
         Population Mortality:
            Density-Dependent Mortality : fj,D = fiD(PB), PB =  total population biomass.
                                        PB = Po
                                        PB>PC
                     continuous and linear,  elsewhere
         Figure  4.   Mortality functions used  in population model.
                        dmL
                         d\
                        "dA  "*s
                         dp
                         dX
                        'n>L
     Our approach is  to  solve a transformation of this  system of codes numeri-
cally.   Examples of graphical representations of these  numerical solutions are
given later for several  situations of  interest.

     The behavior of  the population in the absence of a toxicant is oscil-
latory on several scales.   There are oscillations on the time scale of the

                                       311

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(assumed periodic) reproductive time (gestation period) of the species.  There
are longer term oscillations that do not seem to be related to mortality but
rather to the rate at which the organisms grow.  This longer term oscillation
is not apparently due to the dynamics of the resource, which is assumed to be
at constant density.  In particular, this is not a typical predator-prey
oscillation where both predator and prey have oscillatory behavior.  However,
this oscillatory behavior of a consumer when the resource is at a relatively
constant level is characteristic of some aquatic populations. (Murdock and
McCauley 1985) that do oscillate in the presence of a nonoscillatory resource.
The cause of the longer term fluctuations is apparently not a result of the
assumed density-dependent regulation.  We mention this because the folklore in
rudimentary nonstructured models indicates that oscillations are often caused
by inclusion of density dependence  (Hallam 1986.).

     Our numerical procedure essentially follows cohorts of individuals along
characteristics.  This allows effects of toxicant exposures to be assessed at
the individual level, a desirable attribute as we have indicated above, but
the overall effects on the population can still be determined by an accumula-
tion of individual effects.  The addition of a toxicant leads to another type
of mortality  assessment.  This mortality due to toxicant exposure  is assessed
at the individual level according  to  lipid  content of the  individual.  Hence,
in the model,  there  are four  essentially distinct causes of death:  mortality
due to the physiological  process of aging,  mortality due to size  (as,  for
example,  determined by predation),  mortality due to  density dependence (as,
for example,  determined by  total population size), and mortality  due to
toxicity  (as,  for example,  determined by the lipid distribution in the popula-
tion) .

      The  population structure and  analysis  as  reported here is based upon  27
different types  of individuals.  These  individual ecotypes (morphs) are
determined by the constant  level of resource at which they feed,  the quality
of that resource as indicated by its  lipid  content,  and  the gut clearance  rate
of the organism.   Each  of these  three individual characteristics  ranges
through three levels for  the  total of 27.   Any number of individuals can be
employed  in the  model.  We  employ  27  because  the diversity generated by the  27
ecotypes  is  considerable.  Figures 5  and 6  indicate  the  graphs  of several  of
the  ecotypes  of  individuals.   The  parameters we employ are selected from the
literature on trout although  there are  several requiring estimation (Lassiter
and Hallam,  1989b).

      The  population model records  the dynamics of  cohorts  of  individuals in
 the  population,  assesses  mortality in these cohorts, and indicates births.
 Organisms are assumed to  be clones of their female parent; this may not be a
proper hypothesis for fish, which  are not parthenogenetic.

      Our objectives are to indicate the processes  that affect studies  on
 effects of toxicants on populations and,  although  the importance  of many
 environmental parameters  on ecological systems is  recognized,  we  have  not
 included important relationships such as seasonality, temperature, and pH in
 process formulations.  In general, these environmental variables  affect only
 the parameter values in our process representations  and not the formulations
 themselves.
                                      312

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                 o.o
                                                  219.0
                                                  219.0
                                 days
                                                  21'S.O
Figure 5.   The  graphs of  nine  ecotypes of fish utilized  in structuring
  the population.  [The differences between the ecotypes are the resource  level at
   which they fend and the fraction of lipid in the'food.  Another variable used to
   generate variation in the population is the gut clearance rate; the variation caused
   by changes in this parameter are not presented  here  but they are used in the popula-
   tion model (smallest mg).]
                                        313

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Figure  6.  The Climax Population. [A. The dynamic lipid density  function of the
   climax population on the interval 2000 to 2737.5 days.  B.  The dynamic  structural
   mass density function of the climax population on the interval 2000 to  2.737.5 days.
   C. The dynamic age density function of the climax population on the interval 2000
   to 2737.5 days.]
                                       314

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                     TOXIC EFFECTS AT THE POPULATION LEVEL

     As we have noted,  survival  of  the  fattest  is a  theory obtained by con-
 sidering  toxic exposures  to  a  static population-exposures that are necessarily
 acute.  We have demonstrated in  previous work that this theory is not valid
 when the  hypothesis of  a  static  population  is violated.

     Survival of a population  after chronic exposure is determined not only by
 the lipid distribution  as  in the survival of the fattest but now, in a dynamic
 setting,  also by the growth  rate of the individuals  in the population.  When
 the model population was  stressed by a chronic  exposure at a concentration
 where there was some mortality but  also some cohort  survival, the particular
 ecotypes  of the surviving  individuals depended  upon many factors including
 biological aspects of the  population and toxicological aspects of the chemi-
 cal.  The biological features  were  related  to the intrinsic oscillations of
 the population, to the  assessment of mortality, and  to the reproductive
 characteristics.  Toxicological  features of importance include the length of
 exposure, the initial time of  exposure, as  well as the strength of the chemi-
 cal.

     The  fact that exposure may  come from both  the environmental and the food
 chain pathways complicates determination of cause of death or, equivalently,
 reason for survivorship in a dynamic population.  We shall focus upon a single
 aspect of the exposure, namely its  duration.  This approach will illustrate
 our procedure to assess the effects of a toxic chemical on our model fish
 population.  In each instance  below we shall stress the population employing
 the same  chemical but we investigate the outcomes of different durations of
 exposure.
     The dynamics of the physiological variables--lipid,
as expressed through the respective distributions of the
on the time interval from 2007.5 days to 2737.5 days are
6a, b, c.  This and all subsequent population graphs are
physiological variable size determined by the ecotypes.
scaling is to allow viewing of the larger individuals in
with this scaling, the smaller individuals still dominate
of their numbers.
structure, and age--
unstressed population
presented in Figure
scaled by a maximal
The purpose of this
the population.  Even
 the graphics because
     A 4-8 day exposure, initiated on day 2025, to a 3.2 parts-per-million
concentration of a chemical having an octanol-water partition coefficient of
105 decreases the number of cohorts of fish in the stressed model population
from 85 down to 32.  The surviving cohorts primarily consist of older in-
dividuals because no individuals in the younger age classes survive the
exposure.  The population continues to persist until day 2502, at which time
it goes to extinction.  The reason for this extinction is that the numbers of
individuals are small and the types of individuals grow sufficiently slowly so
that a threshold biomass in the density dependent mortality function is not
exceeded.  Figure 7a, b, c represent the lipid, structural, and age distribu-
tions in this stressed population.

