EPA-600/3-83-065
August 1983
THE POTENTIAL FOR BIOLOGICAL CONTROLS OF
Cladophora glomerata
by
Ruth Patrick, Charles F. Rhyne, R. William Richardson, III,
Richard A. Larson, Thomas L. Bott, arid Kurt Rogenmuser
Academy of Natural Sciences of Philadelphia
Philadelphia, PA 19103
EPA Grant No. R-805106
EPA Project Officer
Nelson Thomas
Grosse lie Laboratory
Grosse lie, MI 48138
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MM 55804
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technical report data
{I'lvasc read Instructions on the wwii be/are completing)
1 REPORT NO. 2..
EPA-600/3-83-065
3 RECIPIENT'S ACCESSXON NO V
psS3 ¦,)- ..s / :xi r
4, TITLE AND SUBTITLE
The Potential for Biological Controls of
Cladophora glooerata
5. REPORT DATE
Auqust 1983
6, PERFORMING ORG ANI2AT1GN CODE
7 authors) Ruth Patrick, Charles F. Rhyne. R. William
Richardson, III, Richard A. Larson, Thomas L, Bott,
and Kurt Roaenmuser
H PERFORMING ORGANIZATION REPORT NO,
9 PERFORMING ORGANIZATION NAME AND AOORESS
Academy of Natural Sciences of Philadelphia
Philadelphia, Pennsylvania 19103
10. PROGRAM ELEMENT NO,
11, CONTRACT/GBANT NO,
Grant R-805106
12, SPONSORING agency name and address
U.S. Environmental Protection Agency
Environmental Research Laboratory-Ruluth
6201 Congdon Boulevard
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PE RIOO COVEfiiD
14, SPONSORING AGENCY CODE
EPA/600/03
15, SUPPLEMENTARY NOTES
16, ABSTRACT Th« p*rpo*a o> tt>f » r«s#arch program «a& to ottara loa what?»®r cr not ttiar a vara plural biolog I eal
controls o# CUdggftora glonwrata that ooyld ba davalopad, Tao avaiwaa o# r#s*arch aara purfuad. CHa *as
to study Macroscopic organises that «r« known to occur In tt»a Gr«et lakas srae to &»« If my of Wwaa
would or#far C« qioraarata a* a tcsod soy res ana under what conditions tfte desirability ef C» qlame&fa,
cou?d ba Incraasad. Tha second approach was Ho study mlcroargafilsffls, particularly Iuk^I, which wars of
ccflwoft occurranoa 1ft acual'lt acosys tarns to datamtl r* If thay function a* controls for C* q trcmaratfl ¦
rtia results of that* studies show that £« gl qftftrofe mb* a poor food for Phyta hatarovtropha,
Qrcortactaa proplnpuus, flans plplans, Ictalurus punctstus, and Pltwaphalas promalas. Mot only would thasa
spociaiTrwt i»t ttia food, but'if thgy did *flt tVa fori th©ra s,o«»w*J to to a dafetarlou* affect. In tf>a
stud las with snails It was avldant that agg product Ion *»as $raatly curtailad on tha C. qlomerata 0 iat»
Tha> unialgaS C, qlomarata was lass prafarrad than ftuC. qlonarata with apiphytas. Th® affects <*v
th» organisms; othar than Pftysa haterosfropha «&rs mainly losses In *alght. TN» crayfish did not «»©lt avan
*h#n tK# ayastai kjs «ar« nwnovod, and tt»a frog Tad pota* did not vtov any Slgnk of matamerphoslfig.. AH
organ I whs vtaadl I y lost wofgut during tha #*.pw J-wants*
Ari cm am J na1 Ion ol tt»a tactical cxxi&T I tuaot& of tfaMs of £. qlc rf or«rrcas wara mainly that tha diatoms had larger amounts of ami no acids, such 3*
»«- I oa,. aspar ? ic acf d, and tji utamlc acid. AJ so tha irm and oonbi/iad fatty acids as d J a tons war a
d! f far ant than those of CIadoflhora^ The diatoms war* dcnl natad by Cjg ®nd Combl nad tatty
acids* tfharaas His unsaturetad C«o 'atty acld^ particularly IInolanlc acid, m* common In C.
glgweitg. Of tin fr»« fatty ©elds ttit dlato^-domliieted p«r iphyton c&ntBlnad I ft I y Cj| and
lsu>»arst kMI« C. qlomarpfa ccf\tqlngd nast|y It *ai Intarastlrtg to nofa tfiat of *h* 5^>P«*T~cf»aly too. It l& known Ircm othw a
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DISCLAIMER
Although the research described in this article has been funded
wholly or in part by the United States Environmental Protection
Agency through Grant #80 5106 to the Academy of Natural Sciences,
it has not been subjected to the Agency's required peer and policy
review and, therefore, does riot necessarily reflect the views of
the Agency and no official endorsement should be inferred.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
ii
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The purpose of this research program sponsored under Grant R-805106
of EPA was to determine whether or not there were natural biological controls
of Cladophora qlomerata that could be developed. Two avenues of research
were pursued. One was to study macroscopic organisms that were known to
occur in the Great Lakes area to see if any of them would prefer C_. qlomerata
as a food source and under what conditions the desirability of C. qlomerata
could be increased. The second approach was to study microorganisms, par-"
ticularly fungi, which were of common occurrence in aquatic ecosystems.
The results of these studies show that C. qlomerata was a poor food
for Physa heterostrooha, Orconectss propinquus, Rana pipiens, ictalurus
punctatus, and Pimephales promelas. Not only would these species not eat
tne food, but if they did eat the food there seemed to be a deleterious
effect. In the studies with snails it was evident that egg production was
greatly curtailed on the C, qlomerata. diet.
The unialgal C. qlomerata was less preferred than the C. Qlomerata with
epiphytes. The effects on the organisms other than Physa hetarostropha were
mainly losses in weight. The' crayfish did not mo 11 even when the eyesialks
were removed, and the frog tadpoles did not show any signs of metamorphosing.
All organisms steadily lost weight during the experiments.
An examination of the chemical constituents of cells of C, qlomerata
and of diatoms showed that they differed considerably from diatom-dominated
peripnyton, which was the preferred diet in all experiments. These differ-
ences were mainly that the diatoms had larger amounts of amino acids, such
as serine, aspartic acid, and glutamic acid. Also the free and combined
fatty acids of diatoms were different than those of Cladophora. The diatoms
were dominated by Cl6 and C?Q combined fatty acids, whereas the unsaturated
CIS fatty acid, particularly 1inolenic acid, was common in C. qlomerata. Of
the free fatty acids the diatom-dominated periphyton contained mainly C]4
and C]6 isomers, while C_. glomerata contained mostly C]g. It was interest-
ing to note that of the short-chain fatty acids, C. qlomerata contained Cf2
acids, particularly lauric acid, and this was absent from the perichyton.
It is known from other experiments that lauric acid is toxic to several
organisms.
The second avenue of approach was to study the effect of fungi as para-
sites on C_. qlomerata. One fungus, Acremonium ki 1 iense (Fungi Imperfacti )
was found to have an antagonistic effect on C. alomerata. The effect varied
in various experiments; chose carried out in the summer months showing the
i i i
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greatest antagonistic effect. It may be that the lesser effect in the fall
was due to temperature and other environmental conditions or to the fact
that A, ki1iensa had reduced virulence. This toxin is present in the super-
natant derived from cultures of C_. dor,erata that had been damaged by this
pathogen. It is water soluble and can withstand heat up to 30°C.
i v
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)
CONTENTS
Page
Disclaimer -j
Foreward. i •}
Abstract. ill
Tables. vi
Figures vi i i
Acknowledgements. ix
Executive Summary ]
Introduction 5
Recommendstion. 6
Part I - The potential of Cladophora qlomerata as a satisfactory
food for various invertebrates and fish. . . 7
Introduction 7
Review of Pertinent Literature ........ , 8 )
Methods and Procedures. . 14
Results . 23
Tables. 33
Literature Cited 134
Part II - Chemical composition of Cladophora. qlomerata (L.) Kutz.
cells 7 . ............. 137
Introduction 137
Methods and Procedures. ........ .... 138
Results 140
Tables 146
Literature Cited. . 150
Part III - A fungal pathogen for possible biological control of
Cladophora qlomerata . . ........... 15d
Introduction. . 154
Literature Review . 154
Methods and Procedures. ... ..... 155
Results 157
Tables. .163
Figures . 175
Literature Cited. ........................ 181
v
)
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)
TABLES
Number Page
Part I -
1. Data on snail tissue, 1978 experiments 33
2. Data on which calorie calculations are based, 1378 snail
tissue experiments 37
3. Data on snail tissue, 1977 experiments, 39
4. Data on which calorie calculations are based, 1977 snail
tissue experiments, . , ... 43
5. Data an snail tissue, 1976 experiments. 45
8, Data on which calorie calculations are based, 1976 snail
tissue experiments, 47
7. Snail respiration, 1978 experiments , » 49
8. Snail respiration, 1977 experiments 50
9. Snail food consumption, 1978 experiments. 51
10. Data for basis of conversions from dry weight to calories,
1973 snail food experiments 53
11. Snail fecundity, 1978 experiments 55
12. Snail fecundity, 1977 experiments 56
13. Snail fecundity, 1976 experiments 57
14. Numbers, weights, and caloric content of snail eggs,
1978 experiment 59
15. Snail feces, 1977-73 experiments. , . 60
16. Utilization of calories ingested per snail, 1978 experiment ... 62
17. Utilization or calories assimilated per snail, 1977 experiment. . 63
18. Individual weights and caloric values, 1977 crayfish experiment . 64
19. Data for basis of conversion from dry weight to calories,
1977 crayfish tissue experiment ....... ... 83
20. Respiration, 1977 crayfish experiment ...... ... 35
21. Food consumption, 1977 crayfish experiment. ........... 86
22. Data for basis of conversions from dry weight to calories,
1977 crayfish food experiment - 97
23. Cladoohora breakage, 1977 exDerimen t. 38
24. Crayfish faces, 1977 experiment 101
25. Utilization of calories per crayfish, 1977 crayfish experiment. . 103
26. Crayfish molting experiment, 1977-73. ^05
27. Individual tissue weights, 1977 tadpole experiment. ....... 106
28. Respiration, 1977 tadpole experiment. .............. 109
29. Tissue weights, 1977 catfish experiment ............. 110
30. Fathead minnow tissue weights, 1978 experiment. ......... H2
)
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Number Page )
31. Data for basis of conversions from dry weight to calories,
1978 fathead minnow tissue experiment 12!
32. Respiration, 1978 fathead minnow experiment ........... 123
33. Food consumption, 1978 fathead minnow experiment. ........ 125
34. Data for basis of conversions from dry weight to calories,
1978 fathead minnow food experiment 130
35. utilization of calories per fathead minnow. . 13
36. Radioactivity in body parts and calculated assimilation ratios
on successive days for fathead minnows fed three diets. . . , . . 132
37. Partial radioactivi ty budgets for diet studies of fathead
minnows ........ ............ 133
Part II -
1. Nutrient content of filamentous algae, periphyton, and diatoms. . 146
2. Amino acid composition of proteins of some filamentous algae
and diatoms 147
3. Free fatty acids of CIadophora and mixed periphyton ....... 148
4. Growth of Saccharomyces cerevisiae exposed to algal extracts. . . 149
Part III -
1. Viruses, bacteria, and fungi with antagonistic activity against
algae (other than CI adophora) 163
2. Assay of effects of isolate PR-1 on C_. qlomerata in various
incubation media and in the presence of antibiotics .(strepto-
mycin sulfate, final concentration 0.06 mg/ml, and penicillin g,
0.01 mg/ml, 1625 units/mg). 167
3. Effect of A. ki1iense on £. qlomerata. Results of assays with
positive results. .168
4." Occurrence and condition of CIadophora and Ulothrix following
exposure to A. kiliense in flowing water microcosms, July 17-31,
1978. Inoculated microcosms received additions as in Figure 4. . 169
5. Occurrence and condition of Cladophora and Ulothrix following
exposure to A. ki1iense in flowing water and static microcosms,
August 11-31 , 1978 170
6. Concentration of selected chemical parameters in Lititz Creek
water added to microcosms .................... 171
7. Concentrations of selected chemical parameters in water removed
from microcosms, August 11-31, 1973, Microcosms 1 and 3 were
inoculated with A. ki1iense: 2 and 4 were controls. ....... 172
8. Concentration of selected chemical parameters in Lititz Creek
water and water removed from microcosm, October 10-20, 1978 . . . 173
9. Occurrence and condition of Cladophora and U1othrix following
exposure to A. ki1iense in flowing water microcosms, October
10-2C, 1973 174
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)
)
FIGURES
Number Page
Part III j.
1. Effects of A. kitiense (8.32 x 10 conidia/ml U) on cell
condition, chlorophyll a_ content and dry weight of £. glomerata
from stock culture arid Lititz Creek . 175
2. Effects of fiiter-steri1ized supernatant fluids from assay
flasks in which Cladophora was damaged on £. g1omerata from
stock culture 176
3. Effect of supernatant from A, ki1iense grown in Czapek-Dox
broth at 22°C for 5 days on C. glomerata call condition,
chlorophyll _a content and dry weight. 177
4. Condition of Cladophora cells in flowing water microcosms
inoculated with A. ki1iense conidia; 113, 6.25, and 29,2 x
104/ml on 8/14, 8/17, and 8/21 respectively, ana with mycelium
and conidia after that (0,32 and 0.39 g wet weight/1 to stream I,
0.02 and 0.39 g/1 to stream III on 8/24 and 8/28 respectively . , 178
5. Condition of Cladophora eel 1s in statis microcosms inoculated
with A. ki1iense conidia; (113, 6.25, and 28.8 x 104/ml on
8/14, 8/17, and 8/21 respectively) and with mycelium and conidia
after that (0.64 and 0.78 g wet weight/1 to lake I, 0.02 and
0.78 g/1 to lake III on 8.24 and 3/28 respectively) 179
6. Condition of Cladophora cells in flowing water microcosms
inoculated with A. kiliense mycelium and conidia; 0.07-0.21 g
wet weight/I added to microcosm I, 0.03-0.10 g/1 to microcosm
III, 0.01-0.02 g/1 to microcosm IV, C (II) = control 180
)
vi ii
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ACKNOWLEDGEMENTS
This research was made possible by grant £R-805106 from the Environmental
Protection Agency, We acknowledge with thanks the constructive help of our
project officer, Dr. Nelson Thomas.
The authors wish to express their appreciation to Dr. Raymond Jezerinac,
who provided the crayfish for the study; Dr. E. M. Zipf, who aided in the
removal of crayfish eyestalks for the molting studies, and Or, Walter Gams
of the Institut fur Schimmelspi1ze, Baarn, the Netherlands, who confirmed
our generic identification of Acremonium ki1iense. Mrs. Lee Anderson has
carried out the editing and preparation of the manuscript.
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EXECUTIVE SUMMARY
In this research two avenues were followed in trying to identify a bio-
logical control of Cladophora qlomerata, One of these was to study macro-
organisms common to the Great Lakes region which previous research has shown
select algae as a considerable part, if not the main part, of their diets.
The other avenue of research was to find a microorganism which by parasitism
would destroy C. glomerata. It was realized that this organism must be
fairly specific for C,. qlomerata, and if present in fairly large amounts
'would not destroy other algae that were desirable food sources.
Studies with Macrooroanisms
The organisms selected for the macroorgani sm studies we re the pond
snai 1, Physa heterostropha; the crayfish, Orconectes prooinquus; the tadpole,
Rana pioiens; the channel catfish, Ictalurus punctatus; and the fathead min-
now, Pimephales promelas.
All of these organisms feed more or less extensively on algae. We did
not want to use any exotic species to the Great Lakes, as these mignt pose
other problems if introduced. Some of these, such as Tilapia aurea, are
known to be prodigious algal feeders. In these experiments most of the
work was done on Physa heterostropha, Orconectes propinquus, and Pimephales
promelas.
All of the studies showed that Cladophora qlomerata was not a satis-
factory diet. Qiatom-dominated periphyton was a more satisfactory diet, as
the organisms had a better rate of growth on that diet than on Cladophora
plus epiphytes. The Cladophora plus epiphytes tended to be a better food
source than unialgal Cladophora, In the experiments using Physa hetero-
stropha some snails were starved. Their decline in weight and their respir-
ation rates tended to be greater when starved than when fed unialgal £,
qlomerata.
The results of the experiments with Physa heterostrooha showed that the
relative value of the three diets presented were diatom-dominated periphy-
ton > Cladophora plus epiphytes > unialgal Cladophora, The respiration
rates of snails fed Cladophora or Cladoohora plus epiphytes were less than
those on the periphyton diet. There was not much difference between diets
of Cladoohora plus epiphytes and unialgal Cladophora,
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The reproductive success was much greater on the diatom-dominated peri-
phyton diet and was very poor on the unialgal Cladoohora diet. Egg-laying
greatly decreased in a relatively few days (11-15 dayiT~aftar the experiments
started. However, the eggs produced from all three diets had similar caloric
content. These experiments were repeated at various seasons of the year and
showed similar results.
The experiment with Orconectes propirtquus showed that none of the diets
were very satisfactory, but that the diatom-dominated periphyton was much
better than the other two diets. The crayfish molted for only a short time
after the experiment started on July 28. This may he due to the normal life
cycle trends as crayfish tend to hibernate in the cooler months. However,
the temperature at which the experiments were run was similar to summer
temperatures. Molting was forced by removing the eyestalks and associated
X organ-sinus gland. Following this operation in January the females on the
periphyton diet all molted.
The respiration rates on all three diets declined during the experiments,
and the rates of the females tended to be greater than the males.
The experiments with the tadpoles of Rana pipiens resulted in increases
in size, ana there were signs of metamorphosis on the diatom-dominated peri -
phyto" diet and on Cladophora plus epiphytes diet. Those fed on unialgal
Cladophora grew very little and showed no signs of metamorphosis. The respir-
ation rates for tadpoles were similar on the periphyton and Cladoshora plus
epiphytes diets, and were much lower for tadpoles on the Claaoonora diet.
These tadpoles were very small and weighed very little, as they had lost
weight.
The experiments with the channel catfish (Ictalurus punctatus) showed a
similar pattern. The diatom-dominated diet was more satisfactory than the
unialgal Cladophora diet. However, when compared with the fish fed on pre-
pared fish food, none of the diets were as satisfactory.
The fathead minnow (Pimephales promelas) ingested and assimilated much
more of the diatom-dominated periphyton than the Cladophora plus epiphytes
or the unialgal Cladoohora. The fish fed the last two diets decreased in
weight,with those on unialgal Cladophora showing the greatest decrease. The
respiration rates were variable for fish on the periphyton diet. It was
highest for those fed on Cladophora plus epiphytes. On both the Cladophora
diets the cal/gio/hr (Table 32} were higher than on the periphyton diet.
These results led us to an examination of the relative food value of
Cladophora qlomerata and diatom-dominated periphyton. Oetermination of the
calories/mg showed similar amounts.
An analysis of the chemical composition of diatoms versus Cladoohora
showed some interesting results. The amino acids of diatoms that were in
2
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larger amounts were serine, aspartic acid, and glutamic acid, and a small
amount of cystine which was absent in C. glomerata.
The combined fatty acids in diatoms were dominated by Cjg and C20
acids. Unsaturated C1s fatty acids, particularly linoienic acid, was much
lower in diatoms. C. glomerata was more like green plants, having Cl8 un-
saturated fatty acids, particularly linoienic acid, and low concentrations
of C15 and C72 acids.
Periphyton contained more free fatty acids than C. glomerata, with the
majority being Ci4 and C]g isomers, while those present in Cladoonora were
mostly C]g.
Of the short chain free fatty acids CIadophora contained C12 (lauric
acid), which was absent in periphyton, and C]q (caoric acid) was in peri-
phyton but not in CIadophora. Lauric acid has been shown from many reports
to be toxic.
Experiments with yeasts (Saccharomyces cervisiae; showed that the extract
of CIadophora containing this acid was toxic to yeast. This suggests that
the presence of this acid may be in part the reason why C. glomerata was an
unsatisfactory food.
As to soluble carbohydrates, C. .glomerata contained 4,51, 'whereas the
periphyton contained 21.62. There was also more phosphorus in the periphyton.
The results of the studies show that there are several differences in
the chemical composition of the periphyton dominated by diatoms and unialga1
£• glomerata. These differences would result in differences in nutrient value
of these two food sources. In C. glomerata lauric acid was present, which
may be toxic to the organisms studied. Further research is needed to verify
this potential toxicity.
Studies with Microorganisms
The second avenue of approach was to determine whether fungi commonly
occurring in the aquatic environment would have an antagonistic effect on
C.. glomerata. One isolate, Acremonium ki 11 ense (Fungi Imperfect!) was found
to have an antagonistic effect on C. glomerata. This fungus was isolated
from the stock culture of Cladophora where it was present in small amounts.
This fungus in flask cultures deleteriously affected the call condition of £.
glomerata and caused a reduction in chlorophyll a_ when the stock cultures of
CIadoohora were used. Cladoohora that was isolated from tititz Creek was
more susceptible to the fungus than Cladophora from stock cultures. In the
Lititz Creek community experiments there was not as good a correlation of
loss of chlorophyll a_ with damage to the cells. This may be due to the fact
that small amounts of algae were present that were not damaged by A. kiliense
and thus would obscure the loss of chlorophyll a.. Therefore, it was decided
that direct microscopic examination of the cells was the most reliable way
to determine the damage of this fungus.
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Supernatant culture medium from the flasks where Cladophora had been
damaged when added to other undamaged Cladophora cells also had deleteri-
ous effects on the algae. This antagonistic substance from A, ki7iense is
water soluble and does not seem to be damaged by heating to 80°C. Cultures
of A. ki1iense were raised on tzapek-Qox broth, and Cladophora was inoculated
with conidia, but these did not produce damage.
Microcosm experiments were then sat up in which the C. glomerata-domin-
ated communi tes were exposed to varying concentrations of A. ki1iense. The
first experiment was run from July 17 to August 1, 1978, and the second was
run from August U to August 31, 1978. In both cases there was a striking
effect of these fungi on the Cladophora, although diatoms and Ulothrix
living in the microcosms were unaffected. In fact, the Ulothrix divided
rapidly and occupied the habitats formerly occupied by Z. glomerata. Scene-
desmus and Soirocyra were also present, as were protozoans and rotifers, in
the experiment carried out August 11-31. They did not seem to be affected
by this fungus. Static microcosms produced similar results.
These experiments were then repeated in the fall of the year during the
period from September 18-October 4. In these experiments the effect was not
as apparent. The experiment was again repeated October 10-20 in a flowing
stream microcosm, and although there was some effect it was not nearly as
dramatic as in August. It may have been the difference in day length, in
temperature, or a variety of environmental conditions that reduced the an-
tagonistic effect of the fungus. It also may be that the fungus in culture
loses its virulence, and therefore is not as affective as fleshly isolated
culturas.
More studies need to be carried out to determine just what the chemical
is that is causing this effect, whether the fungus loses its virulence on
cultaring, and/or what other environmental conditions may affect its virulence.
An experiment was run toward the and of the study using 100-400 mg/1 of
cephalosporin. This produced some chlorosis, but in concentrations of 10-
50 rng/1 had no effect. Whether this is the ccmoound that produced the an-
tagonistic effect in the previous experiments is not known, and further studies
need to be carried out.
From these various types of studies it would appear that microscopic
organisms have greater promise as effective biological controls than the
larger organisms.
4
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SECTION 1
INTRODUCTION
The purpose of this research program was to determine if biological
controls of the growth of CIadophora qlomerata could be developed. This
alga is becoming a nuisance growth in many of the Great Lakes. It is also
a nuisance growth in many of the rivers of the United States. This alga
belongs to the Family CIadophoraceae, Order CIadophora1es, in the Division
Chlorophyta.
Two methods of approach have been used in trying to answer this question-
One was an attempt to identify one of the naturally occurring species in the
Great Lakes that would preferentially feed on CIadophora g1omsrata. In
other words, are there species in this area for which C, qlomerata is a
satisfactory food?
The second approach was to determine if there was a microbial parasite
naturally occurring in the area or commonly occurring in natural environ-
ments that if encouraged could become a control of this alga.
The need to determine the chemical characteristics of C. glprorata
became evident during the course of these experiments-
Si nee the procedures and methods of study were so different for these
two approaches to the problem, the report is divided into three sections
for discussion. The executive summary contains a general discussion of
these sections.
The three sections are:
I, The potential of CIadophora qlomerata as a satisfactory food
for various invertebrates and fish, by Ruth Patrick, Charles
F. Rhyne, and R. William Richardson, III.
II. Chemical composition of Cladophora qlomerata eel Is, by Richard
A. Larson
III. Fungal pathogen for possible biological control of Cladophora
qlomerata, by Thomas L. Bott and Kurt Rogenmuser
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SECTION 3
RECOMMENDATIONS
For developing a biological control the results of this research indi-
cate that the fungus Acremonium ki1iense may be a potential biological
control. However, a good deal more experimentation has to be done to under-
stand the conditions under which it devsloos and what the toxin is to
Cladophora glotnerata. The fact that it does not seem to damage diatoms,
Ulothrix, Scenedesmus, Spirpgyra, protozoa, nor rotifers indicates that it
may be confined in its effects to a relatively few species,
The results from the experiments with megaorganisms as biological con-
trols of C. qlomerata produced negative results. However, they did turn
up some very interesting information as to the nutritional value of C.
glomerata. This alga has a very different assortment of free and combined
free and fatty acids and different amino acid proportions than diatom-
dominated periphyton, which was obviously the preferred diet. It also con-
tains C]2 (lauric acid) which has been shown to be toxic to some aquatic
organ isms.
Further studies should be carried out to determine whether it is the
lauric acid that is producing the effect that inhibits the eating of this
algae by various mega invertebrates and whether it also produces a toxic
effect on the organisms. An attempt should be made to determine its mode
of action—that is, whether membranes or damaged, enzyme systems inhibited,
etc. If further research clearly shows that this alga inhibits the repro-
duction of many snail species, particularly those that are vectors of
schistosomiasis, this alga might be used as a control mechanism for this
disease, £. qlomerata is a very tolerant alga and can grow under a great
variety of conditions. The question also of interest is whether other algae
that develop large populations under eutrophic conditions contain toxic
substances.
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I, THE POTENTIAL OF CLADOPHQRA GLOMESATA AS A SATISFACTORY
FOOD FOR VARIOUS INVERTEBRATES AND FISH
INTRODUCTION
In order to determine whether Cladoghora glomerata is a satisfactory
food for a predator, the growth and, if possible in the time allotted, the
reproductive success of the species fed only Cladophcra should be compared
with other available algal diets. It was noted that C. qlomerata in nature
has many epiphytes growing on the filaments, and most of these are diatoms.
Therefore it was decided that the three food sources to be studied were a
mixed periphyton dominated by diatoms, C_. qlomerata with epiphytes, and
unialgal C. qlomerata. We had hoped to raise axenic cultures of the alga
but as pointed out by Gerloff and Fitzgerald (1) and others, no one has
succeeded in doing this.
The naturally occurring species selected for study were as follows.
The snail, Physa heterostropha, was chosen because it is a widely distributed
pulmonate and can live in water of varying oxygen concentrations. It has
been found ( 2) to have a toxicity tolerance intermediate between diatoms
and bluegill sunfish, Leopmis macrochirus, and it is an excellent food for
many fish. The crayfish,Orconectes propinquus,was chosen because it is
native to the Great Lakes area and is an important part of the food web in
the area. Tadpoles, Rana pipiens, were chosen for a similar reason. The
fathead minnow, Pimephales promelas, was chosen because it is commonly
occurring in this area, and because its general physiology is well known
through the work of Mount, Brungs, and others. The tests on channel
catfish, Ictalurus punctatus, were to determine if this fish would eat and
grow on this food, and were only carried out for a short period of time.
The most extensive tests have been on _P. heterostrocha, 0. propinquus, and
P_. promelas. Time did not allow more extensive tests on the otner organ-
i sms.
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REVIEW OF PERTiilEilT LITERATURE
)
SnaiIs
There are numerous articles in the literature concerning the effects
of various types of algal and invertebrate foods on the growth and repro-
duction of various pulmonate molluscs. Bovbjerg ( 3 ) studied the food
preferences of the snail Staqnicgla reflexa Say. He found that the snail
preferred the alga Spirog.yra sp. over the higher plants Ceratoonvi1um demer-
sum, Myriophyllum exalbescens, and Ranunculus lonqirostris. He found that
from a distance of 1-2 cm the snails did not perceive this food by any chemo-
reception. Only when the snail physically encountered the food it preferred
would it stop and eat, otherwise it would continue to move. In other studies
with different plants the snail moved three times as much on green and dead
cattails, bladderwort, buttercups, liverworts, and duckweed than it did on
the algal food.
Bovbjerg ( 3 ) in his studies with the lymnaeid snails Staqnicola re-
flexa , S. exilis, 5. elodea, and Lymnaea staonalis,found that these snails
preferred pellets .made from crayfish over those made from plant food and
that they could detect from some distance the pellets of crayfish, whereas
they could not detect those of plant "food. He found there was also little
response to the plant filtrate of the pondweed, Potamogeton richardsoni i.
For growth the best diet was a mixture of plant and animal material, and
only on this mixed diet did the animals reproduce.
Calow (4) studied the snail, Lymnaea perecer, to determine its prefer-
ence for various types of food sources. This snail is commonly found grow-
ing on El odea. An examination of the guts showed that some of the Elodea
tissue 'was in the gut, but that it was mostly filled with epiphytes. He
incorporated this in an agar block, and in other agar blocks he incorporated
singly crushed Elodea, mixed diatoms, and a mixture of filamentous algae.
From this study he found that the greatest preference was for the epiphytes
taken from the Elodea and for the filamentous algae. He states that the
work of Paine and Vedes ( 5 ) shows that the ash-free dry weight of the dia-
toms have the greatest potential energy stored—that is, the kcal/g ash-free
dry weight is greater in diatoms than in the other algae. The reason why
this snail seemed to prefer the mixed culture of algae was that it was
easier to eat, there being a harder call wall about the Elodea and silicious
walls on the diatoms. He states that the preference of this snail was first
for the filamentous algae and the extract of epiphytes from the Elodea,
secondly for the diatoms, and thirdly for the Elodea.
Eisenberg ( 6 }, working with the pond' snai1 Lymnaea el odes, found that
the amount of food is of major importance in determining the growth and
8
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fecundity of this snail. Growth, weight, and total eggs were strongly corre-
lated with the food supply. The eggs/egg mass did not change very much with
food supply, but did increase somewhat. The total number of egg masses,
however, increased greatly with food supply.
Hunter ( 7 ), working with Lymnaea palustris, determined that the stage
of the life history was very important in regard to the physiological con-
dition of the snail. He found that this snail had the greatest amount of
nitrogen and carbon (ug/snail) at the mid-breeding period, and that there
was considerable variation from snail to snail in the amount of these ele-
ments. The mean dry weight was greatest at this period. He placed this
snail in two different envi ronments-~one designated as flAR and the other
as FAY. He found that the snails that typically grew in the MAR environment
when placed in tne FAY environment, and those snails that typically grew in
the FAY environment were much more fecund in the FAY environment. He did
not state just what the characteristics of the environment were that he
believed produced this.effect.
Studies of Laevaoex fuscus and Lvmnaea palustris indicate that they
prefer an intermediate carbon content diet which is high in nitrogen and
probably protein, and this is best for their growth and reproduction. Dia-
toms, bacteria, and blue-green algae have a high nitrogen content. Hcf'ahon,
at al. (8) state that the qualities of food are more important than the
quantities.
Elwood and Goldstein ( 9 ), working with Goniobasis clavaeformis, examined
the 32p which was sorbed onto the periphyton and then ingested by the snail.
He found that the food ingestion increased with increased temperature up to
14°C. The elimination of absorbed phosphorus also increased with increased
temperature, so that the shortest retention time was 6 days at 19.3gC as
compared with 34 days at 1Q°C. The absorption efficiency was fairly con-
stant and was about 39%; they speculate that perhaps the efficiency may be
related to gut clearance,
Paine and Vedes (5 ) studied the relative food value of green, red,
and brown algae and the diatom Nitzschia paraaoxa. They found that the
broad-leaved algae, such as Laminaria macrocystis and Gicartina sp.
were the most preferred foods and had intermediate food value. They found
the highest food value in Nitzschia paradoxa, which was 5.47 kcal/gm ash-
free dry weight and the caloric value in the marine form of CIadoonora sp.
was highly variable and averaged about 5.17 Kcal/gm ash-free dry weight.
Spight and Emlin {10} studied two marine snails, Thais larr.el losa which
spawns annually and T. gmarqinata which spawns many times during the year.
They found that growth was correlated with rood supply, and that clutch size
J
9
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increased exponentially with shell length, for example, T. emarqinata
triples its fecundity as it moves from minimum mature size to average mature
size; whereas T. laroellosa doubles its fecundity as it moves from minimum
mature size to average nature size. In the larger animals there are larger
capsules or egg masses produced and more eggs in both species. It is inter-
esting to note that T. laroellosa, which reproduces once a year, produces
about the same number of eggs as X- emarqinata which spawns many times during
the year.
Nicotri (11) studied the diets of the marine molluscs Col 1isella pelta,
C.. striqatella, Notoacmaea scutum, and Littorina scutulata, He found that;
all of these snails selectively removed and seemed to prefer three species
of diatoms. These were Melosira monilifprim's, M. nummuloides, and Fraqilaria
striatala var. californica. These were among the largest and most common
diatoms. He found a great many frustules of Nitzschia frustuluro in the guts
of these organisms, but he felt that because of their small size they probably
did not contribute much to the diet. He found that I. scutulata was less
efficient at digesting Achnanthes brevipes and Synedra tabu!ata.