     An exposure with the same chemical at the same concentration but of
duration 3.8 days instead of 4-8 days leads to a completely different con-
                                     315

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Figure 7.   Extinction caused  by chemical stress.    [Duration of exposure is
   4.8 days  initiated on day 2025.   See text for details  about chemical properties.  A.
   The dynamic lipid density of the  stressed model population.  B.  The dynamic strusture
   density of the stressed model population.  C.  The dynamic age density of the stressed
   model population.]
                                         316

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 elusion.  This population is now persistent over the study period.  The number
 of remaining cohorts that survive are fish that grow relatively fast.  It is
 their presence that allows this population to survive and their absence in the
 above stressed population that contributes to its demise.  Figure 8 demon-
 strates the evolution of this stressed population.  A comparison with the
 unstressed population of Figure 6 shows relatively small differences in the
 stressed and the unstressed populations.
                                     SUMMARY

      To demonstrate a proposed protocol for assessing the effects of a toxi-
 cant on a fish population, we have developed a model of an individual fish and
 incorporated these individual dynamics into a population model.  Accordingly,
 our approach focuses upon the individual -- the interface between the biology
 and the chemistry.  This allows investigation of the effects of toxicants on
 populations by accumulating the effects on individuals and reinforces our
 premise that the basis for studies in ecotoxicology should be the individual.

      The model fish population was stressed by a chemical and the effects of
 the duration of exposure studied.   The ecotypes of the surviving individuals
 were determined by both biological succession in the population and toxico-
 logical aspects of the chemical.   Biological succession proceeds to the
 dominant- -fas test growing- -ecotype among all ecotypes that structure the
 population.   Toxic stress can alter the successional dominant ecotype from the
 fastest grower ecotype (Hallam et  al.  1989).   Here we have seen that the
 duration of exposure can also drastically influence the population evolution
 A situation was presented where the faster growing morphs were eliminated from
 the population and the population  eventually went to extinction (Figure 7)    A
 shortening , of the duration of exposure allowed the population to persist
 (Figure 8).   Even though the  surviving cohorts were not the fastest growing
 morphs  they grew sufficiently fast to  overcome the threshold of mortality
 present in the density dependent mortality representation.

     As we have previously demonstrated for Daphnia. in a dynamic population
 model,  survival of the fattest is  not  valid,  even when exposure is  only from
 the environmental pathway.  For fish this  statement is  also valid   Both the

 ind^HeiTSented ^ FiSUreS  ?  Snd 8 indlcate   -ettlng where  the surviving
 individuals were  not the  fattest;  they were the slower or the  faster growers
 of  the  ecotypes.   The  "survival of the fastest" grower is characteristic^
 the  evolutionary  behavior of our model.  The surviving ecotypes with the
 largest growth  rate will eventually dominate the population
         experiments necessary to study our hypotheses and our conclusions
Hor  then T?     /^ in some instances, not even conceptualized.
However  the models studied, here are well grounded in biological and toxico-
anfre1! 1^^ *?* ^^ credible.   It is a clear implication of tnis
attribute   f ^^      ?* assessment should not be based solely upon
fundi  Tl  5   t T1C Chemica1'   The bilgy f the exposed organisms is
fundamental to the determination of the effects of the toxicant on a popula-
                                     317

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Figure 8.   Population persistence following a chemical  stress  of 3.8
  days duration  initiated on  day 2025.   [The same  chemical is used as in the
  situation represented by Figure  7.  A.  The dynamic lipid  density function of the
  stressed model population.   B.   The stressed density function of the stressed model
  population.  C. The dynamic age  density function of the stressed model population.]

                                          318

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Barber, M. C., L. A. Suarez, and R. R. Lassiter.  1988.  Modeling bioaccumula-
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Lassiter, R. R. 1986.  A theoretical basis for modeling element cycling.  In:
     Mathematical Ecology:  An Introduction.  T. G. Hallam and S. A. Levin
     (eds.).  Vol. 17, Biomathematics,  Springer, Berlin, 341-380.

Lassiter, R. R. and T. G. Hallam.  1989a.  Survival of the Fattest:  A Theory
     for Assessing Acute Effects of Hydrophobia Chemicals on Populations.
     Environ. Toxicol. Chem.

Lassiter, R. R. and T. G. Hallam.  1989b.  A physiological model of a fish.
     In preparation.

Lomnicki, A.  1988.  Population Ecology of Individuals Monographs in Popu-
     lation Biology.  Vol 25.  Princeton Press, Princeton, N.J.

Menzel, D. W.  1960.  Utilization of food by a Bermuda reef fish Epinephlus
     gullalus.  J. Cons. perm. int. Explor. Mer.  25:216-222.

Metz, J. A. J. and 0. Diekmann.  1986.   The Dynamics of Physiologically
     Structured Populations.  Lecture Notes in Biomathematics, Vol. 68.
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Murdock, W. W. and E. McCauley.  1985.   Three distinct types of dynamic
     behavior shown by a single planktonic system.  Nature.  316:628-630.

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     quantitative structure-activity to comparative toxicity of selected
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Stewart, D. J., D. Weininger, D. v. Rottiers, and T. A. Edsall.  1983.  An
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                                     320

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                      TOXICITY OF  ISOPRQTHIOLANE TO  FISHES

                                      by

             Zhai Liangan1, Zhao Xiaochun, Yao Aiqin, and Li Jifang


                                  INTRODUCTION

     It is an inevitable trend that new pesticides will be substituted for
organic chlorine  ones because  of  the toxicity of the latter's residuals to
environments, animals, fishes  and even human beings, although they have played
an important role in  the control  of pests and insects in China.  In recent
years, we have produced and imported organophosphors, pyrethrin, and other
pesticides but reports about their toxicity to fish are quite scarce.  This
paper reports a toxicity experiment using a bacteriocide--Isoprothiolane. The
goal is to provide a scientific base for pesticides application, environmental
protection, and criteria and standards development for fisheries water
quality.
                             MATERIALS AND METHODS
     Isoprothiolane (IPT),  i.e. Fuji--one, NNF--109, is a heterocyclic
pesticide that was experimentally produced by Japan Pesticides Company in
1968.  Since 1983, we have imported 2500 tons (95% emulsifiable concentrate).
The name of its effective chemical composition is, Ms(isopropyl)-1.3-dithio-
lane-2-ylidene-malonate, with a structure of
                       (CH3)2 CH - 0 - C
                                         \
                                           C -  C

                       (CH3)2 CH - 0 - C             s I
                                      0
         Molecular  formula:   C12 H18 04 S2,  molecular weight:   290.4.
    Yangtze Institute of Fisheries, Chinese Academy of Fishery Sciences,PRC.