Runham (12) points out that as yet the nutritional requirements of
pulmonate snails are virtually unknown. We do not know, for example, their
requirements for proteins, fats, carbohydrates, vitamins, and minerals. Some
species such as Limax flay us (L,} can live completely on wheat flour;
however, the growth rate and size were reduced. For some snails the require-
ments of vitamins have been demonstrated. Blomphalaria ~!abratum, when reared
under axenic conditions and fed on a diet of Escherichia coli and yeast, grew
well but did not lay eggs unless vitamin E was added to the food (Runham,
quoting Vieira, 12). Runham also pointed out that to be a suitable food the
texture of the cell wall, its smell, and its taste are important as well as
its caloric value. Thus we find a great variation in the kinds of preferred
foods of this group of organisms.
Crayfish
Crayfish are known to be scavengers and to feed on a wide variety of
foods. Typically they are detritus feeders and seem to be more or less
carnivorous. However, Budd, et al. (13) have shown that they could maintain
a breeding population of crayfish by supplying them with a rich green-algal
flora, Bovbjerg (14) determined that when the crayfish become independent
of the yolk sack at the time of the fourth ins tar, they are filter feeders
and feed upon algae.
Magnuson, et al. (15) summarized the data on the ability of various
species of crayfish to control aquatic vegetation. All of this vegetation
is higher plants, and no control of algae by the use of crayfish seems to be
evident in the literature, excapt for Chara sp. and Nitella sp. It has been
10
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shown that Qrconectes rusticus will control Pot arose ton, Valliseriaria,
Ceratoohy11 urn, Myriophyllum, Megalodonta, fluphar, and Nitella. It should
be pointed out that the introduction of crayfisri for the control of macro-
phytes may produce a bad side effect in that they are known to feed upon
the eggs of several species of fish.
In studies conducted by Monot, et al. (16), ft was evident that the
crayfish Qrconectes prppj nguus and £. virilis fed largely on the benthos,
and the algae was a large part of this benthos. Analyses of the stomachs
of the young-of-the-year of £. virilis indicate that the highest amount of
any one food was plant detritus that occurred in all the stomachs examined.
The next highest was pine pollen that occurred in 82.9% of the stomachs ex-
amined, and diatoms that occurred in 45.1% of the stomachs analyzed. Twenty-
nine percent was in the form of chi ronomid eggs, and 25.5% was fragments of
arthropods. These data indicate that the crayfish 0. virilis is an omnivore
feeding on many different kinds of organisms and that the type of organism
it seems to prefer is the benthos.
Momot {17} found tnat 0. virilis was primarily an herbivore in marl
lakes, feeding chiefly on algae and the "aufwuchs" with marl incrustations.
In the marl lake, West Lost Lake, the biomass of crayfish was nearly 10-fold
that of other invertebrates, and it was believed that this was due to their
ability to utilize algae.
The crayfish CL virilis usually goes into hibernation about the first
of October. The maturation of eggs takes place during the winter months.
This maturation process is affected by the X organ-sinus gland complex of
the eyestalk. Temperature and photooeriod are necessary for the proper
maturation of the eggs (18).
The work of Aiken (19) shows that the molt cycle of 0. viri1 is is regu-
lated by environmental factors, especially photoperiod, and by certain
endocrines. The endocrine system modifies the inducive effect of the photo-
period. The photoperiod seems to control the interaction of two hormones
designated as MH and MIH, Proecdysis (molting) is induced when the influence
of MH exceeds that of MIH. If after the onset of proecdysis the titer of
MIH interferes with oremolt preparation for one or more of these molting
steps, the molt attempt is terminated at that point where the affected step
occurs. A lesser amount of MIH may simply prolong proecdysis, whereas a
larger amount may suspend it indefinitely. The photoperiod necessary for
molting to proceed is 15-16 hours for subthreshold lengths in November and
December, marginal lengths in January, and clearly above the threshold
lengths in February. The MH hormone seems to be produced from the paired
endocrine organs in the thorax, whereas the molting-inhibiting hormone MIH
is from the X organ-sinus gland complex of the eyestalk.
11
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fish
Fathead Minnows (Pimepha1es oromelas) - There is a great diversity of
opinion as to what is the food of this fish. Smith, et al. (20) noted in
his studies that they could grow and reproduce when fed brine shrimp. Held
and Peterka (21) state that the main food is Crustacea, particularly Cladocera,
and that algae furnish only a small percent of the ingested food (0.2* by
volume were mainly diatoms), lord (22) stated that these fish are mainly
algal feeders and that they feed upon the filamentous green algae, Oedoaonium
and Soiroqyra, and probably other filamentous algae, which he does not name.
They also eat a great many diatoms. Coyle (23) noted that fish ranging in
size from 25-^-0 mm feed upon diatom slime and small algae, which they nibble
off the stems of submerged and emergent aquatic plants.
Channel Catfish (Ictalurus punctatus) - The food habits of blue and
channel catfish have been studied. The channel catfish feeds primarily upon
amphipods, small insects, algae, and various types of organic matter (24).
Devaraj (25), in his studies of Ictalurus punctatus, found that 47.7" of
their food were dipteran larvae, and that the algae were 1-23% of the gut
content. These were relatively small fish. When they became larger--!.e.,
300 mm "long—fish formed about 87* of the volume of food in the gut, accord-
ing to Ambrose and Brown (26, cited by Devaraj, 25). Perry (24) found that
the channel catfish up to a length of 376 mm fed primarily on amphipods,
small insects, algae, and undetermined organic material.
Similar results as to the value of the blue-green algae as compared
with the green alga Soiroqyra so, were found by Stanley and Jones (27) 'in
their studies with the bigmouth buffalo, Ictiobus cyprinellus, and Tilapia
aurea. They found that the conversion rate when these fish were fed on
blue-green algae was 2, whereas when the grass carp, Ctenopharynaodon idella,
was fed on Spiroqyra sp. the conversion rate was about 10 and poor growth
resulted.
Repsys, et al. (28), in their studies of the black bullhead, ictalurus
melas, found that 32% of the food in the gut was planktonic Cruatacea, 25X
chironomids, 27% fish, 9% crayfish, 2% filamentous algae, and 5* miscellane-
ous organisms. However, in the young bullhead 94.4% was planktonic Crustacea
and 5.6X was chironomids. No algae were noted.
The results of feeding studies on the brown bullhead, Ictalurus nebu-
losus, indicate that 23* of the carbon presented as Soiroqyra sp. and 671 of
the carbon presented as Anabaeoa flos-aguae was assimilated by this fish in
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24 hours. These data indicate that Anabaena flos-aquae is a better food
source than Spiroqyra (23). ——
Tadpoles
Various tadpoles have been studied as to their preferred food. It is
well known that tadpoles eat largely algae before they metamorphose and
develop their hind legs. Jenssen (30) found that the green frog tadpole,
Rana clamitans, ingested algae in proportion to their number in the environ-
ment. He found that they ingested Pithop.hora and Cladophora. However,
Kamat (31) found that they ingested aigae in proportion to its frequency
in the pond water but that they did not ingest Pithophora, Cladophora, and
Chara. Thus there seems to be a difference in the results cf these two
studies.
Richards (32) found that algal-like cells inhibited the growth of Rana
pipiens. The algal cells were not positively identified and May belong to
the genus Prototheca, These cells seemed to enlarge in the gut of the tad-
pole and when passed in the feces divide in the water. In concentrations
of 1 x 1Q6 - 4 x 1Q7 cells in the tadpoles' environment, they will inhibit
growth,
Sin and Gavrfla (33) in the studies of the tadpoles of Rana ridibunda,
found that the main food was aquatic macrophytes and secondarily food of
animal origin.
Dickman (34) found that the tadpoles in Marion Lake, British Columbia,
fed largely on Mougeotia, a filamentous green alga, shortly after they
hatched from the eggs. Thus their predation greatly altered the algae forming
the periphyton.
Franz (35) studied the feeding habits of the larvae of the tailed frog,
Ascaphus truei, from two streams in northwestern Montana. Coyle Creek was
dominated by diatoms. The analyses of the guts of tadpoles from this stream
showed them to consist primarily of diatoms. In Ward Creek, where Spiroqyra
and Monostroma were the dominant algae, examination of the guts of the tad-
poles showed that they were filled mostly with diatoms with a few strands of
Ulothrix. Although Spiroayra and Monostroma were the most common algae in
the stream, no filaments of these algae were found in the guts. He then
conducted an experiment placing five larvae into plastic cartons containing
a pure culture of Spiroqyra, another five placed into a carton containing
a pure culture of Monostroma, and a third group with only Ulothrix. After
four hours the intestines of the larvae were examined and no algae were
found in the tracts of these organisms. Only a few diatoms were present.
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METHODS AND PROCEDURES
Obtaining and Culturing of Unialgal Cladophora
Cladophora qlomerata was initially obtained from nearly streams. The
identification of this species was carefully checked in order to make sure
that it was the same species as commonly found in the Great Lakes area.
The isolation of this C. qlomerata was achieved by allowing filaments
to grow up to approximately 1 cm on 1% agar made with the culture medium of
Fitzgerald and Gerlofr (Medium II, 1). Several filaments were laid out on
the surface of the agar and the petri dish was inverted below an artificial
light source. In two to three weeks filaments had grown sufficiently so that
filament tips could be aseptically removed. These filament tips were placed
on fresh agar, and the procedure was repeated several times. The entire
procedure was carried out in a hood using aseptic techniques. Finally the
unialgal filament tips, free from epiphytes, were transferred to liquid
culture Medium II ( 1 }, This medium was used throughout all experiments for
the growth of _C. qlomerata under unialgal conditions.
The culture vessels used were 4-liter aspirator bottles and 2.8-liter
Ferback flasks containing 2 liters and 1.5 liters of culture medium respec-
tively. The cultures were illuminated from below by 40-watt Vita-lite
fluorescent tubes, and moderate aeration was continuous. A 15-hour photo-
period at 100 u einsteins/m2/sec was used, and temperatures were maintained
between 21° and 23°C.
Culturinq of Periphyton
The periphyton cultures were maintained in a greenhouse by seeding large
plexiglas plates (13 cm x 23 cm x 2 mm), with natural periphyton from White
Clay Creek, Chester County, Pennsylvania. These were maintained by allow-
ing stream water of suitable velocity to flow continuously over the plates.
Within about two weeks the plates were covered with a well-developed peri-
phyton growth. The growths were examined periodically under the microscope
and were found to be diatom-dominated, although small amounts of algae and
some protozoans occurred from time to time. The dominant diatoms varied
seasonally, but Melosira varians was generally dominant with smaller popula-
tions of Synedra spp., Ni tzschia spp., Navicula sop., Achnanthes sop.,
Cocconeis spp., and Gomphonema spp.
Plates seeded with periphyton were allowed to sit in inclined trays for
20 minutes before weighing in order to remove any excess water. The bottoms
of the plates were then wiped clean and dried, and wet weight of the plate
plus the periphyton was determined. When the plates were removed after
they had been preyed upon, they were dried in a similar manner, and then
reweighed, The periphyton was then scraped from the plates and the plates
weighed. In this way an estimate of the periphyton consumed was obtained.
14
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Qraanisms usad in the Experiments
Physa hetarsostrooha - This pulxonate snail was originally collected
from nearby sections of Buck Run Creek in East Marlborougn Township, Chester
County, Pennsy1 vania. The snails were subsequently reared in the greenhouse.
Succeeding generations of these snails, reared on periphyton in a continuous
flow-through system, were used in these tests. They were maintained at
temperatures of 20° + "PC in a 12-hour photoperiod.
Orconectes propinquus - This species of crayfish was collected in
Erie and Geauga Counties, Ohio. They ware collected by Or, Raymond
Jezerinac. Over 200 individuals were separated by size (eliminating very
small and very large crayfish) and by sex. The males were all in form 1
and were on an average larger than the females. All crayfish were put into
SSOgallan glass aquaria containing rocks which had bean previously scrubbed.
These were to provide a satisfactory habitat with hiding areas until the
experiment started. Males and females were kept in separate tanks.
Pimephales promelas - The fathead minnow was obtained from the Newtown
Fish Toxicity Station of EPA in Cincinnati, Ohio. These were roughly
cm long when the experiment started.
Rana pi pi ens - The tadpoles were purchased from the Carolina Biologi-
cal Supply Company at Burlington, North Carolina. These tadpoles were
placed in a large 55-galIon aquarium until the experiments started.
Ictalurus punctatus - The fingerlings of the channel catfish were
supplied by the Fish and Wildlife Service at Orangeburg, South Carolina.
These fish averaged approximately 3.5 cm in length, and weighed approximately
0.8 gm each when received,
Conditions of Experiments
1. Snail Experiments
The snail experiments were carried out in 20-liter capacity polyethylene
trays (18 an x 45 cm x 23 cm). Two closed recirculating water systems were
used in parallel. These were positioned over 90-liter reservoirs. One
submersible pump at the bottom of each reservoir delivered water to the trays
at the rate of 1-2 liters/min/tray. Water flowing out of each tray was re-
turned by gravity to a reservoir through a loose cellulose filter. Weekly
50-60% of the water in each recirculating system was replaced by fresh stream
water at experimental temperatures. The snails being fed on unialgal
Cladophora were in one recirculating system, while the snails fad on diatom-
dominated periphyton were in the other.
During the winter experiments in which we tested four diets, the
setup was somewhat changed. The starvation and unialgal Cladophora diets
were in one recirculating system, while the snails being fed Cladophora
with epiphytes and those being fed the aiatom-dominated periphynon were
15
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in the other, Watar in the unialgal Cadophora system was initially
filtered through a 10-15 u filter, and such filtering was carried out
during the weekly watar rep 1acarnents. It was found that such filtering
greatly reduced any chance of contamination of the system with other algae.
In the 1977 spring-summer experiments, 27 snails of fairly uniform size
that had been reared in the laboratory were placed in each animal tray.
Three trays of snails were olaced on each of the two diets--unia!gal CIad-
ophora and diatom-dominated periphyton, In the 1973 winter experiments 20
snails of fairly uniform size that were reared in the laboratory were placed
in each animal tray. Two trays of snails were placed on each of the follow-
ing diets: urn"alga] Cladoohora , diatom-dominated periphyton, CIadophora
with epiphytes, and no food at all. Two arbitrary size classes of snails
were used--7-8 mm and 0-9 mm total shell length. Each diet had 20 individ-
uals of each size class in separate trays.
Introduction of Food
In the 1977 experiments food was continually present and renewed as
needed to insure tnat a source of food was never limiting, Cladoohora was
replaced periodically*al though very little had been eaten.
Through observation it was learned that these snails typically feed
at night. It was therefore decided in the 1978 experiments that food would
be introduced at 5:00 one afternoon and removed at 12 noon on the following
day. The wet-weighed amounts of periphyton and Cladophora with epiphytes
were renewed daily, while the unialgal Cladophora was renewed only twice a
week because it was not eaten. At all times more food was present during
the feeding periods than could be consumed, and the remaining food was re-
moved and the wet weight determined. These estimates were only made during
the 1978 experiments.
The purpose of the feeding procedures in 197? and 1978 were to make
sure that food was not a limiting factor.
The periphyton was introduced as growth on Plexiglas plates, and the
snails were allowed to feed directly on the plates. The unialgal Cladophora
and the Cladophora with epiphytes were anchored to the bottom of the tray by
short glass rods.
Growth Studies
The estimates of growth were made by determining the change in weight
of the snails throughout the experiment. To accomplish this a specific
number of snails was removed at random initially and periodically throughout
the experiment from each test. In those cases where the wet weight was ob-
tained, the snails were dried on a paper towel to remove all loose moisture
and then weighed. Following weighing they were returned to the container.
16
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Dry weignt was obtained by immersing the snail in boiling water for 15
seconds and extracting the tissue with Forceps. The dry weight was obtained
by drying at 60°C for 24 hours, and the ash-free dry weight by drying at
500°C for two hours, The caloric values were determined"by the use of a
Parr Bomb Calorimeter (36),
Respiration Tests
The oxygen uptake was measured at 20°C using a Gil son GRP 20 Differ-
ential Respirometer (37). This temperature was used because the average
temperature in the test tanks during the summer was 22°-23QC and during
the winter was 19.9°C.
Individual snails were taken from each experimental tray and placed in
a tast vessel with 7 ml of filtered (0.45 u) stream water and acclimated to
test conditions for 1 hour. Oxygen consumption was measured every thirty
minutes for a 2-3 hour period, and the carbon dioxide that evolved was
absorbed by KOH. Similar sized individuals were placed in each vessel,
although the number per vessel varied from 1 to 5 depending upon the snail
size. This assured that measurable quantities of oxygen would be consumed
in each vessel while depletion would not occur prior to the end of the ex-
perimental test period. The test animals were free to move in the vassal
and had not been previously starved.
Fecundity Studies
Each test tank was examined for egg masses and eggs almost daily except
for weekends in the 1976 and 1977 experiments. In the 1973 experiments the
egg mass coll actions were done at less frequent intervals.
Data concerning the number of egg masses and the number of eggs in
each mass were recorded only on days after which eggs had been removed from
the tanks. By this means one can be sure that the estimates are fairly
correct.
Upon removing the egg masses the eggs were examined under the micro-
scope to see if they were viable, A very few instances of non-viable eggs
were recorded. Data concerning the caloric content of the eggs were obtained
by the use of microoomb calorimetry (38).
2. Crayfish Experiments
The crayfish experiments were carried out in 55-galIon glass aquaria.
On the bottom of each aquaria were placed rocks of approximately 3 x 4-5
inches in order to afford a natual habitat and protection from the other
crayfish at the time of molting, Twenty crayfish were placed in each
aquarium. Each individual was marked in the aerola section of the cara-
pace, using a combination of three different fingernail polishes. No
detrimental effects were observed. Three diets were used in these experi-
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merits; the same diet was put into two of the aquaria. The Cladoohora and
periphyton were cultured as described in the snail experiments. The Clad-
oohora with epiphytes was collected from a holding pond at the laboratory
and allowed to stand in trays of the stream water for 24 hours. Diurnal
temperature increases arid low oxygen levels forced the many insect larvae
arid Planaria out, leaving the Cladophora less contaminated. Upon examination
this Cladophora not only contained epiphytic diatoms, as in the case of the
snail experiment, but also had associated with it small amounts of Spi rooyra
and Stiqeoclonium,
Introduction of Food
A review of the literature and observations in nature indicated that
these crayfish feed mostly at night. Therefore, the food was wet-weighed
and introduced at 5:00 p.m. daily and taken out at 8:00 a.m. the following
day. During the feeding period the aquaria were without light. The CI ad-
os ho ra was placed on filter paper to drain off all the excess water and the
wet weight obtained. In the morning when the Cladophora was removed it was
again dried in a similar manner and weighed.
It was found that a considerable amount of the CIadcohora was dislodged
and broken by the movement of the crayfish. An experiment was conducted to
study the amount of Cladophora filament breakage caused by crayfish feeding
during 4- and 16-hour periods over a 5-day interval. This was done by
using six polyethylene tanks containing rocks in a closed water circulating
system. Two grams of Cladophora were introduced at 5:00 p.m. into each
test tank containing four crayfish. At the end of the 4-and 16-hour periods
the Cladophora filaments found in a form large enough to pick up with for-
ceps were termed retrievable. Filaments that were too small to pick up and
lying on the bottom were referred to as broken. The percentage weights of
the original weight introduced were added to that which was retrieved in
order to get a more accurate estimate of the food intake of the crayfish.
The periphyton plates were introduced at 5:00 p.m. and removed at 8:00
a.m. the following day. They were dried and weighed as set forth in the
general methods.
Growth Studies
All of the crayfish were collected and weighed every ten days. Dry
weights were obtained by drying at 60°C for 24 hours, and ash free dry
weights by drying at 500°C for two hours. The calories were determined by
the use of a Parr Bomb Calorimeter.
IE
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Moltinci Studies
The crayfish were observed daily for evidence of molting. Exoskeletons
were removed if found before they had been eaten, and frozen for future
analysis if desirable.
Molting was a very infrequent occurrence in the experiments, 'here-
fore it was decided to try to force molting to see if the different diets
had an effect upon the ability to molt. With the aid of Or, E. M. Zipf,
the eyes talks were carefully removed by the use of a scalpel. It is well
known that removing the ayestalks will often force the crayfish to molt.
Following the removal of the eyestalks the crayfish were imnersed in chilled
stream water in order to prevent bleeding. Twelve crayfish were used in
this experiment, and after their eyes were removed they were placed back
in their respective aquaria along with the normal crayfish- An effort was
made to be sure that there were plenty of crevices among the rocks for pro-
tection of these eyeless forms from cannibalism. The longevity, weight
change, and molting were followed until the crayfish died.
Feces Studies
The feces were obtained by isolating 5 to 10 crayfish in approximately
10-liter containers with aerated stream water for a period of 8 hours. The
fecal material was collected and dried at 60°C for 24 hours and stored for
later analyses. This method for obtaining feces was carried out at vary-
ing times during the course of the experiment. Microscopic observations
were made of the feces. Those feces from the crayfish fed on periphyton
contained a great many dead diatoms. The protoplasm had been removed. The
feces of those fed on CIadoohora contained both green filaments and those
that had only the cell walls remaining, Attempts were made to determine •
the calories in each of the types of feces by the use of a microbomb
calorimeter.
Respiration Studies
Respiration studies were carried out every ten days in Plexiglas res-
pi rometer chambers. The chambers were similar in design to tnose of Mac
Intire, et al. (39). The whole unit was housed in a plywood water jacket.
Ten crayfish were placed in each chamber, which was lined wi tn clean rocks.
The dissolved oxygen concentrations in the chambers were recorded in dark-
ness without experimental animals in order to obtain any background error.
It was realized that this method is only a rough means of determining res-
piration rates, as inevitably during the time in which the crayfish are in
the tank bacterial growth occurs and error is introduced. However, since
all the experiments were carried out in the same manner, the estimates are
good for comparative purposes and believed to be fairly accurate.
19
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Fish Experiments
3. Pimephales oromelas Experiments
The diets of the fish In these experiments were unialgal Cladophora and
Cladoonora with epiphytes. The experiments were conducted in a controlled
temperature room with a temperature between 20°-2S°C, and a phctoperioa of
12 hours.
A closed recirculating water system was built for each diet. This
closed system consisted of two 200-liter glass aquaria, each with a water
filter, attached in parallel to a 90-liter reservoir. One submersible pump
at the bottom of each reservoir delivered the water to the aquaria at the
rate of 2-3 1iters/min/aquarium. The water was returned to the reservoir
through a filter of loose cellulose fiber by gravity flow. Each week 30"
of the water in each recirculating system was replaced by fresh stream water
which was at the experimental temperature. One month after the start of
the experiment the glass aquaria were replaced by 20-liter polyethylene
tanks (IS cm x 45 cm x 23 an). This was done to facilitate the collection
of faces which was very difficult in the glass tanks. The replacement water
and the original water in the unialgal Cladophora and the fish food diets
were filtered through a 10-15 u filter in order to keep down contamination
by other organisms.
In these experiments two size classes were used. The fish were sorted,
and 20 fish of each size class were placed on each diet. Each fish was given
an identifiable mark by injecting India ink from a 1 cc syringe with a 26
gauge needle into the caudal fin. The tattoos were performed while the fish
was held on its side with 1ight fine mesh nettings in approximately 0.5 cm
water, which allowed the fish to continue breathing. The marks remained
visible for approximately 3 weeks, whereupon they were remarked,
Food was introduced at 5:00 p.m. and removed at noon the next day.
Wet-weighed amounts of periphyton and Cladoonora with epiphytes were renewed
daily (see general methods). The unialgal Cladophora was reused as so little
was eaten, and replaced with new algae twice a week. Always more food was
introduced than could be consumed and the remaining food was removed and
weighed. Peri phyton was introduced as growths on Plexiglas plates (13 cm x
23 cm x 2 um) while the unialgal and Cladophora with epiphytes were anchored
to the bottom by short glass rods.
Growth Studies
The fish were wet-weighed individually in a small transparent plastic
bag partially filled with water, at periodic intervals during the experi-
ment. Dry weights were obtained only on fish used to determine respiration
rates. After the fish were killed in hot v/ater they were dried at 60°C for
-------
48 hours, and ash free dry weights were obtained by drying at 5Q0°C for two
) hours. Individual and average wet weight values of fish are used to assess
growth over time.
Respiration Studies
Respiration determinations were conducted on fish selected at random
from each experimental diet. One-quart Mason jars served- as the test chambers.
The"jar lids were modified to accommodate two 0.5 cm diameter glass tubes
through which water was delivered to the bottom of the jar and exited at
the top. The flow rata was regulated with a stopcock and set to deliver
between 6 ana 8 ml/minute/'jar. The water was delivered by gravity directly
to each jar through Q.5 cnt diameter rubber tubing from a reservoir located
above the test chambers. The flow rates remained relatively constant by
maintaining a constant volume or head pressure in the reservoir, A sub-
mersible pump and several 100-1 reservoirs at ground level supplied the head
tank. Black plastic oags enveloped each jar in darkness to prevent algal
growth.
Two fish were placed in each test chamber 72 hours prior to the test
with test diet food. Twenty-four hours prior to the determinations all feces
and remaining food was removed, and time was allowed for more than one com-
plete replacement of water. The lids were examined for leaks and the desired
experimental flow rates were set (6-8 ml/minute/jar), During the acclimation
period, flow rates were 20-30 ml/minute/jar.
At the start of the test water samples (approximately 130-140 ml) were
collected from the head tank and the water leaving the test chambers. Water
was siphoned separately out of the head tank,and in all collections approxi-
mately 30-50% of the total sample volume was allowed to overflow, in an
effort to minimize the error introduced by sample aeration. Given a con-
stant known flow rate and knowing the oxygen concentration entering and
leaving the test chamber, the oxygen consumption by the fish can be calcu-
lated. Oxygen determinations -were performed using the standard Winkler
method.
Well water was used during the respiration determinations to minimize
the background respirations.
Caloric determinations were made by the use of a Parr Bomb Calorimeter
(36) and a microbomb calorimeter (38).
'4C Uptake Studies
Diets were labeled by exposing Cladophora, Cladophora plus epiphytes,
and periphyton (approximately 12.5 g/wet weight each) to 15 p Ci MaH1^003
for photosynthetic incorporation. After 4-hour incubation at E0°C, in-
)
21
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corporate^ radioactivity was determined. Fifteen samples of each algal
type were taken, dried, exposed to HC1 fumes to remove sorted 14C} and
weighed. The samples (approximately 5 mg dry weight) wera combusted in a
sample oxidizer (Packard Inst., Napervi1:e, IL) and radioactivity was deter-
mined using a liquid scintillation counter, The bulk of the algae was
transferred to fresh water and held overnight to permit release of labile
excretion products and stabilize radioactivity levels. Having verified that
the diets were sufficiently labeled for feeding studies from the initial
check of radioactive content, the diets were again sampled and the radio-
active content used as the initial level for each diet. Fish wera fed as
in feeding studies with unlabeled algae. Each day feces were collected,
dried, weighed and oxidized as described for algae. Each day fish wera
transferred to clean water, killed, and blotted dry. The fish heads, guts,
and viscera were dissected and the musculature was cut into several pieces
of appropriate size for oxidation. All samples were dried, weighed, and
oxidized for radioactive content as described. At the end of the experiment
the diets were again sampled for radioactive content. The ash content of
all materials was determined from weights after combustion at 550°C for four
hours.
4, Ictalorus punctatus Experiments
Twenty fish were placed in each of six 55-galIon glass aquaria to
acclimate for four days before the start of the experiment. Fish in two
aquaria were placed on each of the following diets: periphyton and unialgal
Cladopnora. Fish in two aquaria were sacrificed for starting weights.
Temperature and pH were recorded daily; oxygen and ammonia, one to three
time a week; nitrate and nitrite, on two occasions.
From time to time microscopic observations were made of the periphyton
on the plates that were supplied as food. The periphyton was dominated by
Melosira varians with large populations of Navicula, Nitzschia, and Svnedra,
and smaller populations of Oiatoma, Gcmohonema, and other genera. Ciliated,
fI age 11a ted, and amoeboid protozoans were represented in varying densities.
Filamentous green algae such as Soirocyra and Stiqeoc Ionium were occasionally
observed and removed prior to introduction into the aquaria.
Fecal material was collected by isolating individuals in containers
with filtered stream water for several hours. Feces were collected and
microscopically observed for contents and algal condition.
Growth was determined by weighing the fish in tared plastic vessels con-
taining a minimum of water on a triple beam balance. Wet weights were re-
corded to the nearest one-hundredth of a gram. All fish in each aquarium
were removed and weighed at approximately one-week intervals.
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5. Tadpole Experiments, Rarsa pipiens
These studies were carried out in polyethylene 20-liter trays. The
diets consisted of cultured Claodp'nora, Cladophora plus epiphytes, and diatom-
dominated periphyton. Determinations of pre-experiment weight and dry weights
were carried out on a random sample of 40 tadpoles from an initial 280 indi-
viduals. Twenty-five tadpoles in each of two trays per diet were maintained.
During the first four weeks of the experiment water was metered in by
a syringe pump at a rate of approximately 8 liters per hour. Overflow was
returned to a 100-liter reservoir. Turnover time in the trays was approxi-
mately 2.25 hours. The closed system used during the last four weeks of the
experiment was described in the methods for the snail experiments. Water in
the trays was analyzed for temperature, NH„, dissolved oxygen, and pH.
Periphyton plates were replaced daily. Cladophora was introduced daily
in the Cladophora-pi us-epiphytes diet and two to three times a week in the
Cladophora-diet trays.
Every two weeks four or five tadpoles per tray were removed to deter-
mine wet and dry weights. Feces were collected daily for microscopic obser-
vations and later chemical analyses.
Two respiration measures are reported. A GRP 20 Gi1 son differential
respirometer was used, and a temperature of 18°C was maintained during the
test. Mean temperature in the experimental trays was 20°C.
RESULTS
All of these experiments were run in the natural soft waters of White
Clay Creek, Pennsylvania. The oxygen was maintained above 7 ppm, the pH was
circumneutral (7-8.2) and the ammonia was less than 40 pg/1.
Snail (Physa heterostropha) Studies
These studies to determine the suitability of Cladophora qlcmerata as
a food for the snail Physa heterostropha were carried out over a three-year
period, 1976-1978. The most extensive studies were made in 1978, when an
attempt was made to relate the amount of food taken in and assimilated to
that dissipated in respiration and excretion and utilized in growth. The
experiments in 1976 were to determine the best design of experimentation,
and were part of a series of preliminary studies on several organisms.
In 197S four sets of experiments were performed: in one series the
snails were fed on periphyton; in a second, the food was Cladophora glomerata
plus epiphytes; in a third, unialgal Cladophora glomerata was the food; and
in a fourth, the snails were starved. In the January 25-February 10 experi-
23
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merits the mean weight of the snails decreased in all experiments except those
in ^which periphyton was the food {Tables 1,2). The variance was large and
this obscured the trend. The starved snails had an average decrease in mean
ash free dry weight of 58.'!% by the end of the experiment; the unialgal C laa-
QDhora-fed snails, 50.IS; and the snails with Cladooriora p"1 us epionytes as a
'cod source, 21%, The decrease in caloric value per snail showed a similar
trend with the greatest loss in the starved and the unialgal CIadophora-fed
snails. As seen in Tables 1 and 2, the calories per mg of tissue were quite
similar on all diets, which indicates the correlation of increased caloric
values with growth (Table 2).
A study of the shells showed a similar trend in that the increase in
weight of the shell was greatest for the snails on the periphyton diet.
On all diets the weight of the shell increased, but this increase was very
little for the starved and Cladophora-fed snails.
A statistical analysis (40) of the dry weights of the snails on January
31 and February 10 showed the following relations of the tissue weights on
the following diets: starved < Cladophora < Cladophora + epiphytes < peri-
phyton (significant at P< 0.001). On January 31 lower weights of the starved
snails (P 0.05 > P > 0,01) and higher weights of the periphyton-fed snails
(P< 0,001) were significantly different from the pre-experiment weights.
By February 10 the starved and Cladophora-fed snails were significantly
lower (? < b.001) in weight, the Cladophora-plus-epiphytes-fed snails were
slightly but not significantly lower in weight, and the periphyton-fed snails
continued to he significantly higher in weight than the pre-experiment
snai1s.
The May 19 to July 14, 1977, experiment was much longer than the 1978
experiment (January 25 to February 10.) However, there were similar trends
with a great increase in weight of tissue of those snails fed on periphyton
and a decrease in tissue in those fed on unialgal CIadoohora. Over a similar
time span (14 days) the 1977 periphyton-fed snails increased 11,3 times in
wei ght and 11.8 times in caloric value, as compared to 1 978 with 2.1 times
weight increase and 2 cimes increase in calories. This greater increase in
1977 was probably due to better environmental conditions for growth in May-
Julys and to the smaller size of the May-July snai1s. The caloric values
over a similar time period averaged per mg ash free dry weight was 5.8 calories
in 1977 and 5.24 calories in 1978 for the periphyton-fed snails, and 5.3 in
1977 and 5.63 in 1978 for the Cladophora-fed snails. There was not as much
variability in the calories per mg of snail tissue as there was in the whole
snail. As in the 1978 experiments, the value differences in the whole snail
were due to growth (Tables 3, 4).
The loss in weight of the Cladophora-fed snails for the above time
period was 15.IS in 1977 compared to 50.1% in 1978, and the decrease in caloric
content of the snail tissue was 10" in 1977 as compared to 4-7.6% in 1978.