                                    321

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 Purified isoprothiolane is a white crystal,  and its  melting point is  54C.
 Its  steam pressure is 1.41 X 10~4mmHg,  and it is slightly dissolved in water
 and  easily in benzene,  acetone,  alcohol and  other organic solvents.   Its
 solubility in water at 20C is 48 ppm.   Isoprothiolane  is a systemic  bac-
 tericide for the control of rice blast,  and  can prevent physiological dis-
 orders  caused by low temperature, over-moisture in soil,  etc.
 TABLE 1.   TEST ORGANISMS
  Species
   Standards
Simple description
                                                 Source
 Common  carp
 Average body
 length, 1.72 cm;
 weight, 0.0368 g
Omnivore, living in
the lower layer
From the experi-
mental fish farm
of our institute
 Silver carp
 Average body
 length, 1.80 cm;
 weight, 0.046 g
Feeder on phyto-
plankton, living
in the upper layer
From the experi-
mental fish farm
of,our institute
 Fish eggs of
 grass carp
                     From fertilized
                     eggs td gastrula
                     stage
                        From the experi-
                        mental fish farm
                        of our institute
 Daphnia
 roagna
2 to 3 days age
A kind of fish
food feeding on
phytoplankton
Cultured in
laboratory
     The water used in this experiment is tap water that has been exposed to
air.  Its turbidness is 3.7; pH 7.57; total hardness is 8.6 (Germany Hard-
ness); dissolved oxygen meets fisheries requirements.


ACUTE TOXICITY EXPERIMENT WITH FISH

     Concentrations of isoprothiolane for experiments with common carp
(Cyprius carpio") were determined to be 8.0, 2.7, 0.9, 0.3, and 0.1 ppm by the
equidistant transformation method of logarithm; those for silver carp (Hypo-
thalmichthys molitrix") were 4.5, 2.5, 1.4, 0.8, and 0.45 ppm.   Each group had
a replicate and a control.  Poisoning symptoms and the mortality rate of
fishes were noted, then the 96-hour LC50 was obtained by the Karber method.
The results were multiplied by 0.1 to find the safety concentration.
                                    322

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 GROWTH EXPERIMENT WITH FISH

      The  experiment  carried out by  stocking  10  fish  in  0.4 ppm  (safety
 concentration for common carp), 0.85 ppm,  0.45  ppm,  0.25 ppm  (survival
 concentration for silver carp) and  in pesticide free water (control).  Fish
 were  fed  in fixed quantity  for 28 days.  The growth  rate was  determined by
 measuring the body length and weight at  the  end of the  experiment  in
 comparison with  that at the beginning.


 EMBRYONIC DEVELOPMENT EXPERIMENT

      Concentrations  of isoprothiolane for  experimental  groups were determined
 to be 8.00,  4.50,  2.50,  1.40 and 6.88 ppm  by equidistant transformation method
 of logarithm.  Twenty eggs  at the gastrula stage were stocked in experimental
 groups  and a control group.  Embryo lethality,  hatching rate and survival rate
 were  noted periodically and compared with  that  of the control group.


 EXPERIMENT WITH  DAPHNIA MAGNA

      Seven isoprothiolane concentrations were determined between 0.15 and 2.5
 ppm,  and  a control and a replicate were  set  up.  Ten Danhnia magna was stocked
 in each group.   Mortality was noted at 24, 48 and 96 hours, and the LC50 was
 solved  from those  counts.
                            RESULTS AND DISCUSSION

     In the acute toxicity experiment, the fish appear to be uncomfortable and
to swim at the water surface and to be sluggish in behaviour at the beginning
of the experiment.  At high concentrations of isoprothiolane, fish would be
blackened in body color and some even died.  Results in Table 2 show that
isoprothiolane has moderate toxicity to fish.

     In the growth experiment (Table 3), isoprothiolane at 0.40 ppm had no
effect on the growth of common carp, but 0.80 ppm had a distinct effect on the
growth of silver carp.  0.45 ppm, 0.25 ppm and control had no effect on the
growth of silver carp, so the maximum functioning concentration of isoprothio-
lane on silver carp growth is 0.45 ppm.

     In the embryonic development test (Table 4),  it was shown that 2.50 ppm,
4.50 ppm and 8.00 ppm could affect development.   The embryonic body in 1.40
ppm, 0.80 ppm and control group begins to wriggle slightly after 12 hours.
Hatching rates from high to low concentrations are 20%,  30%,  85%,  90%,  80% and
75%, respectively.  Obviously,  only 4.50 ppm and 8.00 ppm have a distinct
effect on embryonic development.   Fingerlings in those concentrations with
high deformative rate all died after 96 hours.  There is no significant
difference among 2.50 ppm, 1.40 ppm, 0.80 ppm and control group.   The maximum
no-effect concentration for the embryo was 2.50  ppm.

     In the daphnia experiment (Table 5),  there  was no significant difference
among 0.15 ppm,  0.27 ppm,  0.47 ppm,  0.84 ppm, 1.50 ppm and control group.
Daphnia magna should not be higher than 0.178 ppm.

                                    323

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TABLE 2.  ACUTE TOXICITY EXPERIMENT RESULT OF IPT TO FISH


Species
Common
carp




Silver
carp





Logarithmic
Cone en. value of
ppm Concen . (X)
8.0 0.903
2.7 0.431
0.9 -0.046
0.3 -0.523
0.1 -1.000
Total 	
4.5
2.5
1.4
0.8
0.45
0.25
Total
No. of Mortality
fish (r) r2 96 -hr LC50
10 10 100 4.138
10 11
10 0 0 95% confidence
10 0 0 limits
10 0 0 =4.13810.888
Sr=ll Sr2-101 SE=0.0477
3.180

95% confidence
limits
=3.1800.360
SE=0.025

TABLE 3.  GROWTH EXPERIMENT RESULT OF IPT TO COMMON CARP AND SILVER CARP
                    Beginning          Ending           Increase
Species  Concen. Length,  Weight,  Length,  Weight,  Length,  Weight,  Expla-
          ppm      cm       g       cm       g        cm        g      nation
Common
Carp
Silver
Carp


0.40
0.00
0.80
0.45
0.25
0.00
1.72
1.72
1.80
1.80
1.80
1.80
0.0368
0.0368
0.046
0.046
0.046
0.046
2.03
2.02
2.35
2.46
2.44
2.54
0.085
0.072
0.0742
0.0940
0.0990
0.1175
0.31
0.30
0.55
0.66
0.64
0.74
0.0482
0.0352
0.0282
0.048
0.053
0.072

27 days



28 days
                                     324

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  TABLE  4.   EMBRYONIC  DEVELOPMENT  RESULT  OF  IPT  TO  GRASS  CARP


Concen. ,

8.00

4.50
2.50
1.40
0.80
Control
Number
of Eggs

20

20
20
20
20
20
Survival number3

6hr
20

19
19
20
20
19

9hr
20

19
19
20
20
19

12hr
18

18
19
20
20
18

24hr
18

17
19
18
17
16

28hr
4

6
17
18
17
15

34hr
4

5
17
18
17
15

72hr
4

5
15
17
17
15

96hr
All
died
5
14
17
16
15
 aAt 110 hours,  all had died.
                                    SUMMARY

1.  96-hour LC50 values  of isoprothiolane for common carp  and silver carp
    are 4.138 ppm and 3.180 ppm, respectively.  96-hr LC50 value  multi-
    plied by 0.1 are 0.4138 ppm and 0.1380 ppm, respectively.

2.  0.45 ppm of iosoprothiolane is the maximum no-effect concentration for
    silver carp growth.