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In 1976 similar experiments were performed in August-October. The size
) of the snails was iintermediate between those used in 1977 and 1978. The per-
centage increase in dry weight over a similar period of time {20 days) was
5,6 times for the periphyton-fed snails and 5.3 times in caloric value. The
C, adoohora was not entirely free of epiphytes, but snails fed on this food
produced similar results to those fed on unialgal CIadoohora in 197S--1hat
is, a loss of 42.4% in 1976 and 50.IS in 1978. The average calories lost
per snail was 32% as compared to 47.8% in the 1978 Cladoohora feeding experi-
ments (Tables 5, 6).
Respiration Studies
For the 1978 experiments we have two sets of respiration studies—one
set before the experiments started and one set on all diets during the ex-
periment. An analysis of variance of the January 31 data shows the follow-
ing ordering of differences among the groups (P 0.05 > P 0,01); starved <
Cladoohora < Cladophora plus epiphytes < periphyton (Table 7).
In the 1977 experiments we have four sets of data, all gathered during
the experiment. Snails fed on Cladophora had significantly lower (P < 0.05),
respiration rates tnan those fed on periphyton on June 2, 16, and 30
(Table 8).
An examination of the 1978 data shows an increase in respiration rates
on all diets with the highest rate being on those snails fed on periphyton
and the least increase in those snails that were being starved. In the 1977
) and 1978 experiments the snails feeding on Cladoohora had a lower respiration
rate than those feeding on periphyton. The four sets of data obtained in
1977 are somewhat irregular. The rate of the periphyton-fed snails seemed
to tend to decrease as the snails grew, whereas in the CIadoohora-fad snails
the trend was not as evident, as there was one very low rata on June 30 and
a very high one on July 14. This may well be due to experimental error (Table 8).
Mo respiration studies were made during the 1976 experiments.
Food Intake
An effort was made to try to determine the food intake per day and the
relative caloric value. It was realized that caloric value is only one
measure of the nutritional value of food. For example, the caloric value
of two very different carbohydrates might be very similar but the digest-
ibility of the compounds very different.
One of the main problems was to determine the amount the snails actually
ate. Wet weights of the food were carefully determined, but once it was
presented to the snails considerable amounts may have been lost and not eaten.
Cladophora often broke up into very short filaments which were practically
;
25
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unco 11ectab1e, and they were very difficult to sea or to filter out of the
feeding tanks. The diatoms were sometimes loosened in the feeding process
and lost- Estimates of how much the snails ate each day are very rough.
The caloric determinations were made by the micrcbomo calorimeter tech-
nique. These are set forth in Tables 9 arid 10. Our estimates of the
caloric value of the diatoms are a little low as compared with some values
in the literature. Our low values are probably due to fine silt which we
could not separate from the diatom mat-
Two sets of data were obtained for Cladophora plus epiphytes and one
set for unialgal Cladophora, As seen in Table 9, many periphyton values
were determined. These data were only collected for ]978, but it is very
probable that the caloric values for Cladoohora (the same strain) and diatoms
were very similar in 1977. As found by Paine and Vedes(S) the caloric values
of diatoms and Cladophora are not very different.
Fecundity
In order to estimate the fecundity of the snai1 eggs and egg masses,
they must be collected the day previous to the study period and, if possible,
during the study, but always at the end of the period. When one examines
Tables 1 and 11 and 3 and 12 it is evident that the difference in dry weight
of tissue, egg production, and egg mass production are correlated,
In 1978 tissue weights only extend to February 10. Within that period
the greatest increase >n eggs/snail on the periphyton diet is correlated
with the greatest increase in tissue weight (January 25 - 31, Tables 2, 11).
However, the largest numbers of eggs per snail were in the periods February
12-14 and February 17. The weight increase was much less between January
31 and February 10. The egg masses per snail were largest on February 17,
but the percentage increase was greatest between January 25 and January 31
(Table 11). On the Cladophora plus epiphytes diet the eggs per snai1 were
much less,and usually less than one egg mass per snail was produced. The
snails on the Cladophora diet and those that were starved produced very few
eggs.
Viability tests were run on eggs collected on January 27, February 1,
and February 6, 1978. In all cases only a few eggs did not hatch.
The caloric value of the eggs as determined from the 1978 experiments
showed little variation on the different diets {Table 14).
In 1977 (Tables 3, 12) when one compares increased tissue weight with
increased production on the periphyton diet, the greatest increase in
tissue weight occurred between May 19 and June 2. This was also the period
of greatest increase in eggs and egg masses. There was a steady increase
in tissue weight per snail through July 14, but the increase between June 30
26
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and July 14 was only 4 percent. The egg production per snail increased
through June 30 and showed a 14% decrease on July I-. The production of
egg masses per snail did not show such a consistent pattern of increase
until June 30, but did show a decrease toward the end of the experiment.
In contrast, the snails fed unialgal CIadophora showed a decrease in
weight and very few eggs and egg mass production as of June 2, and no eggs
were produced after that date except for eight eggs on July 13.
The 1378 experiments (Table 13) showed the same general pattern of the
1977 and 1978 tests. The periphyton-fed snails increased in tissue weight,
eggs, and egg masses, whereas the snails on the Cladophora plus epiphytes
laia a few egg masses irregularly for a few days, and no eggs were laid after
September 3, the 14th day of the experiment. The tissue dry weight of the
snails steadily decreased.
Feces
In Table 15 is given the dates on which feces were collected in the
1977 and 1978 experiments. An inspection of this table shows that the
feces of periphyton-fed snails had a mean ash free dry weight which was
84.5% (1973) to 86.2% (1977) of the dry weight. For the unialgal Cladophcra-
fed snails the mean percent varied from 54.4 (1977) to 50.6'i (1 978T* Thi
snails fed on CIadophora plus epiphytes had a mean percentage ash free dry
weight of 73.7%, and for the starved snails 73-5% of the dry weight. These
data indicate that the greatest amount of organic matter was in the feces
of the periphyton-fed snails and the least in those snails fed on uni algal *
Cladophora.
Because there was very little organic matter in the faces, we were not
able to determine the caloric content using the microbomb calorimeter.
Utilization of Calories
An attempt has been made to determine how the calories assimilated were
partitioned in the 1378 and 1977 experiments (Tables 16, 17).
In 1978 the total calories assimilated by snails on the periphyton diet
between January 25 and 31 were 52.35 ca!/snail. Of this amount 27.7% was
weight gained, 28.3% respired, and 44% was used in egg production. Only 17.5%
of the food ingested on a calorie basis was assimilated (Table 16). On the
Cladophora plus epiphytes diet, the amount of calories ingested was only
21* of those ingested on the periphyton diet. The snails lost weight. If
we add calories in lost weight to those utilized in respiration and egg
production, the partitioning is 33.3% for weight loss, 41.4% for respiration,
and 25.3% for egg production. On the Cladophora diet and for those snails
that were starved the calories used in respiration and egg production were
less than in the periphyton and Cladoohora plus epiphytes diets.
27
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In the 1977 experiments we can only examine the partitioning of assimi-
lated calories into growth or loss of weight and respiration (Table 17), On
the periphyton diet the snails increased in weight throughout the experiment,
but the calories partitioned to weight gain decreased from June 2, and
those used in respiration increased. On the Cladophora diet the calories
assimilated were much less. There was a decrease in caloric value of the
snail tissue except in the period of June 30 to July 14, when there was a
slight gain in weight from the June 30 weight. The percent of calories
utilized in respiration showed an irregular trend, but were generally high
which perhaps indicates that the snails were stressed by this diet. The
data for June 16-30 is quite anomalous to the findings on the other dates.
Crayfish (Qrconectes propinouus) Studies
The results of the crayfish experiments showed from a study of the mean
weights that the crayfish fed on periphyton gained weight whereas those fed
on Cladophora plus epiphytes and on unialgal Cladophora lost weight. How-
ever, in all cases the standard deviation was large enough to mask: any real
trend {Tables 18 and 19). The males tended to be heavier throughout the
experiment. Very little molting occurred. It was quite evident that these
three diets were not satisfactory and that the periphyton was a little
better diet than the other two.
Respiration rates on all three diets declined during the course of the
experiments (Table 20). This may have been at least in part due to the fact
that the crayfish had recently molted when the experiment started and they
molted very little during the course of the experiments. It is also known
that crayfish often go into hiberation during the cooler months. The respir-
ation rates were always higher in both males and females fed on periphyton.
Respiration was a much larger percent of the food ingested on Cladophora and
Cladophora plus epiphytes than it was for those crayfish fed on periphyton
(Table 26).
The food consumed on a caloric basis was much greater on the periphyton
diet than on the other two diets (Table 21, 22). On all diets the amount con-
sumed decreased over the experiment. The females tended to ingest more
food than the males, although this was not always true. It is very diffi-
cult to obtain an accurate estimate of the amount of food ingested due to
breakage. An experiment was carried out to determine just how much this
breakage was {Table 23). It was apparent that the amount of breakage was
very variable, being 1-21% of the food presented.
Studies were made of the dry weight of the feces collected on any one
day (Table 24), It was evident that the feces per crayfish was greater from
those crayfish fed on periphyton. This one would expect, because of the
much larger amount of food ingested. The average amount of feces produced
28
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from the CIadoohora plus epiphytes diet and the Cladoohora diet were not
significantly di-ferent because of the very large variance which occurred.
One can only say from these studies that the mean weights of the feces were
less in these diets than in the periphyton diet; however, the large variance
may negate this statement,
A study was made to determine how the calories ingested -..ers partitioned,
A much larger percentage of the food ingested seemed to have been assimi-
lated on the Cladophora plus epiphytes and the CIadoohora diet than in the
experiments where the crayfish were fed periphyton {Table 25). The only data
we have for all three diets is on October 3-4. The male and female crayfish
lost weight on the Cladophora plus epiphytes diet but not on Cladophora, It
Is probable that on the"Cladophora plus epiphytes diet they assimilated most
of the food they ingested. On the Cladophora diet the females assimilated
only 57.5% of the food ingested, whereas the males assimilated most of the
food ingested.
On October 3 and 4 the crayfish on the three diets respired a similar
percentage of the calories assimilated, but on December 13 and 14 the per-
centage of calories assimilated which were respired was less on the Cladophora
plus epiphytes diet than on the other two diets.
At the end of the experiment the X organ-sinus gland of the eyestalk
was removed to see if we could force molting. Molting did occur in the
females on the periphyton diet (Table 26), and there was a gain in weight
of the crayfish. It was difficult to determine whether the crayfish did not
molt because of the period in their life cycle when the experiments were
run or whether it was because of unsatisfactory conditions in the test experi-
ments. Both factors might have been involved. However, it is evident from
these experiments that the periphyton was a better diet for the crayfish
than was the CIadophora or the Cladophora with epiphytes, If one bases his
conclusions on weignt, it was quite evident that they ingested more food on
the periphyton diet than on the Cladophora plus epiphytes, and the latter
was the better diet than Cladoohora alone.
Tadpole (Rana pipiens) Studies
The results of the feeding experiments with the tadpoles Rana pipiens
are set forth in Table 27. It is evident from Table 27 that those
tadpoles fed on diatom-dominated periphyton grew much faster than those on
Cladophora. The periphyton-fed tadpoles continued to increase rapidly in
weight throughout the experiment. The tadpoles fed on Cladopnora plus
epiphytes grew at about the same rate as those fed on diatom-dominated peri-
phyton alone. There is a little evidence that they even grew somewhat better
when one compares the actual weights; however, the standard deviation is so
great that these differences are really not significant. In contrast, the
tadpoles fed Cladoohora decreased from their starting weights until May 18
.)
29
-------
when they grew between Hay 6 and 13 to be at about the same weight as they
were at the start of the experiment.
The tadpoles fed on periphyton showed signs of metamorphosing toward
the end of the experiment. This was also true of those fed on Cladoohora
plus epiphytes. Those fed on Cladoohora showed no signs of metamorphosing.
It was also noted that the tadpoles feeding on the Cladoohora with
epiphytes were just feeding on the epiphytes, as an examination of their
intestines showed that diatoms were the main food passing through the gut.
Because the diatoms were rather plentiful on the CIadophora and the tadpoles
have the ability to scrape the diatoms from the Cladoohora, it is under-
standable that these growth rates should not be different.
An examination of their respiration rates (Table 28} showed that the
tadpoles fed on diatom periphyton has a similar respiration rate to those
fed on Cladoohora plus epiphytes, and these rates were lower than those fed
on Cladoohora alone. Those tadpoles fed on peripnyton and those fed on
CIadophora plus epiphytes had a decrease in respiration rate between April
5 and 18, whereas those fed on Cladophora alone had an increase in respira-
tion rate. When we look at the calories consumed per tadpole in respiration
those fed on Cladoohora having very little weight consumed the greatest
amount of oxygen, These small tadpoles which were fed Cladophora had a
much higher respiration rate than those which were on a satisfactory diet,
which may be correlated with the fact that they were under stress due to
starvation.
Catfish (Ictalurus punctatus) Studies
These experiments were started on June 17 and terminated on July 15.
They were cursory experiments simply to determine whether Ictalurus punctatus
would eat Cladophora.
The experiments (Table 29) showed that the catfish on the periphyton
diet had gained weight on July 1, continued to gain weight on July 8, but
lost weight on July 15, so that their weight was similar to that at the
start of the experiment.
On the Cladoohora diet there were only two weighings after the start of
the experiment, because there was a steady decrease in the size of the fish
and they were about half their weight at the termination on July 8 than they
were at the beginning of the experiment.
Fathead minnow (Pimeohales promelas) Studies
The results of the experiments (March 7-May 23, 1978) in which peri-
phyton, Cladophora and epiphytes, and unialgal Cladoohora were fed to the
30
-------
fathead minnow, Pimephales promelas, produced several interesting results
as to growth, assimilation of food ingested, and respiration.
In Tables 30 and 31 the growth of the fish throughout the exoeriment is
given. The variance of the mean is fairly large, but it is clear that the
fish on the periphyton diet grew throughout the experiment. Those fish on
unialgal Cladoohora or on Cladophora plus epiphytes lost weight throughout
the experiment with the greatest loss in terms of calories between April 14
and May 23. This difference in growth is probably due to the difference in
food consumed which was measured by food loss between the food presented and
the food remaining after feeding (Tables 33, 34), and by experiments using
'4C-labeled food (Table 37), The average calories of food ingested in the
two trays on the periphyton diet was approximately 23 times that ingested on
the Cladophora plus epiphytes diet and 227 times the calories ingested on
unialgal Cladophora.
As seen from Table 35, the average assimilated calories of periphyton as
estimated from gain in weight and respiration was 6.2% of the food ingested
March 7 to April 14, and 6.3% of ingested food from April 14 to May 23. This
was determined by estimating the food consumed on a daily basis from Table 33
and the food assimilated for the number of days in the time interval from
data in Table 35. In the CIadophora plus epiphytes diet these percentages
were 2215 by April 14 and 246" for April 14-May 23. For the CIadophora diet
the calories assimilated of those ingested averaged 1717% as of April 14 ana
2079% between Apri1 14 and May 13, when al1 fish died. This is to a large
measure the cause of the great weight losses on the two CIadophora diets.
These data can only be considered as approximate, for although several
attempts were made to determine respiration, reliable data were only obtained
for one date (Table 32).
The assimilation of food as determined by experiments with (Tables
36, 37) show data which support the above relationship between food assimilated
on the various diets. The uptake of the tagged food was less on an ash
free dry weight (AFDW) basis than for the two C1adophora diets, The assimi-
lation ratio—i.e., counts in muscles / counts in guts—was 0.14 for Cladoohora
and epiphytes versus 0.83 for periphyton (May 25) and 0.19 versus 0.42 (May 26)
(Table 36). The intake of food (DPM/mg AFDW) on unialgal Cladoohora was much
less and so variable that not much significance can be attached to the data.
In Table 37 an attempt is made to show the distribution of the food consumed
in the fish. Generally the largest QPM/mg is in the muscle tissue and the
least amount is in the viscera. It is interesting that the feces have a higher
count in the Cladophora plus epiphytes diet than in the periphyton diet, and
would indicate that of the food consumed much lass was digested. As one
would expect (although the data are not very reliable) much less food is
ingested in the Cladoohora diet.
The respiration rates from the data ii rable 32 are least for fish
on the unialgal Cladoohora diet and greatest for those on the Cladoohora
)
31
-------
plus epiphytes diet. The increase in weight (calories) on the periphyton
diet as a percent of the calories respired varied between 25.8-30.5';'; (Table
35). The loss in weight (calories) as a percent of the calories respired
ort the Cladophora plus epiphytes diet was 34.1-72%, and on the unialgal
Cladophora diet, 58-102.5%
The results of these experiments clearly showed that unialgal CIadophora
and Cladophora plus epiphytes were unsatisfactory diets for the fathead min-
now, Pimephales promelas. These data plus those on snails showed the im-
portance of the chemical analyses of the food values of C. glomerata and the
identifications, if they existed, of the presence of toxic chemicals. The
studies of Larson (Part II) elucidate these concerns.
32
-------
TABLE 1. DATA ON SNAIL TISSUE, 1978 EXPERIMENTS
Ash Free 3
Ash Free b
Dry Weights
Dry Weight
Dry Weight
mq/snail
ma/snail
cal/snail
Start of Experiment 3.484
4.434
3.205
4.079
17.256
21.961
Jan. 25
6.2 Id
2.122
5.717
1.952
30.780
10.510
4.270
4.852
3.928
4.464
21.148
24.034
3.176
7.276
2.922
6.694
15.732
36.040
3.490
3.408
3.211
3.135
17,288
16.879
4.966
4.156
4.569
3.824
24.599
20.588
5.114
5.068
4.705
4.663
25.332
25.106
10.216
2.992
9.399
2.753
50.604
14.822
4.240
4.572
3.901
4.206
21.003
22.645
6.216
4.788
5.719
4.405
30.791
23.717
5.412
3.052
4.979
.2.808
26.807
15.118
6.120
3.626
5.630
3.336
30.312
17.961
& Tap
4.084
4.046
3.757
21.784
20.228
3.328
3.840
3.062
3.533
16.486
19.022
7.160
6.552
6.587
6.028
35.464
32.455
5.824
5.176
5.358
4.762
28.84 7
25.639
X
4,
800
4.
417
23.
78
SD
I.
61
1.
47
7.
93
N
32
32
32
Periphyton
Jan, 31
X
SD
N
® AFQW based on 92,0% of dry weight
" 5.3841 cal/mg AFDW, see Table 2.
see Table 2
12.108
10.982 C
52.725
11.148
10.111
48.544
8.800
7.982
33.322
10.364
9.400
45.130
8.856
8.032
38.562
4.560
4.136
19.857
7.816
7.089
34.035
6.648
6.030
28.950
8.790
7.970
38.258
2.46
2.234
10.801
8
8
a
^ AFDW based on 90.7% of dry weight, see Table Z
d 4.8011 cal/mg AFDW, see Table 2.
(continued)
33
-------
TABLE 1, continued.
Ash Free 6
Ash Free
Dry Weight
Dry Weight
Dry Weight
mq/snai1
mq/snai1
cal/snai1
Periphyton
8.882
46.579
Feb. 10
9.723
6.020
5.496
28.822
10.280
9.386
49.222
7.333
5.700
35.136
11.652
10.638
55.788
10.240
9.349
49.028
• 21.688
19.801
103.840
4.336
3.959
20.762
X
10.160
9.276
43.647
SD
5.269
4.811
25.258
N
8
o
Q
o
® AFDW based on 91.3% of dry weight.
see Table 2.
T 5.2442 cal/mg AFDW,
see Table 2,
Hadoohora
4.100
3.485 9
18.701 h
+¦ epiphytes
4.628
3,934
21.no
Jan 31
3.252
2.764
14.832
3.592
3.053
16.382
7.068
6.008
32.239
3.696
3.142
16.860
4.264
3.624
19.446
3.500
3.060
16,420
SD 1.21 1.029
19.499
^AFDW values based on 35,01 of dry weight, see Table 2,
"5.3664 cal/mg AFDW, see Table 2,
Feb. 10 4,400 4.083 1 21.617 J
5.112 4.744 25.117
2.528 2.346 12.421
4.704 4.365 23,110
3.908 3.627 19.203
1.792 1.663 8.805
X 3.741 3.471 18.379
5D 1.307 1.213 6.420
N 6 8 6
i AFDW based on 92.8* of dry weight, see Table 2,
J 5.2944 cal/mg AFDW, see Table 2.
(continued)
34
-------
TABLE 1, continued.
Ash Free
Ash Free ^
Dry Weight
Dry Weight
Dry Weight
mq/1 snail
mg/l snai1
cal/snaiI
CIadoohora
2,346
2.1721
10.4312
Jan, 31
4.830
4.473
21.481
4.438
4,110
19.738
2.782
9 q7g
12.371
5.004
V.63A
22.254
3.590
3,324
15.963
3.146
2.913
13.989
4.198
3.887
18.667
X
3.792
3.513
16.862
SO
0.978
0.904
4,351
N 8 8 8
ifAFDW values based on 92.5% of dry weight, see Table 2
1 4.8024 cal/mg AFOW, see Table 2.
Feb. 3
1.400
1.295 m
7 290
2.376
2.198
12.373
2.7G6
2.503
14.090
3.000
2.775
15.621
2.636
2.438
13.724
2.184
2.020
1 i .371
X
2.384
2,205
12,412
SO
0,558
0.516
2.904
N
6
6
6
mAFDW values based on 92.5% of dry weight, see Table 2.
n5.6292 cal/ma AFDW, see Table 2.
(continued)
35
-------
TABLE 1 , continued.
Ash Free 0
Ash Free P
Dry Weight
Dry Weight
Dry Weight
mg/1 snail
mq/1 snail
cal/snai1
Sudr/ed
(Day 6) Jan, 31
2.482
2.336
11.809
2.146
2.019
* 10.206
5.304
4.991
25.231
2.230
2.145
10,843
2.9*40
2.267
13.988
3.472
3.267
16.515
4.370
4.112
20.787
2.062
1.940
9.807
X
3.130-
2.947 -
14.898
SD
1.180
1.106
5.591
N
8
8
8
Q-AFDW values
based
on 94.11 of dry weight, see Tab
P 5.0552 cal/mg AFDW,
see Table 2,
(Day 15) Feb. 9
1.792
1.636 R
8.780 p
2.406
2.197
11 .791
2.884
2.633
14.131
G.930
0.849
4.557
1.370
1.251
6.714
2.592
2.366
12.698
X
1.996
1.822
9.779
SD
0.759
0.693
3.719
N
6
6
6
^ AFDW values
based on 91 3% of dry weight, see Table
r 5.3671 cal/mg AFDW,
see Table 2.
36
-------
TABLE 2. DATA ON WHICH CALORIE CALCULATIONS ARE BASED,
1978 SNAIL TISSUE EXPERIMENTS
Percent Ash Free
Ash Free Ash Free Dry Weight
Dry Weight* Dry Weiabt Dry Wei grit cal/mg
Start of Experiment
Jan,
24
9.30
8.76
9^.2
5.1133
6.48
5.94
91 .7
5.2559
10.52
9.68
92.0
5.2581
7.67
7.19
93.7
5.3631
8.34
7.36
88.2
5.7003
8.58
7.91
92.2
5.6089
I
92.0
5.3841
SO
2.11
0.2253
N
6
6
Periphyton
Jan. 31
7.76
6.88
88.7
4.3416
12.26
11.02
89.9
5.0948
10.32
9.64
93.4
<1.9668
X
90.7
4.8011
SD
2.44
0.4030
N
3
3
Feb. 9
9.12
8.26
90.6
5.6160
10.74
10.08
S3.9
5.3502
9.08
8.12
89.4
4.7664
I
91.3
5.2442
50
2.33
0.4346
N
3
3
Cladophora
Jan, 31
X
SD
N
+ epiphytes
'.72
10.80
8.00
7.09
9,22
6.22
91,8
85.4
77.7
85.0
7,1
3
5.0661
4.8669
6.1662
5.3664
0,6997
3
Feb. 9
X
SD
N
6.36
8.56
8.28
5.90
7.98
7.64
92.8
93.2
92.3
92.8
0.45
3
5.4955
5.0390
5.3487
5.2944
0.2330
3
(continued)
37
-------
TABLE 2, continued.
Dry Weight
Ash free
Dry Weiaht
Percent
Ash Free
Dry Weiqht
4sh Free
ury Weigl
cal/mq
CI adoohora
Jan. 31
7.00
6.28
89.7
4.4470
a,24
7.80
94.7
4.6904
6.74
6.30
93.5
5.2699
X
92.6
4.8024
SD
2.61
0.4227
N
3
3
Feb. 9
6.53
6,03
92.3
5.4796
7.10
6.58
92,7
5.7788
X
92.5
5.6292
SD
0.28
0.2115
N
2
2
tarved
Jan. 31
6.68
6.42
96.1
5.2041
8.46
6.04
93.5
4.7585
6.34
5.88
92.7
5.2029
I
94.1
5.0552
SD
1.78
0.2569
N
3
3
Feb. 9
6.18
5.74
92.9
5.6544
4.68
4,20
89.7
5.0798
X
91.3
5.3671
SD
2.26
0.4063
N
2
2
* Number of snails not known.
38
-------
)
TABLE 3. DATA ON SNAIL TISSUE, 1977 EXPERIMENTS
Start of Experiment
May 19
X
SD
N
Periphvton
June 2
X
SD
N
Ash Free a
Ash Free b .
Dry Weight
Dry Weight
Dry Weight
mq/snai1
mq/snai1
cal/snail
1.919
1,626
1.764
1.494
9.649
8.166
0.168
0.788
0.154
0.724
0.842
3.957
1.362
0.512
1,252
0.471
6.848
2.574
0.204
0.588
0.187
0.540
1.023
2.952
1.490
1.460
1.369
1.342
7.488
7.335
0.422
1.818
0.388
1.671
2.121
9.134
1.328
0. 656
1.220
0.603
6.669
3.296
0.808
0.758
0.743
0.697
4.061
3.810
0.440
1.072
0.404
3.985
2.208
5.384
0.208
0.672
0.191
0.618
1.044
3.378
0.806
0.278
0,741
0.255
4.050
1.394
0.718
0.600
0.660
0.551
3.608
3.012
0.466
0.700
0.428
0,643
2.339
3.515
0.840
0.
773
4.
225
0.
510
0.
468
2.
562
26
26
26
AFDW values based on 91.9S of dry weight,
see Table 4.
5.4660 cal/mg AFDW, see Table 4.
6.688 8,996
5.364 15.353
6,876 11.360
II.800 10.424
8.916 13.328
12.096 8.648
10.616 9.136
7.324
5.959 8.015C
4.779 13.630
6.127 10.122
10.514 9.288
7.944 11.875
10.778 7.705
9.459 8.140
6.526
34.373 46.233 d
27.567 78.911
35.343 58.387
60.6*8 53.576
45.824 68.499
12.171 44.445
54.563 46.954
37.644
9.79 8.727 50.041
2.70 2.420 14.437
15 15 15
CAFDW based on 92.8% of dry weight, see Table 4
d5.8082 cal/mg AFDW, see Table 4.
)
39
-------
con tinued,
Dry Weight
mq/snail
Ash Free e
Dry Weight
mq/snail
Ash Free f
Dry Weight
cal/snai1
June 16
June 30
July 14
14.316
13.392
18,856
11 .712
12.396
13.608
6,332
15.696
17.248 13,371 16.110 68.851 82.955
20.600 12.503 19.240 64.407 99.073
17.336 17.612 16.192 90.689 83.377
10,620 10.939 9.919 56.328 51.076
22.032 11.578 20.578 59.619 105.960
14.700 12.710 13.730 65.448 70.700
15,584 6.381 14.555 32.858 74.948
14.660 75.489
15.0 14.006 72.113
3.92 3,665 18.870
15 IS 15
®AFDW based on 93.4% of dry weight, see Table
f 5.1493 cal/mg AFDW, see Table 4,
21.850
32.800
29.800
20.300
32.050
44.500
30.200
23.000
26.500
26.650
29,000
24.400
22.500
15.000
21.600
20.124
30.209
27.446
18,696
29.518
40.985
27.814
21
24.407 9 99.854 121.110 h
24
i? a r*
545
26.709
22.472
20.723
13.815
19.894
149.900 121.790
136.190 132.530
92.769 111.500
146.470 102.830
203.370 68.549
138.010 98.713
105.11
24.569 121.91
6.94 6.390 31.71
15 15 IS
9AFDW based on 92.1% of dry weight, see Table
h 4.9620 cal/mg AFDW, see Table 4.
53.100
13.000
34.792
26.976
22.192
20.400
37.400
47.400
6,636
6.142
51.650
16.052
32.904
41,000
46.728
11.440
30.617
23.739
19.529
17.952
32.912
41
5
5
45
14
28
7121 260.320
840 63.730
405 170.570
452 132.250
126 108.800
956 100.010
080
232.380J
32.535
30.111
253.210
78.696
161.310
181 3 St'
I U * V <•*
-------
TABLE 3, continued.
Cladophora
June 'I
X
SD
N
Dry Weight
mq/snaiI
Ash Free ^
Dry Weight
ml snail
Ash Free ^
Dry Weight
cal/snai1
0.468
0.338
0.434
0.314
2.521
1.824
0.708
0.572
0. 657
0.531
3.816
3.084
1.098
1.034
1.019
0.960
5.918
5.576
0,572
0.802
0.531
Q. 744
3.084
4.321
0.700
0. 658
0.650
0.611
3.775
3.549
0.292
0.718
0.271
0.666
1.574
3.868
0.964
0.923
0.895
0.861
5.198
5.001
0.754
0.700
4.066
0.707
0.240
15
0.656
0.221
15
3.812
1.281
15
* AFOW based on 92.8% of drv weight, see Table 4.
1 5.8082 cal/mg AFDW, see Table 4.
June 16
0.898
0,788
0.823
0.722 111
4.654
4.084 n
0.844
0.426
0.773
0.390
4.372
2.206
0.642
0.372
0.588
0.341
3.326
1.929
0.934
0.544
0.856
0.498
4.842
2.817
0.346
0.418
0.317
0.333
1.793
2.166
0.274
0.832
0.251
0.762
1.420
4.310
0.746
0.256
0.683
0.234
3.863
1 .323
I
0.594
0.
544
3.
079
50
0.
.240
0.224
1.
269
N
14
14
14
June 30
X
SD
N
m
n 5.6556
cal/mg
AFDW,
0.250
0.300
0.231
0.550
0. 600
0.509
0.650
0.400
0.601
0.400
0.350
0.370
0.470
0.100
0.435
0.700
0.250
0.648
0.150
0.350
0.139
0.400
0.350
0.278 0
0.555
0.370
0.324
0.093
0.231
0.324
1 .239
2.731
3.224
1 .985
2.334
3.477
0.746
1.878
1.491 p
2.978
1.985
1.738
0.499
1.239
1.738
0.40 0.364 1.952
0.18 0.162 0.872
15 15 15
0 AFDW based cn 92.5?S of dry weight, see Table 4
P 5.3651 cal/mg AFDW, see Table 4 .
(continued)
41
-------
TABLE 3, continued.
Ash Free q Ash Free r
Dry Weight Dry Weight Dry Weight
mq/snai1 mg/snail cal/snai1
July 14 0.474 0.762 0.416 0.669 2,033 3.270
0,400 0,368 0.351 0.323 1.716 1.579
0.488 0.804 0.428 0.706 2.092 3.451
0.340 0.264 0.299 0.232 1.462 1.134
0.184 1.000 0.182 0.878 0.792 4.292
0.480 0.264 0,421 0.232 2.053 1.134
0,348 0.524 0.306 0.460 1.496 2.248
X 0.480 0,418 2.054
SD 0.23 0.204 0.991
N 14 14 14
3 AFDW based on 87.8% of dry weight, see Table 4.
r 4.8877 ca1/mg AFDW, see Table 4.
)
42
-------
TABLE 4. DATA ON WHICH CALORIE CALCULATIONS ARE BASED,
1977 SNAIL TISSUE EXPERIMENTS
Ash Free
Dry Weight*" Dry Height
Percent
Ash Free
Ash Free
Dry Weight
Start of Experiment
May 19
7.16
6.58
91.9
5.6703
5.88
5.40
91.8
5.2616
I
91.9
5.4660
SO
0.07
0.29
N
2
2
Periphyton
June 2
8.53
7.98
93.6
6.0491
10,13
8.69
35.8
6.0332
11,68 "
10.26
87.8
5.2227
X
89.1
5.7683
SD
4.1
0.47
N
3
3
June 16
18,24
17.26
94.6
5.0270
13,34
12.08
90.6
5.0209
10.86
10.30
94.9
5.4000
X
93.4
5.1493
SO
2.4
0.22
N
3
3
June 30
12,22
10.00
81.8
5.1832
25,06
24.18
96.5
4.6084
17.44
16.46
94.4
4.9204
8.26
7.90
95.6
5.1358
I
92.1
4.9620
SD
S.9
0.26
N
4
4
July 14
20.12
19.14
88.0
5.0179
12,34
10.86
88.0
5.4432
19.42
17.10
88.1
6.2510
X
88.0
5.5707
SD
0.06
0.63
N
3
3
(continued)
43
-------
TABLE 4, continued.
Percent Ash Free
Ash free Ash Free Dry Weight
Dry Weiqht Dry Weiqfit Dry Weight cal/mq
Cladophora
June 2
3.38
3.12
92.3
5.4799
4.12
3.84
93.2
6.1365
f
92.8
5.8082
SD
0.64
0.46
N
2
2
June 16
6,18
5.66
91.6
5.6556
June 30
4.82
4.46
92.5
5.3651
July 14
6.40
5,62
87.8
4.8877
* Number of snails not known.