3.  4.50 ppm of isoprothiolane is the maximum no-effect concentration for
    embryonic development.
4.
96-hour LC50 value  of isoprothiolane  for  Daphnia magna  is  1.780  ppm.
In conclusion, 0.318 ppm of isoprothiolane is a safe level with
respect to acute toxicity, growth embryonic development of Daphnia
rcagna survival.  Therefore, it is advisable to recommend that 0.318
ppm (0.3 ppm)  of isoprothiolane should be the water quality standards
for fisheries.
                                    325

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TABLE 5.  RESULT OF EXPERIMENT WITH Daphnia magna  (96-hr)'

Concen. ,
ppm
2.50
2.00
1.50
0.84
0.47
0.27
0.15
Control
Daphnia magna
number
10
10
10
10
10
10
10
10

24 hr
10
10
10
10
9.5
10
10
10

48 hr
10
10
10
9
9
10
9
10
Result
72 hr 96 hr 96 hr LC50
0
3
9 8
9 9 1.7826168
8.5 7.5 ~ 1.80
8 9
8 9
9.5 9.5
 "Water temperature 205C.  Results are  for  two  replicate  experiments.
                                      326

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                FISH  ENZYMATIC  INDICATORS  IN AQUATIC TOXICOLOGY

                                      by

                  Xu  Lihong1, Zhang Yongyuan, and Wang Deming




                                 INTRODUCTION

     The response of an intact animal, such as death or a change of reproduc-
tive behavior, has been used as an index in classical toxicological tests.
Such responses, however, occur only when a toxicant concentration is relative-
ly high.  The indices are comparatively simple and approximate.   More and more
physiological and biochemical indices are being introduced into toxicology
now.

     By measuring changes in the activities of different enzymes, changes of
metabolic or hematological parameters, or changes in the components of tissue
of animals inhabiting a certain water body or being exposed to toxicants, a
series of biochemical parameters can be obtained.   Similarly,  when selected
organ or tissue homogenate is exposed to toxicants, in vitro biochemical
parameters can be obtained.  With these biochemical indices, toxicant action
mode could be explained, health of animal in the water could be evaluated,
early signs of potential pollution could be provided,  fast screening of
toxicity of chemicals could be made,  and effects of long-term exposure to low
toxicant dosage on an animal could be detected.  Also,  the biochemical indices
are helpful for determining the target organ of toxicant action.  Some indices
can be used as specific indicators for the existence of certain pollutants.

     Enzymes are one of the most important macromolecular proteins in the
body.  Some enzymes have already been used as toxicological indices, and
others are potentially useful.   The reaction between key enzymes of organisms
and toxicants has become a topic of mutual interest to toxicologists and
biochemists.

     Adenosine triphosphatase (ATPase) plays an important role in ion trans-
portation and osmoregulation of fish.  Acetylcholinesterase (AChE) inactivated
acetylcholine is released from the synamses of cholinergic nerves preventing
"Institute of Hydrobiology,  Academia Sinica,  Wuhan,  PRC

                                     327

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tetanic firing of the post-synaptic nerve.  The effects of toxicants on ATPase
and AChE are presented in this paper.  The possibility of the two enzymes
being used as toxicological indices and their mode of action are discussed.


                             MATERIALS AND METHODS

     Grass carp (Ctenopharyngodon idellus). 11 to 14.5 cm long, were caught in
a fish pond and acclimated in dechlorinated tap water for 3 days.  Paradise
fish (Macropodus opercularis) . 1-year old and 5.9 to 7.8 cm long, were
cultured in a laboratory, where temperature was kept over 15C.  For ATPase
assay (Paxton and Limminger 1983), the relevant tissue was homogenized in cold
buffer (containing 40 mM imidazole, 250 mM sucrose, and 5 mM EDTA at pH 7.1
and 25C).  The homogenate was centrifuged at 3000 rpm for 5 minutes and the
supernatant was kept for enzyme assay.  Supernatant (0.2 ml) and 0.3 ml of
tested toxicant stock solution (0.3 ml of double-distilled water substituted
for the stock solution in the control and in in vivo tests) were incubated in
a reaction mixture at 25C for 20 minutes, then 0.1 ml Na2 ATP was added to
the mixture.  The reaction mixture in a final volume of 3.0 ml contained 40 mM
imidazole, 100 mM Na+,  20 mM K+,  2  mM Mg+  (without Na+ and K+ for NaK-ATPase
assay) and 2 mM NaaATP (pH 7.1,  25C).  After 15 min,  the reaction was stopped
by 30% cold TCA, and the reaction mixture was centrifuged (3000 rpm) for 5
minutes.  The supernatant was kept for inorganic phosphate determination.
Protein concentration of the enzyme was estimated with the Lowry method (Scope
1982).  The specific activity of the enzyme was recorded in terms of micro-
molar Pimg"1 protein hr"1.  For AChE assay, brain tissue was homogenated in
0.01M. phosphate buffer (pH 7.2).  In the final homogenation, 2 mg of brain
tissue per milliliter of homogenate was obtained.  One milliliter of this
homogenate was added to 1 ml of 0.004M bromine acetylcholine, then the mixture
was incubated for 20 minutes at 30C.  The remaining bromine acetylcholine was
measured according to the Hestrin method  (Hestrin 1949).  The specific
                          3 recorded in terms of micromolar bromine
activity
                enzyme
choline mg'1 brain tissue hr"1.
jtyl-
     In the Hg2+ treatment of grass carp,  the fish were exposed to 0.2 ppm Hg2+
(15C, pH 7.8).  After 24 hours, NaKMg-ATPase activity of various tissues were
measured for both the experimental group and the control group.

     In the Cu2* treatment of paradise fish,  the fish was  exposed to 0.1 ppm
Cu2* in a flow-through system (21-24C,  pH 7.2-8.0).   After  4 weeks,  brain
AChE activity was measured.   Control  (2.8 ^igCu2+/l) was measured at the same
time.

     LAS (linear alkyl benzylsulfonate) and TCB  (tri-chlorinated biphenyls)
were used as environmental analysis standard reagents.  Others were  analysis
reagents.
                                    RESULTS
     NaKMg-ATPase activities of grass carp caught at different times and
accli-ated 3 days in the laboratory were measured.  Table 1 shows NaKMg-ATPase
activities of gill; Table 2 shows that of kidney.  One-way variance analysis

                                     328

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TABLE 1.  GILL NaKMg-ATPase ACTIVITIES OF GRASS CARP CAUGHT AT DIFFERENT TIMES

Body length, cm Acclimated temperature, C ATPase activity,
mean  SD
11.5-12 19.3-20.5
12.5-13.5 13.0-13.5
12-12.5 13
11.5-13.5 12.2-14.0
12.5-13.5 14.5
13-13.5 17.2-19.0
11-12 19.2-20.2
13.5-14 15.5-18
18.69  1.09
19.69  1.00
17.41  3.15
15.44 1.12
15.57  1.07
16.25  2.33
18.28  1.95
19.58  3.48
 was  carried out.   When the significance level was 0.05,  there is no sig-
 nificant difference in activities of NaKMg-ATPase for both gill and kidney
 between different groups of fish.