44
-------
TABLE 5. DATA ON SNAIL TISSUE, 1976 EXPERIMENTS
Ash Free a Ash Free ^
Dry Weights
Dry Weight
Dry Weight
mg/snail
mq/snaiI
cal/snai1
Start of Experiment
August 24
1,476
1.105
1.312
0.982
5.865 4.390
2.070
2.067
1.840
1.838
8.225 8.216
3.000
2.412
2.667
2.144
11.921 9.584
2.686
1.188
2.388
1.056
10.674 4.720
1.630
2.391
1.494
2.570
6.678 11.488
2.188
1.851
1.945
1.646
8.694 7.358
1,607
2.447
1 .429
2.175
6.383 9.722
2.559
3.522
2.275
3.131
10.169 14.000
2.969
2.536
2.629
2.255
11.796 10.080
1.423
1.251
1.265
1.112
5.655 4.971
2.973
1.832
2.643
1.525
11.814 7.282
1.157
0.818
1.029
0,727
4.600 3.250
I
2.
07
1.
841
8.231
SD
0.
73
0.
653
2.918
N
24
24
24
a Actual dry weights and wet weights of snail
0 4.4742 cal/mg AFDW, see Table
¦ 6.
Periphyton
Sept.
3
A
A
X
6.23c
5.538
24.778
N
12
12
12
Sept.
13
,4
X
11.42
10.358
55.712d
N
12
12
12
Sept.
22
A
X
12.49
11.553d
57.640
N
12
12
12
Oct^ 5
X
N
(continued)
18.20
12
14.724d
12
78.7 42d
12
Sno individual weights for snails
See Table 6 for values on which calculations are based.
45
-------
TABLE 5, continued
Ash Free Ash Free
Dry Weights Dry Weight Dry Weight
mg/1 snail mq/1 snail cal/snai1
Cladophora
Sept. 3
X i .46s 1.348 4.839
N 12 12 12
Sept. 13
X 1.30 1.060 5.53
N 12 12 12
Sept. 22
X 0.79 0.639 3.281
N 12 12 -12
Oct-_ 5
X 0.81 0.710 3,76
N 12 12 12
eNo individual weights for snails
46
-------
TABLE 6. DATA ON WHICH CALORIE CALCULATIONS ARE BASED,
1976 SNAIL TISSUE EXPERIMENTS
Ash Free
Percent
Ash Free
Ash Free
Dry Weight
Dry Weiaht
Dry Weiqht
Dry Weight
cal/roq
Start of Experiment
Aug. 24
no data
Periphyton
Sept. 3
16.34
15.94
97.6
4.1248
14.78
14.16
95.8
4.6326
8.58
6.30
73.4
4.6651
X
•
88.9
4.4742
SO
13.5
0.30
N
3
3
Sept, 13
11,08
9.98
90.1
5.3786
7.78
7.10
91.3
5.1714
X
90.7
5.2750
SO
0.9
0.15
N
2
2
Sept. 22
21.82
19.64
90.0
5.1124
18.28
17.34
94.9
4.8659
I
92.5
4.9892
SD
3.5
0.17
N
2
2
Oct. 5
12.88'
10.02
77.8
4.9616
11.28
9.46
83.9
5.7341
I
80.6
5.3479
SD
4.3
0.55
N
2
2
Cladophora
9,38
8.96
95.5
3.4595
Sept. 3
8.02
7.48
93.3
3.8957
6.98
6.15
88.1
3.4132
X
92.3
3.5895
SO'
3.8
0.27
N
3
3
(continued)
)
47
-------
TABLE 6t continued.
Percent Ash Free
Ash Free Ash Free Dry Weight
Dry Weight Dry Height Dry Weight caT/mg
Cladophora
Sept. 13
6.28
4.80
76.4
5.0175
4.92
4,26
86.6
5.5146
X
81 .5
5.2661
SD
7.2
0.35
N
2
2
Sept. 22
6.28
5.08
80.9
5.1354
Oct, 5
3.76
3.34
88.8
5.8183
3.22
2.78
86.3
4.7908
I
87.6
5.3048
SD
1.8
0.73
H
2
2
* Number of snails not known.
48
-------
TABLE 7, SNAIL RESPIRATION, 1978 EXPERIMENTS
Dry wei ght of snai I
tissue (mg)
X SO N
ul 02/mg snail/hr
X SD N
cal / *
mg snail/
hr
cal/
snai1/
hr
cal /
snai1 /
day
Jan. 2k
Start of Experiment
4.8o
1.61
32
1,2:5
0.20
8
.0059
0.028
0.672
Jan. 31
Periphyton
8.79
2.46
8
2.%
0.11
2
.0117
0.103
2.472
CIadophora + Epiphytes
if. 28
1.21
a
1.8%
0.64
2
.0086
0.037
0.888
Cladophora
3-79
0.98
Q
1.65
0,11
2
.0078
0,029
0,696
Starved
3.13
1.18
8
1.51
0.22
2
.0071
0,022
0.528
* 1 ul 02 = 0.00472 calories
-------
TABLE a, SNAIL RESPIRATION, 197? EXPERIMENTS
Periphyton
June 2
June 16
June 30
July 14
U%
O
Cladophora
June 2
June 16
June 30
July 14
Dry weight of
tissue (iiif)}
X SO
snail
II
ul Q«/mg snail/hr
X * SO N
ca1/mg *
snail/hr
cal/
snail/hr
9 80
2.70
15
2.S4
1.16
3
0.012
0.118
15.00
3.92
15
1.99
0.46
3
0.0094
0.141
26.68
8.94
15
2.27
0.25
3
0.011
0.293
29.30
15.85
14
2.09
0.36
3
0.0099
0.290
0.71
0.24
15
1.29
0.07
3
0.0061
0.0040
0.59
0.24
14
1.28
0 b4
3
0.0060
0.0035
0.40
0,17
15
0.67
0.24
3
0.0031
0.0010
0.48
0.23
14
4.13
1,27
3
0.0194
0.0930
cal/
snail/day
2.822
3.304
7.044
6.962
0.096
0,004
0.024
0.232
* 1 ul 02 = 0,00472 calories
-------
TABLE 9. SNAIL FOOD CONSUMPTION, 1978 EXPERIMENTS
Wet Wt.
Dry Wt,a
mg k
Mg
Calorie;
nig
mg
Ash Free
No. of
AFDW/
consumec
consumed
consumed
Dry Weight
Snails
snail
snai 1
Peri phyton
Jan. 29 -
Tray
5
4790
2031
280.3
20
14.014
55.333
Tray
6
5100
2162
298.4
20
14.921
58.905
Jan. 30 -
Tray
5
3900
1654
2.28.2
20
11.410
45.046
Tray
6
4100
1738
239.9
19
12.626
49.849
Jan, 31 -
Tray
5
3100
1314
181.4
16
11.337
42.205
Tray
6
800
339
46.81
14
3.344
12.447
¥
11.275
43.964
SO
4.136
16.641
N
6
6
Feb, 1 -
Tray
5
3720
1577
220.8
15
14.719
54.795
Tray
6
1200
509
71.23
10
7.123
26.518
Feb. 3 -
Tray
5
2700
1145
160.3
15
10.685
39.777
Tray
6
2300
975
136.5
10
13.653
C A Q97
3U *OC/
Feb. 4 -
Tray
5
2300
975
136.5
15
9.100
33.877
Tray
6
3100
1314
181.4
10
18.140
67.532
Feb. 5 -
Tray
c,
1900
806
112.8
15
7.519
27.991
Tray
6
2000
848
118.7
10
11.870
44.190
Feb. 6 -
Tray
5
3100
1314
181 .4
15
12.093
43.360
Tray
6
2500
1060
148.4
10
14.840
56.891
Feb. 8 -
Tray
5
2900
1230
172.1
14
12.296
47.138
Tray
6
2100
890
124.7 *
9
13.851
53.098
Feb. 9 -
Tray
5
1300
551
77.17
10
7.717
29.583
Tray
6
700
237
41.55
5
8.310
31.859
Feb. 10 -
Tray
5
1400
594
83.10
9
9.234
35.399
Feb. 13 -
Tray
5
900
382
53.42
8
6.673
25.601
Tray
6
1000
424
59.36
4
14.840
56.891
I
11.333
42.843
SD
3.372
12.685
N
17
17
a Dry
weight
based on
42.41 of wet weight.
b Ash
Free Dry Ueiqht
based on 13.4% of dry weight for Jan.
29-31;
14,0% of dry weight
on Feb. 1.
c Jan.
. 29-31
= 3.9484 cal/mg AFDW; Feb, 1-5
= 3.7228 cal/mg AFDW;
Feb.
. 6-10 :
3 3.8336 cal/mg AFDW
, see Table
10.
(continued)
51
-------
TABLE 3, continued.
y I
Met Wt. Dry Wt. mg mg Calories
mg mg Ash Free No. of AFDW/ consumed
consumed consumed Orv Weight Snails snail snail
Cladophora +"Epiphytes
Jan. 29 - Tray 7
36.82
25.37
25.37
20
1.269
5.594
Jan. 31 - Tray 7
2.10
1.45
1 .45
13
0.112
0.494
Tray 8
67.33
46.39
46.39
n
4.217
18.589
Feb. 1 - Tray 7
JO . DO
23.19
23.19
9
2.577
11.359
Tray 8
31.56
21.74
21.74
11
1-976
3.710
X
2.03
8.949
SD
1.528
6.737
N
5
5
dDry weight based on 10.52% of wet weight.
|AFDW based on 68.3% of dry weight, see Table 10.
f4.408 cal/mg AFOW, ses Table 10.
Cladophora h -
Jan. 29 - Tray 4 13.40 15.499 15.49 19 0.815 3.2301
9Dry weight based on 18.4% of wet weight.
^AFDW based on 34.2% of dry weight, see Table 10.
13.9615 cal/mg AFDW, see Table 10.
)
52
-------
TABLE 10.
DATA FOR BASIS OF CONVERSIONS FROM DRY WEIGHT
TO CALORIES, 1978 SNAIL FOOD EXPERIMENTS
Periphyton
Jan. 30
X
SD
N
Dry Weight
mq/1
943.9
522.9
196.3
Ash Free
Dry Weight
mq/1
129.4
67.7
26.8
% Ash Free
Dry Weight
13.7
12.9
13.7
13.4
0.46
3
Ash Free
Dry Weight
cal/mq
3.6909
4.2059
3.9434
0.364
2
Feb.
X
SO
N
915.5
914.6
312.6
124.2
126.6
189.7
13.6
13.8
14.5
14.0
0.47
3
3.4361
3.9595
3.7228
0.335
2
Feb. 10
X
SO
N
727.9
624.1
351.5
96.0
35.2
54.5
13.2
13.7
15.1
14.0
0.98
3
4.0036
3.1842
4.0745
4.0722
3.8336
0.434
4
Cladophora + Epiphytes
Feb." 1979
X
SD
N
12.72
13.74
17.94
19.30
12.80
14.98
20.28
19.24
9.64
10.18
10.48
10.94
10.40
12.06
13.14
11.50
75.8
74.1
58.4
56.7
81.3
80.1
64.8
59.8
68.9
10.1
8
4.480
4.358
4.167
4.406
4.308
4.470
4.379
4.699
4.408
0.1536
8
(continued}
53
-------
TABLE 10, continued.
Ash Free Ash Free
Dry Weight Ory Weight % Ash Free Dry Weight
mq/1 mq/1 Dry Weight cal/mq
CIadophora
Jan. 30 428.1 354,1 82.7 4.044?
385,3 321.4 83.3 4.1909
311,9 270.3 86,7 3.6489
I 84.2 3.9615
SD 2.16 0.280
N 3 3
54
-------
TABLE 11. SNAIL FECUNDITY, 1978 EXPERIMENTS
No.
No.
No. egg
No.
Eggs/
Egg masses/
Days
Eqqs
Masses
Snails
snail/day
snai1/day
Periphyton
76
0.48
Jan. 24-27
4
998
40
6.24
Jan. 28-30
3
2832
138
39.67
23.80
1.16
Jan. 31-Feb. 1
2
1328
88
27.5
33.24
1.60
Feb. 2-3
2
1827
83
25
35.54
1.66
Feb. 12-14
3
6559
113
25
87.45
1.51
Feb. 17
1
1136
34
14
81.14
2.43
Cladophora + Epiphytes
11.5
0.86
Jan. 31-Feb. 1
2
506
38
22
Feb. 2-3
2
250
25
20
5.25
0.63
Feb. 12-14
3
427
29
19
7.49
0.51
Feb. 17
1
125
6
5
25.0
1.20
Cladophora
Jan. 31-Feb. 1
2
83
11
20.5
2.02
0.27
Feb. 12-14
3
47
S
9
1.74
0.19
Starved
Jan. 31-Feb. 1
2
82
12
22.5
1,82
0.27
Feb. 12-14
3
26
6
13.67
0.63
0,15
)
55
-------
TABLE 12.
SNAIL FECUNDITY, 1977 EXPERIMENTS
1
No,
Ho.
No. Egg
No,
Eggs/
Egg Masses/
Dates & Diets
Days
Eaas
Masses
Snai1s
Snai 1 /Day
Snai1 /Day
Periphyton
May 23
1
129
11
81
1.59
0. 14
May 24-27
k
3357
186
81
10.36
0.57
June 7-8
2
5941
79
59
50.35
0.67
June 9-10
2
6378
123
59
54.05
1 ,04
June 11-13
3
9769
179
58
56.14
1.03
June 14-15
2
6384
93
58
55.03
0.80
June 16-17
2
5720
84
42
68.10
1 .00
June 18-20
k
10369
152
41
63-23
0.93
June 22-24
3
1 1 221
145
40
93.51
1.21
June 25-27
3
10834
145
40
90.28
1.21
June 28-July 1
k
15687
184
32
122.55
1,44
July 8-8
3
10442
100
20,33"
171.21
1.64
July 9-11
3
7421
83
'20
123-68
1.38
July 12-13
2
5022
55
20
125-55
1.38
July 14
1
2103
19
20
105.15
0.95
CIadoohora
May 23
1
0
0
0
0
May 24-27
k
0
0
81
0
0
May 31
1
33
6
79
0.42
0.08
June. 3
1
24
6
64
0.38
0.09
June 7-8
2
0 ,
0
63
0
0
June 9-10
2
0
0
63
0
0
June 11-13
3
0
0
63, 62
0
0
June 1*4-15
2
0
0
61
0
0
June 16-17
2
0
0
45, 44
0
0
June 18-21
k
0
0
43* 42
0
0
June 22-24
3
0
0
42
0
0 "
June 25-27
3
0
0
41
0
0
June 20-July 1
1+
0
0
41, 40, 25
0
0
July 6-8
3
0
0
22
0
0
July 9-11
3
0
0
22
0
0
July 13
1
8
2
19.
0.42
0.11
July 14
1
0
0
5"""
0
0
)
"Drop in snail numbers due to removal for respiration experiments.
56
-------
25
26
27
28
31
. 1
. 10
. 14
. 15
. 10
. 17
. 20
. 21
. 22
. 23
. 24
. 27
. 28
. 29
. 30
1
4
ora
26
27
28
31
. 1
. 8
. 9
, 10
. 14
. 15
. 16
. 17
. 20
. 21
. 22
. 23
. 24
TABLE 13. SNAIL FECUNDITY» 1976 EXPERIMENTS
No, No. No. egg No. Eggs/ Egg masses/
Days Eggs Masses Snails snail/day snail/day
1 312 16 158 1.97 0,10
1 196 1Q 156 1.26 0.06
1 580 29 155 3,74 0.19
1 1336 53 152 8,79 0.35
1 1113 26 150 7.42 0,17
1 2870 70 149 19.26 0.47
1 3478 72 115 30.24 0.63
1 1894 34 81* 23.38 0.42
1 3526 64 81 43.53 0.79
1 3400 65 80 42.50 0.81
1 2115 50 78 27.12 0,64
1 2184 40 70 31.20 0.57
1 1807 26 69 2.6.19 0.38
1 1183 17 43* 27.51 0.40
1 141 2 43 3.28 0.05
1 1437 17 41 35.05 0.41
1 982 20 39 25.18 0.51
1 1352 23 38 35.58 0.61
1 0 0 37 0 0
1 1712 21 37 46.27 0.57
1 1179 17 35 33.69 0.49
1 1499 27 33 45.42 0.82
1 49 3 157 0.31 0.02
1 0 0 156 0 0
1 0 0 154 0 0
1 0 0 154 0 0
1 21 1 150 0.14 0.01
1 0 0 150 0 0
15 1 119 0.04 0.01
1 0 0 119 0 0
1 0 0 117 0 0
1 0 0 91 * 0 0
1 0 0 88 0 0
1 0 0 87 0 0
1 0 0 87 0 0
1 0 0 82 0 0
1 0 0 78 0 0
1 0 0 52*" 0 0
1 0 0 50 0 0
1 0 0 47 0 0
57
-------
TABLE 13, continued.