      Plotting the probit of percentage of inhibition of toxicants on enzyme
 and logarithmic concentration of toxicants,  we obtained straight lines (Figure
 1).   I50, 50% inhibition concentration  of toxicant on ATPase, was calculated
 from a linear equation.

                a:  Hg2+ 1=4.70+0.51 InC  I50    1.8 ppm (kidney)
                b:  LAS  1=4.14+0.63 InC  I50    3.9 ppm (gill)
                c:  TCB  1=3.39+0.55 InC  I50   18.4 ppm (gill)
                d:  Cd2+ 1=1.56+0.71 InC  I50  127.2 ppm (kidney)

 I is the probit of percentage of inhibition of toxicant.  C is the concentra-
 tion of toxicant.

      Effects of Hg2+  on NaKMg-ATPase, NaK-ATPase,  and Mg-ATPase are shown by
 plotting inhibition percentage and toxicant concentration in Figure 2.  As
 evident from the figure, the inhibition of Hg2+  on NaK-ATPase was the  strong-
 est.

      After 24 hours of exposure  to Hg2+,  gill and kidney NaKMg-ATPase  in grass
 carp were measured and the results are shown in Table 3.  At the significance
 level of 0.05, there is no significant difference between the  test and the
 control groups.
                                      329

-------
 TABLE 2.   KIDNEY NaKMg-ATPase ACTIVITIES  OF GRASS  CARP CAUGHT  AT  DIFFERENT
           TIMES
 Body length,  cm
Acclimated temperature, C
ATPase activity,
    mean  SD
11.5-12
12-125
11.5-13.5
12.3-13
19.3-20.5
12-13
12.2-14
17.2-19
20.25  0.94
26.09  0.75
24.29  4.12
24.61  1.46
     After 4 weeks of exposure to Cu2+,  brain AChE  activities  of paradise  fish
were measured and the results are presented in Table 4.  At the significance
level of 0.05, there is no significant difference between the test and the
control groups.  It seems that the non-neurotoxic chemical has no effect on
AChE.
                                  DISCUSSION

     Since the 1960s, studies of the reaction of ATPase to toxicants,both in
vivo and in vitro have been carried out in large numbers by scientists.
Different organisms were used as experimental materials,  a wide variety of
chemicals were studied, and some work even delved into the study of enzyme
dynamics.  In these studies, the probable mechanisms of the effect of
toxicants on ATPase have been scrutinized, and the mode of intoxication and
the target organ of the toxicants were deduced.  Because quite a number of
chemicals can influence ATPase, such as organochlorine pesticide, heavy
metals, surfactants, DEHP, PCBs and petroleum refinery wastewater (Boese et
al. 1982, Davis et al. 1972, Kuhnert and Kuhnert 1976, Riadel and Christensen
1979, Srivastava et al. 1975, Verma et al. 1979a, Verma et al. 1979b,  Yap et
al. 1971), some investigators proposed that ATPase activity could be used as a
nonspecific indicator in aquatic environment monitoring (Haya and Waiwood
1983).  Because of -the sensitivity of ATPase to toxicants, many investigators
thought that ATPase activity could reflect the damage of a sublethal
concentration toxicant to an organism and predict potential pollution of
chemicals in the aquatic environment.

     There is a major premise  that fish ATPase could be an indicator.  ATPase
plays a key role in .ionic and  osmotic regulation in fish body, so changes in
ATPase activity might reflect  the health or even the survival of the fish.
                                     330

-------
                  p
                  o"'
                  p
                  <0
                  o  o
                  -  

                     1

                  si
                     o
                     o
o    o
CM    i-
                                                               U)
                                                                             I
                                                                             
                                                                            H
                                                                            ft
                                                                            H
                                                                            H

                                                                             OJ
                            331

-------
        TABLE 3.  IN VIVO EFFECT OF Hg2+ ON GRASS CARP GILL AND
                  KIDNEY ATPase (n=4)
                                  Specific activity, mean  SD
                                 gill
kidney
control
0.2 ppm Hg2+
17.05  3.15 \
16.48  4.54
22.38  5.28
24.08  5.83
     After 3 days acclimation in the laboratory, there is no significant
change in ATPase activity of fish caught from fish-pond.  This is important
for the in vivo tests.  When an in vitro test is carried out, only a small
quantity of tissue is needed.  So, from the view of experimental material, it
is practicable to use grass carp tissue as an enzyme source or to carry out in
vivo tests with grass carp.

     All the toxicants studied in our tests have obvious effects on ATPase
under in vitro conditions.  Such regularities were found on the basis of the
effects of Hg2+,  LAS,  TCB,  CDZ+ on ATPase, that is, linear relationships were
obtained from the plotting of probit of inhibition percentage and logarithm of
toxicants concentration.  This is the same as found in biological tests.  This
means that an in vitro test does show some characteristics of the toxic
action.

     Kg2* had greater inhibition on kidney NaKMg-ATPase  than on gill  NaKMg-
ATPase.  In Figure 2, it is demonstrated that Hg2+ had stronger inhibition on
NaK-ATPase than on Mg-ATPase.  NaK-ATPase activity in the kidney is higher
than in the gill in freshwater fish (Jampol and Epstein 1970).. So, when
NaKMg-ATPase was used as an index, the change of kidney NaKMg-ATPase is more
sensitive than that of gill NaKMg-ATPase.  This indicates the need to search
for several tissues in order to select the most sensitive indicator.  For the
same reason, Cd2+ had great inhibition on kidney NaKMg-ATPase than on gill
NaKMg-ATPase.  Considering the properties of NaK-ATPase on the one hand and
metal on the other hand, metal should affect NaK-ATPase more readily than Mg-
ATPase .

     After 24 hours of exposure to 0.2 ppm Hg2"1",  grass carp  gill and  kidney
NaKMg-ATPase were unaffected and death occurred at 0.4 ppm Hg2+ exposure.
Sastry and Sharma (1980) reported the results of this experiment in which
freshwater fish were exposed to 1.8 ppm Hg+ for 96 hours and to 0.3 ppm Hg2+
for 15 days and 30 days.  ATPase activity increased a little in the 96-hour
acute exposure but decreased slightly in the 15-day and 30-day exposures,
although death occurred in all of the test groups.  Maybe the lethal toxicity
                                     332

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        TABLE 4.  IN VIVO EFFECT OF Cu2+ ON PARADISE FISH BRAIN
                  AChE
                                      Specific activity, mean  SD
                                              (mean  SD)
control
100 jugCu2+/l
1.79  0.36 (n=6)
1.47 + 0.20 (n=5)
of Hg2+ to fish was not the inhibition on gill and kidney ATPase but its
effect on other enzymes or tissues.  For example, death probably resulted from
the suffocation of  fish owing to the deposition of Hg2+ on gill epithelia,  a
situation that would not be revealed in the in vitro test.  So  it is helpful
to integrate the in vitro and the in vivo test results in deciding  the
probable mode of toxicity on organisms.

     From previous  reports and the present research, it is obvious  that ATPase
activity is affected by varied chemicals and a consistent pattern can be
obtained.  The 50%  inhibition concentration of toxicant on enzyme (I50) ,
corresponding to LC50,  also could be  determined.   So,  it  is  feasible to  use
ATPase activity as  a quick nonspecific index, which is very helpful in
toxicity evaluation of chemicals.  Although I50  derived from an in vitro test
cannot tell all the characteristics of chemicals, it does provide preliminary
judgment for the toxic properties of chemicals,  When ATPase activity of fish
exposed to toxicants of low dosage is measured, sublethal effects can be
detected, which provides another basis for understanding the toxic  properties
of chemicals.

     AChE has been  the earliest enzyme adopted in environmental monitoring
(Weiss 1989).  In the late 1940s, the action pattern of organophosphorus
pesticide was found to provide specific inhibition on AChE.  Since  then, AchE
has been used as an index showing the presence of organophosphorus pesticide.
Compared with ATPase, AChE is a relatively specific indicator.  It  can only
reflect the action  of neurotoxic materials.  A specific indicator like AChE is
very important in toxicological research and environmental monitoring.

     Certain biochemical indices represent particular functions.  For example,
the change of ATPase shows the status of the energy metabolism system in the
body, AChE reflects the nerve impulse conduction of organism.   Various
physical function are reflected from specific indices.  Life is maintained by
various activities.  Any process may have ill or even lethal effects on an
organism.  Therefore, one or several indices would not be enough to evaluate
the whole effect of a chemical on an organism.  Death and growth retardation,
of course, can explain in some way the toxicity of a chemical, but no com-
prehensive evaluation can be made from them.   Similarly,  the change of ATPase
                                     333

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               100
                                Concentration of Hg  , ppm
              Figure 2.   Inhibition of Hg2+ on grass carp kidney
                NaKMg-ATPase,  NaK-ATPase,  and Mg-ATPase.
and AChE cannot reflect the harmfulness of a chemical in a comprehensive way.
Consequently, a series of indices that could represent different activities of
an organism should be obtained for the comprehensive evaluation of toxicity of
chemicals.  After establishing the indices, chemicals could be classified
according to the extent of their effects on different indices, e.g., chemicals
having greater effects on ATPase could be classified as "ATPase-toxin"
toxicants, those having greater effects on AChE, "AChE-toxic" toxicant, etc.
Then, not only toxic properties will be understood, but also a certain
knowledge of the action pattern can be gained.  Comprehensive biochemical
indices also can provide parameters for water quality criteria, as well as the
basis and method for rapid screening of chemicals.
                                    SUMMARY

     Several kinds of toxicants had effects on grass carp ATPase.  Plotting of
probit of inhibition percentage and logarithm of toxicant concentration
presents a linear relationship.  I50 obtained from the  plotting is a specific
index of toxicant.  Combined with other  indices, I50 can be  used in a screen-
ing test of chemicals.  Cu2+ had no effect on paradise  fish  brain AChE.   AChE
is a relatively  specific  indicator, which  can be used  as an indicator monitor-?
ing pollution  of neurotoxic toxicants.
                                     334

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                                ACKNOWLEDGMENT

     We are especially grateful to Dr.  Jiankang Liu for his directions.   The
technical assistance of Mrs.  Renzheng Zhou who helped to carry out the test of
Cd2"1"  is gratefully acknowledged.


                                  REFERENCES

Boese, B.L., V.G, Johnson, D.E. Cheopman, andJ.W. Ridlington.  1982.  Effects
     of petroleum refinery wastewater exposure on gill ATPase and selected
     blood parameters in the Pacific staghorn sculpin (Leptocattus armatus).
     Comparative Biochemistry and Physiology.  71C:63-67.

Davis, P.W., J.M. Friedhoff, and G.A. Wedemeyer.  1972.  Organochlorinated
     insecticide, herbicide and polychlorinated biphenyls (PCB) inhibition of
     NaK-ATPase in rainbow trout.  Bulletin of Environmental Contamination and
     Toxicology.  8:69-72.          ,

Haya, K. and B.A. Waiwood.  1983.  Adenylate energy change and ATPase
     activity:  potential biochemical indicators of sublethal effects caused
     by pollutants in aquatic animals.  Advances in Environmental Sciences and
     Technology.  13:307-333.

Hestrin, S.  1949.  The reaction of acetylcholine and other carboxylic acid
     derivatives with hydroxylamine and its analytical application.  Journal
     of Biological Chemistry.  180:249-261.

Jampol, L.M. and F.H. Epstein.  1970.  Sodium potassium-activated adenosine
     triphosphatase and osmotic regulation by fishes.  American Journal of
     Physiology.  218:607-611.

Kuhnert, D.M. and R.B. Kuhnert.  1976.  The effect of in vivo chromium
     exposure on Na/K- and Mg-ATPase activity in several tissues of the
     rainbow trout  (Salmo gairdneri).  Bulletin of Environmental Contamination
     and Toxicology.  15:383-390.

Paxton, B. and B.L. Limminger.  1983.  Altered activities of branchial and
     renal Na/K- and Mg-ATPase in cold-acclimated goldfish (Carassius
     auratus).  Comparative Biochemistry and Physiology 74B:503-506.

Riadel, B. and G. Christensen.  1979.  Effects of selected water toxicants and
     chemical upon adenosine triphosphatase activity in vitro.  Bulletin of
     Environmental Contamination and Toxicology.  23:365-368.

Sastry, K.V. and K. Sharma.  1980.  Effects of mercuric chloride on the
     activities of brain enzymes in a freshwater teleost, Ophiocephalus
      (channa) punctatus.  Archives of Environmental Contamination and
     Toxicology.  9:425-430.

Scope, R.K.  1982.  Protein purification:  principles and practice.  Springer-
     Verlag New York, NY.  pp. 265-266.
                                     335

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Srivastava, S.P., P.K. Seth, and D.K. Agarwal.  1975.  Biochemical effects of
     di-2-ethylhexyl phthalate.  Environmental Physiology and Biochemistry.
     5:178-183.

Verma, S.R. , A.K. Tyagi, N. Pal and R.C. Dalela.  1979a.  In vivo effect of
     the syndets Idet 5L and Swanic 6L on ATPase activity in the teleost,
     Channa punctatus.  Archives of Environmental Contamination and
     Toxicology.  8:241-246.

Verma, S.R. , P. Mohan, A.K. Tyagi, and R.C. Dalela.  1979b.  In vivo response
     of ATPase in few tissues of the fish Mystus vittatus (Ham.) to the
     synthetic detergent Swacofix E. (ABS).  Bulletin of Environmental
     Contamination and Toxicology.  22:327-331.

Weiss, C.M.  1959.  Response of fish to sublethal exposure of organic phos-
     phorus insecticides.   Sewage and Industrial Wastes.  31:580-593.

Yap, H.H., D. Desaiah, and  L.K. Cutkomp.  1971.  Sensitivity of fish ATPase to
     polychlorinated biphenyls.  Nature.  233:61-62.
                                     336

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                RIVER NETWORK WATER QUALITY MODELING USING THE
                  ENHANCED STREAM WATER QUALITY MODEL QUAL2E

                                      by

                                  Qian Song1


                            INTRODUCTION TO QUAL2E

      QUAL2E is a comprehensive and versatile stream water quality model.  It
can simulate up to 13 water quality constituents in any combination desired by
the user.  Constituents that can be simulated are:

             1.  Dissolved oxygen
             2.  Biochemical oxygen demand
             3.  Temperature
             4.  Algae as chlorophyll a
             5.  Organic nitrogen as N
             6.  Ammonia as N
             7 .  Nitrite as N
             8.  Nitrate as N
             9.  Organic phosphorus as P
            10.  Dissolved phosphorus as P
            11.  Coliforms
            12.  Arbitrary nonconservative constituents
            13.  Three conservative constituents

      The model is applicable to dendritic streams that are well mixed.  It
assumes that the major transport mechanisms, advection and dispersion, are
significant only along the main direction of flow (longitudinal axis of the
stream or canal).  It allows for multiple waste discharges, withdrawals,
tributary flows, and incremental inflow and outflow.  It also has the
capability to compute required dilution flows for augmentation to meet any
prespecified dissolved oxygen level.

      The computer program permits simulation of any branching, one-dimen-
sional stream.  The first step in modeling a system is to subdivide the stream
system into reaches, which are stretches of a stream that have uniform
hydraulic characteristics.  Each reach  is then  divided into computational
elements of equal length.  Thus, all  reaches must consist of an integer number
of  computational elements.
"Department of Environmental Sciences, Nanjing University, Nanjing, PRC

                                      337

-------
      There are seven different types of computational elements in the
original model:  (1) headwater element, (2) standard element, (3) element just
upstream from a junction, (4) junction element, (5) last element in system,
(6) input element, and (7) withdrawal element.

      By using QUAL2E, the stream can be conceptualized as a string of
completely mixed reactors (computational elements) that are linked
sequentially to one another via the mechanisms of transport and dispersion.

      The basic equation solved by QUAL2E is the one-dimensional advection-
dispersion mass transport equation, which is numerically integrated over space
and time for each water quality constituent.  This equation includes the
effects of advection, dispersion, dilution, constituent reactions and
interactions, and sources and sinks.  For any constituent, C, this equation
can be written as:
aj
8t
           ac
         3x
                dx  _
                     dx
                                            +  s
(1)
where
      M
      x
      t
      C
       u
 mass
 distance  (L)
- time  (T)
- concentration  (ML"3)
 cross-sectional area (L )
- dispersion coefficient (L2T"X)

= mean velocity  (LT"1)

- external  sources  or sinks  (MT"1)
       The constituent reactions ^nd interreactions are linked by dissolved
 oxygen concentration.  Figure 1 \llustrates the conceptualization of these
 interactions.   The arrows on the figure indicate the direction of normal
 system progression in a moderately polluted environment;  the directions may be
 reversed in some circumstances for some constituents.

       The mathematical relationships that describe the individual reactions
 and interactions are presented by Brown and Barnwell (1985).

       The classical implicit backward difference method is used to solve
 Equation 1.  The finite difference scheme is formulated by considering the
 constituent concentration, C, at four points in the mnemonic scheme, as shown
 in Figure 2.  Three points are required at time n+1 to approximate the spatial
 derivatives.  The temporal derivative is approximated at distance step i.
 After two steps differentiations, we obtain the equation:
                                    in-t-l
                                                                          (2)
                                      338

-------
                                  Atmospheric
                                   Reaeration
                                           /fr/w
            Figure 1.  Major constituent  interactions in QUAL2E.
where a, = -[(AD,),,
       i    L \  L/i-1
                      ViAx,
         = 1.0 + [(ADL)i
 At
LAv,
The values of a, bj.,  cit  and z are all known at time n,  and the cirt'ii terms
are the unknowns at time  step n+1.  An efficient method for solving these
simultaneous linear equations is presented by Brown and Barnwell (1985).


                             IMPROVEMENT OF QUAL2E

      One of the limits in using QUAL2E is -that the river system to be
simulated can only have one  last" element.   That means that the river system

                                     339

-------
cannot have branching elements or "fork" elements, from which simulated
tributaries flow out from a main stem.  And on the other hand, to improve the
water quality, we need to know how much pollutant the river system could
receive while the water quality of the river system still meets the required
level, so that we can know at least how much pollutant should be removed from
the waste discharge.

      To model river system with fork elements, we first need to know the
hydraulics of both the main stem and the tributary.  We assume that the
hydraulics of the river network are known.  (For the fork element, the ratio
of the flow of the main stem and the flow of the tributary, FLWRT are given.)
is:
      We know that, for a standard element i, the basic differential equation
                                                                        (3)
in the case of a fork element, the basic equation becomes
where:
          - Ci*[l-FLWRT(I)]
      Two methods can be used to solve these equations .  Because GJ, and f j  are
several orders smaller then ^ and ~bit  the first method is  to assume that Ci+1
equals Cj ,  and divide Equation 4 into two equations :
                     DOWNSTREAM
UPSTREAM
                       element I + 1
 element i
               1+1
               1-1
                                                      N + 1: t + At/2
                                                       N: t - At/2
                                      I
                    Figure 2.  Classical implicit nodal scheme.

                                    340

-------
 and:
                                                                           (5)
                                                                           (6)
 Now it is easy to use the method presented by Brown and Barnwell (1985)  to
 solve the simultaneous linear equations.   When back-substituting the con-
 centration from the last element,  Equation 4  is used to solve the fork element
 concentration.                                                                 .

       The second method does not use the foregoing  assumption because,  in many
 cases,  Ci+1 does not equal (L.

       Mathematically speaking, simultaneous linear  equations  are always
 solvable, but their efficient solution is  not  easy.   In this  paper,  an
 efficient method for solving those equations  is  presented.

       The method presented by Brown and Barnwell (1985)  is applied before the
 fork element(s),  and when a fork element is met  in  the  process of forward
 substitution, we apply a sub-loop of forward and backward substitution  which
 is shown as follows
       Ci-l  + Wi-iCi -  Gi.j.

        aiCi-i+DiCi+Ci  ' Ct+1+f jCj-Zi

substituting  Equation 7  into Equation 8, we  obtain:

       ci + WtC1+1 = Gi -  FiCj

where: W  = G
        F  =
                                                                           (7)

                                                                           (8)"



                                                                           (9)
                - aiGi.1)/(bi -
and  substituting  Equation 9  to the next basic equation and forward substitut-
ing  the obtained  equation, we  have:
where: F =
        k    kk+1  =  k

               ^-a^.J ,  Fk = a^F^

       k = i+1, i+2,  . . .,  n-1, n

       n = the order of the last element of the reach.
                                                                         (10)
when k = n, k+1 = kk, and kk is the element order of  the next  type  3  or type 5
element, Equation 10 becomes:      '

                                      341

-------
                  = Gn  -  FnCd
      The forward substitution stopped when the next type 3 or  type  5  element
is met.  Then back substitution is started from the element.  The back
substitute  equations are shown as follows
            A    i>n    wr                                                 (n)
       Gk - Ak - BfcCj - n^i^^
where: Ak - Gk - Ak+1Wk,  Bk = Fk - Bk+1Wk, Hk = -WkHk+1
       ^ - Gn> Bn = Fn, ^ = n
       k -  n-1,  n-2,  .... i+1, i
when the back substitution stopped at the element i+1, we have:
                     R   r   H  G                                           (12)
       Ci-H ~ Ai+l  "  "i+l^j  "   i+1 *&
Substituting Equations  12 and 11  into the equations  for  the tributary, we
have:
                                                                             (13)
where: W'k - ck/MM, G'k
       MM - Bi^-a,
       G'j -  (Zj-
       k -  j+1, j+2	kk-2,  kk-1
When k - kk-1, we have:
             ,  TT   r*  	 /-<>       v
          t-i +  ^-1^ - t. ujj-i   S ]&-
                                         ,  F'k - -akF'k_i-
                                         -ajF'if
                           c                                                 <14)
 where: EJ^.J,  - G'^-i,  L^-i = F'j^-i + Wj^-i
 The back substitution of Equation 14 obtained the relationship of Cj  and Ckk:
        C -  E-  - L-Ckk                                                      (15)
 where: ^ =  G'k - W'kEk+1,  1^.  = F'k -  W'kLk+i
        k - kk-2, kk-3,  ...,  j-1,  j
        Substituting Equation  15 to Equation 10, we have:
                    - G
                                                                              (16)
  where: W'B = WB-FnLd, G'n - Gn-FnEd
                                        342

-------
 and rewriting Equation 14:
 Now both the main stream and the tributary down  stream from the fork element
 are ready to go back to the main loop of the  forward and backward substitution.

       By adding another two kinds of computational  element --type 9 (fork) and
 type 8 (element just downstream from a fork on the  tributary )-- the QUAL2E
 source code is modified so that it could simulate the net -like river systems.

       To calculate the maximum point source loads ,  we first need to linearize
 Equations 2 .   The process of linearization is shown as follows

       (1).   Assume that in one reach, the concentration of the constituent at
 the last element is equal to the concentration of the first element of the next
 reach:
                cn =  c
                     n+i
                                                                          (17)
       (2).   Let
and
where:
then we have:
where:
                Vl - -(ba+


                An-2 = (Zn-l-bn-lAn-l)/an-l


                Bn-2 - -(bn-lVl+Cn-lVan-l


                 Ai =  (Zi+i-bi+iAi+i-ci+1Ai+2)/ai+1


                 Bi =  -(bi+iBi+i+ci+iBi+2)/ai+i

                 n =  the number of element of the reach;

                 i -. 1,  2,  3,  ...,  n-3
                  ci = the concentration of the constituent at the first element
                     of the  reach

                  Cn = the concentration of the constituent at the nth element
                         of the  reach.
Rewrite Equation 18 as :
                  cn = Ai +
                                                                          (19)
where:
                  At --
                          /%! for reach i

                          !  for reach i

                                     343

-------
      After the linearization,  we put the first basic equation of every reach
together as a set of  simultaneous equations.   Substituting Equation 19 into
these simultaneous  equations, we have:
where:
                    i,i
                   Ci.2
       the concentration  of  the  last element of reach i-1

       the concentration  of  the  first element of reach i

       the concentration  of  the  second element of reach i
Because G is several orders  smaller,  and the  b and  ai( and Citi  and C1(2 are
not of very much difference,  we assume that Cij2 = Cij:L.  Substituting Equation
19 for Equation 20, we  have:
                                                    and
                                                       (21)
or

where :
AA  
                               ,  i =  2,3,...,n

                   BB = bi + cif i = 1,2, . . . ,n

                   ZZi = Z - a^i-i,  i = l,2,...,n

Rewriting  Equation 21  in matrix form,

                   AB * C - Z  + Z
                                                       (22)
where:
   AB -
BI
A/
(
(

r
*l
^2 BB2
) AA3 BB3
) 0 AA4
: 0 -
i n n_--. ----------
          C =  (^iiii C2>ii  -  -  -  )  Cn,
          Zx -  (0,  -az&!, -a3A2,  -  - -
          Z =  (Zj_,  Z2, - - - ,  Zjj)


      Under  steady-state conditions,
                                      344

-------
 where:
                   Si
                  P..
                       external  sources or  sinks
                       internal sources or sinks
Let P = (Pl,  p2,  ...,  Pn)*, Z
                                   + P, S - (Slf.S2,  ...,  Sn)T and
                      v =
                                              V.,
Substituting Z, Z
equation, we have:
                 lf
                            and V into Equation 22 and rearranging the
                      V*AB*C-V*Z,
                                                                          (23)
      When substituting  the  river water  quality  criteria  C* to Equation 23,
we have the maximum point  load allowed in  the river network:

                  S* = V * AB * C* - V *  Z2                               (24)

      The first step in  computing the maximum point source load  is  to
redefine the reaches of  the  river system.  The input element is  the first
element of the reach.

      Using Equation 19  to Equation 24,  an additional subroutine was coded
for QUAL2E, which was called MAXCNT.  Using the  improved model,  a case study
is performed.

      Figure 3 shows the stream network  of computational elements and
reaches.  The river system contains one  fork element.  The input data set for
the simulation is a modification from one  of the demonstration input data
sets shown at the US EPA's QUAL2E workshop at Nanjing University in 1985.
Figure 4 shows the result of BOD simulation.  Figure 5 shows the river system
and the result of the BOD simulation of  the above-mentioned workshop demon-
stration.   Table 1 shows the BOD point source loads (P.L.) and the  simulation
result (concentration)  at the first element of the reach.   When substituting
the concentrations in Table 1 as C*,  the  maximum  point  source  loads  (MPL)  of
the river network should be the point source loads in Table 1.   Table 2 shows
the result.
                                 REFERENCE
 Brown,  L.  C.  and T.  0.  Barnwell.   1985.   Computer Program Documentation
       for  the Enhanced Water Quality Model QUAL2E.   U.S.  Environmental
       Protection Agency,  Athens,  GA.   EPA/600/3-85/065.
                                    345

-------
                    Reach
                    Number
                   Junction
                     1
             Figure 3.  Stream network of  computational elements

               and reaches.
TABLE 1.  POINT  SOURCE LOADS

Reach
1 2
345 6789 10
P.L., kg/day     0     1865    0
0000
BOD, mg/L      2.19   21.58  1.97  18.78  5.26  0.69   0.71   9.79  9.31  9.14
                                      346

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TABLE 2.  RESULTS
                                        Reach
                 12      3     45      6     7     89    10
CA,  mg/L        2.2  21.6   2.0   18.8   5.3   0.7   0.7    9.8   9.3    9.1
MPL kg/day       0   1870    000     0     0     00.0
                   BOD (mg/L)
                         21.58
                                                                  9.79
                    Figure 4.  BOD simulation result.
                                  347

-------
         BOD (mg/L)
               21.58
                                                           8.58
Figure  5.   River system  and BOD simulation results from
  EPA's QUAL2E workshop,  Nanjing, 1985.
                          348
                         U.S. GOVERNMENT PRINTING OFFICE: 1990 748- 159/ 00436

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