No. No. No. egg No, Eggs/ Egg masses/
Days Eggs Masses Sna i1s snails/day snails/day
Sept. 27 1 0 0 41 0 0
Sept. 28 1 0 0 39 0 0
Sept, 29 1 0 0 36 0 0
Sept. 30 1 0 0 33 0 0
Oct. 1 1 0 0 32 0 0
Oct. 4 1 0 0 28 0 0
Snails removed for respiration experiments
58
-------
TABLE 14. NUMBERS, WEIGHTS, AND CALORIC CONTENT OF SNAIL EGGS,
1978 EXPERIMENT
Sample Date Diet # Eggs mg/egg cal/mg AFPW
245
1/27/78
Periphyton
275
0.034
4.966
234
1/27/78
Periphyton
244
0.049
4.327
X
4.647
242
2/6/78
Cladophora +
427
0.010
4.718
Epiphytes
232
1/27/78
Cladophora
192
0.023
4.215
233
1/27/78
Cladophora
170
0.033
3.998
¥
4.106
231
1/27/78
Starved
175
0.04
4.075
235
1/30/78
Starved
408
0.022
4.087
X
4.081
-------
TABLE IS- SNAIL FECES, 1977-1978 EXPERIMENTS
Ash Free
Dry Weight Dry Weight
Date (ma) (ma)
Periphyton
1978 Jan, 25 25.34 21,54
Jan. 26 1+7.28 38.94
Jan. 29 51 .70 *+3-12
Jan. 30 35-80 32,20
Feb. 3 21.90 18.16
Feb. 4 36.52 30.44
X 34.423 30.733
SO 11.708 6.650
N 6 6
Cladophora + Epiphytes
1978 Jan 25 12.18 8,68
Jan. 27 21.92 15-06
Jan. 30 28.34 21.66
Jan. 31 25.98 17.94
Feb. 8 27-62 22.92
X 23.208 17.252
SO 6.648 5.708
N 5 5
CIadophora
1978 Jan. 25 7-48 3.84
Jan. 30 15.62 10.72
Jan. 30 7.06 4.36
x 10.053 6.307
SO 4.825 3»S31
N 3 3
(continued)
% Ash Free
Dry Weight
85.0
82.4
83.4
89.9
82.9
83-4
84.5
2.786
6
71-3
68.7
76.4
69.1
83.O
73,70
6.035
5
51.3
68,6
61.8
60,567
8.716
3
60
-------
TABLE 15» continued.
Oct ts
S tarved
1378 Jan. 25, 27
Jan. 30, 31
Feb, 1* 2
7
so
N
Pen' phy ton
197? May 23
May 25
June 2
June 13
July 8
July 13
X
SO
N
CIadophora
1977 May 25
Hay 27
June 2
¥
SO
N
Ash Free
Dry Weight Dry Weight % Ash Free
(mg) (mq) Dry Weight
17.70 13-54 76.5
6.62 5.00 75.5
5-92 06 68.6
10.08 7.533 73-533
6.608 5-223 4.302
3 3 3
42.20 37-66 89-2
43.80 39-88 91.1
41.48 36.98 89.2
67.98 61.50 90.5
26.60 23-22 87.3
52.88 36.88 69.7
46.157 39.353 86.167
14.41 12.375 8.173
6 6 6
3-08 1.34 59.7
3.70 1.78 48.1
9.42 5-22 55-4
5.4 2.947 54.4
3-495 1.969 5-864
3 3 3
61
-------
TABLE 16. UTILIZATION OF CALORIES INGESTED PER SNAIL, 1978 EXPERIMENT
Periphyton
January 25
(Start of Experiment)
Tissue X cal,/snai1 23.78
January 31
(6-day period)
Tissue X cal./snail 38.26
Gain or loss +14.48
Respiration
X cal./snail 14.03
Eyy production (cal.) 23.04
Total calories
assimilated 52.35
% wt. gain or loss 27.7
% respiration 28.3
% egg production 44.0
Calories ingested 257.06
% wt. gain or loss 5.6
% respiration 5.8
% egg production 6.2
% intake assimilated 17.6
Cladophora +
Epiphytes Cladophora Starved
23.78 23.78 23.78
*19.50 16,86 14.90
- 4,28 - 6.92 - 8.88
5.33 4.18 3.17
3.26 1,39 1,382
12.87 12.49 13.43
(33.3) 55.4 66.1
41,4 33,5 23.6
25.3 11.1 10,3
53.68 19.4
9.9 35.7
9.9 30.1
5.2 14.5
-------
TABLE 17. UTILIZATION OF CALORIES ASSIMILATED PER SNAIL,
1977 EXPERIMENTS
May 19 Peri phyton CIadophora
(Start of Experiment)
Tissue X cal ./snail 4.23 4,23
June 2 __
Tissue X ca!./snail 50*04 3-81
Gain or loss__ 45.81 {-0.42}
Respiration X cal./snail 39*48 1-34
Total calories used in
growth and respiration 85-29 1.76
% in tissue growth or loss 53*7 (23.9)
% in respiration 46.3 76,1
June 2-16 _
Tissue " cal./snail 72.1 3.079
Gain or loss 22,07 - 0.63
Respiration 47-38 1.18
Total calories used in
growth and respiration 69-45 I.Sl
% in tissue gain or loss 31-8 (34.8)
% in respiration 68.2 65-2
June 16-30 _
Tissue X cal./snail 121.91 1 -95
Gain or loss 49.8 (- 1.13)
Respiration 98.6 0.34
Total calories used in
growth and respiration 148.4 1.47
% in tissue gain or loss 33,6 {76.9)
% in respiration 66,4 23<1
June 30-Julx 14
Tissue X cal./snail 143.45 2.054
Gain or loss 21.54 0.102
Respira ti on 97.47 3.248
Total calories used in
growth and respiration 119.01 3•350
% in tissue gain or loss 18.1 3
% in respiration 81.9 97
63
-------
TABLE 18. INDIVIDUAL WEIGHTS AND CALORIC VALUES,
197? CRAYFISH EXPERIMENTS
Dry Weight
Ash Free
Dry Weight
Ash Free
Dry Weight
cal/cravfish
Start of Experiment
July 18
a
Aquarium 7
9
1,08
1.14
0,808
0.853
3635 3837
T
0.93
1.5*+
0.697
1.152
3136
5183
1.65
0.89
1.234
0.665
5552
2992
1-10
1.48
0.823
1.107
3703
4980
1 -39
1,62
1.040
1.212
4679
5^53
1.33
0.86
0.995
0.643
4476
2893
f r\ A
i « ZZ
1.27
0.913
0.950
4107
4274
i ,4o
1.17
1 . Oh?
0.875
4710
3936
i.39
1 .02
1.040
0.763
4679
3433
1.73
1.27
1.294
0,950
5321
4274
X
1.
27
0.953
1
+288
SD
0.
25
0.190
856
N
20
20
20
Aquarium 8
or
1.58
1.18
1.071
0.800
4i?4b 3118
2.46
2.k6
1.663
1.668
6500
6500
2.74
1.86
1.858
1.261
7241
4914
1.35
1.08
0.915
0.732
3566
2853
2.06
2.38
1.397
1,61 4
5444
6290
1.93
2-37
K309
l .607
5101
6262
2.25
0.87
' 1.526
0.590
5947
2299
1 .60
3-2*+
1.085
2.197
4228
8562
1.8**
1.51
1.248
1.024
4Q6 3
3991
2.01
1.19
1.363
0.807
5312
31^5
X
1.
90
1.
287
5016
SD
0»60
0.
416
1621
N
2.0
20
20
Aquarium 9
1 .01
1,14
0.666
0.751
2996
3379
+•
1 .27
0.98
0.837
0. 6k6
3765
2906
1 .36
0.89
0.896
0.587
4031
2641
Q.81
0.80
0.534
0.527
2402
2371
1-23
1.20
0.811
0.791
3649
3559
1.18
1.24
0.778
0.817
3500
3676
\.m
1.11
0.975
0.731
3577
3289
1.%
1.43
0.962
0.942
4328
4237
0,38
0.81
0,580
0-534
2609
2402
1.08
1.19
0.712
0.784
320 3
3527
X
1.
13
0.
743
3302
SD
0.
22
0,
143
600
N
20
20
20
(continued)
-------
TABLE l8, continued.
Dry Weight
Ash Free
Dry Weight
_S_
Ash Free
Dry Weight
cai/crayfi sh
Aquarium 10 cf
1 .63
1.38
l .074
0.909
4135
3542
1.41
1.52
0.929
1 .002
3620
3905
1.28
1.64
0.844
1.081
3289
4213
1.87
1.63
1 .232
1.074
4801
4185
1.41
1.64
0.929
1 .08!
3620
421 3
1.70
1.91
1.120
1.259
4365
4906
1.67
1.47
1. 101
0.969
4291
3776
1.71
2.33
1.1 27
1.535
4392
5982
1,32
1.69
0.870
5.114
3 390
4341
1.28
2.99
0.844
1.970
3289
7677
X
1.67
1.
103
4299
SD
0.
40
0.
261
1019
N
20
20
20
Aquari um TI • cf*
1.58
1.41
1.013
0.904
3948
3523
1.72
1.45
1.103
0.929
4298
3620
1.54
1.86
0.987
1 .192
38^6
4645
1.79
1»34
1.147
0.859
447C
3348
1.85
1.41
1.186
0.90^
4622
3523
0.95
1.38
0.609
0.885
2373
3449
2.06
1.22
1 • 320
0.782
5144
3047
1.32
1.34
0.846
0.859
3297
3348
1.36
1.43
0.872
0.917
3398
3574
1 • 36
1.08
0.872
0.692
3398
2697
X
1.
47
0.
944
3678
SD
0.
27
, 0,
175
680
N
20
20
20
Aquarium 12 Q
0.83
1.04
0.561
0.703
2524
3163
1 .40
1.30
0.946
0.879
4256
3954
1.32
0.79
0.892
0.534
4013
2402
0-72
0.92
0.487
0.622
2191
2798
0.78
Q.85
0,527
0.575
2371
2587
1.42
0,92
0.960
0.622
4319
2798
1.76
0.95
1.190
0.642
5354
2888
1.97
0.59
1.332
0.399
5992
1795
1.06
1.22
0.717
0.825
3226
3712
0.98
1.14
0.662
0.771
2978
3469
X
1.
10
0.
739
3340
SO
0.
35
0.240
1066
N
20
20
20
(continued)
65
-------
TABLE 18, continued.
Periphyton
July 28
Aquarium 7
Dry Weight
Ash Free
Dry Weight
Ash Free
Dry Weight
cal/cravfish
0.88
1.15
0.643
0.860
2893 a
3869
1,04
0.80
0.778
0.598
3500
2690
1,07
1.82
0.800
1.361
3599
6123
1.36
1.71
1.017
1.279
4575
5754
1.22
1.63
0.913
1.219
4107
5484
1.36
0.98
1 .017
0.733
4575
3298
1.75
1.52
1.309
1.137
5889
5115
1.25
1.44
0.935
1.077
4205
4845
1.83
1.11
I !*"T
1«257
0.830
5655
3734
X
SO
N
1.32
0.32
18
0.98?
0.236
18
4440
1063
18
Aquarium 8 - c?
1.61
1.26
1.092
0.854
4256 b
3328
2.56
1.95
1.736
1.322
6765
5152
2.75
1.13
1.865
0.766
7268
2985
1.35
2.41
0.915
1 .634
3566
6368
2.08
1.47
1.410
0.994
5495
3874
2.01
2.44
1.363
1.650
5312
6430
1.62
3.06
1.098
2.075
4279
8086
1.67
2.42
1,132
1 .640
4411
6391
2.04
1.383
5390
X
SD
N
1.99
0.56
17
0.349
0.377
17
5256
1471
17
August 18
Aquarium 7 - <£
X
SD
N
1.12
1
.26
0.838
0
942
3770
0.95
1
.67
0.711
1
249
3199
1.01
1
.68
0.755
1
257
3397
1.52
1
.47
1.137
f
1
100
5115
1.32
1
.58
0.987
1
182
4440
1.69
1
.87
1.264
1
399
5686
1.32
1
.13
0.987
0
845
4440
1,81
;. 354
6091
1.
43
1.
067
0.
29
0.
219
15
15
4238
5619
5655
4949
5318
6294
3801
4801
987
15
(continued)
66
-------
TABLE 18, continued,
Ash Free
Ash Free
Dry Weight
Dry Weight
Dry Weight
g
q
cal/crayfish
Aquarium 8
- cf
I.51 1.66
1.024
1.125
3991 4384
2.41 1.77
1.634
1.200
6368 4676
2.64 1.36
1 .790
1.264
6976 4926
1.32 2.36
0.895
1.600
3*88 6235
2.08 3.06
1.410
2.075
5495 8086
2.28 1.43
1.546
0.970
6025 3780
1.64 2,42
1.112
1.641
4333 6395
I
2.03
1.
377
5368
SO
0.51
0.
343
1356
N
14
14
14
August 29
Aquarium 7
- 0
1.06 1.22
0.793
0.913
3568 4107
1.67 2.IS
1.249
1.608
5620 7234
0.99 1.49
0.741
1.115
3334 5016
1.49 1.85
1.115
1.384
5016 6226
1.67 1.89
1.249
1.414
56 i 9 6361
1.35
1.010
4544
f
1.53
1.
14
5149
SO
0.36
0.
270
1212
N
11
11
11
Aquarium 8
- cf
2.55 1.66
1.730
1.125
6742 4384
3.03 1,77
2.054
1.200
8004 4676
1.35 1.88
0.914
1.275
3562 4969
2-16 2.43
1.464
1.648
570S 6422
2.37 3.31
1.607
2,244
6262 8745
X
2.25
1.
53
5947
50
0.62
0.417
1625
N
10
10
10
September 8
Aquarium 7
- 5
0.99 1.71
0,741
1.279
3334 5754
T
1.63 1.16
1.219
0.868
5484 3905
0.98 1.44
0.733
1.077
3298 4845
«
1.48 1.58
1.107
1.182
£980 5318
1.64 1.88
1.227
1.406
5520 6325
1.34 1.11
1.002
0.830
4508 3734
I
1.41
1.
055
4751
SO
0.30
0.221
997
M
12
12
12
continued)
67
-------
TABLE 18, continued.
Aquarium 8
Dry Weight
-------
TABLE 18, continued.
Aquarium 8 - cT
Dry Weight
2.45 1.70
2.54 2.32
2,24 2.93
1.58 1,41
Ash Free
Dry Weight
q
1.561 1.153
1.722 1.573
1.519 1.987
1.071 0.956
Ash Free
Dry Weight
cal/crayfish
6473 4493
6711 5130
5920 7743
4174 3725
X
SO
2.15
0.53
1.455
0.359
5671
1399
October 13
Acuarium 7
1.00
1.58
0.96
1.44
1.57
1.26
1.67
1.81
1.12
0.748
1.182
0.718
1.077
1.174
0.942
1.249
1.353
0,838
3365 4238
5318 5619
3230 6037
4845 3770
5282
1.38
0.30
9
1.031
0.229
1031
Aquarium 8 - cf
2.42
1 .54
2.50 2.90
2.18 1.37
1.641 1.044
1.695 1.967
1.478 0.929
6395 4063
6605 7665
5760 3620
2.15
0.59
6
1.459
0.400
5686
1559
November 15
Aquarium 7
o
r
1.00 1.56
1.55 1.24
0.88 1.79
1.41 1.07
0.748 1.167
1.159 0.928
0.658 1.339
1.055 0.800
3365 5250
5214 4175
2960 6024
4746 3599
1.31
0.32
0.982
0,237
R
4416
1068
8
(continued)
69
-------
TABLE 18, continued.
SD
Dry Weight
g
Aquarium 8 - cf 2.37 1
2.15
1.33
2.12
0.59
Ash Free
Dry Weight
q
1.609 1.017
1.681 1.939
1.458 0.902
1.434
0.401
Ash Free
Dry Weight
cal/crayfish
6270
6551 7556
5682 3515
5590
1563
December 7
Aquarium 7
X
SD
0.99 1.56
1.56 1,44
0.88 1.79
1,41
1.38
0.33
7
0.741 1.167
1.16? 1.077
0.658 1.339
1.055
1.029
0.244
3334
5250 4845
2960 6024
4746
4630
1099
Aquarium 8 ~ d* 2.45 1.51
1.661
1.458
1.024
6473
3991
X
5D
1.86
0.52
4
1.263
0.355
4
4922
December 14
Aquarium 7 - o
0.99
1.56
0.88
1.40
cs
1.33
1.78
0.741 1
1.167 0.995
0.658 1.331
1.047
3334 5214
5250 ' 4476
2960 5988
4710
1.36
0.32
1 .014
0.241
7
4562
1083
7
Aquarium 8 - cf
2.48
2.15
1.33
1 .681
1.458
0.902
6551
1.99
0.59
1 .347
0.401
{continued)
70
-------
TABLE 18 j, continued.
Cladophora + Epiphytes
July 28
Aquarium 3 o
Dry Weight
-3 —
1.17
0.23
Ash Free
Dry Weight
o
0.J68
0.153
Ash free
Dry Weight
cal/crayfi sh
1.39
1.29
0,916
0.850
3593
3334
1.57
1 .42
1 .035
0-936
4060
3671
1.20
0.83
0.791
0.§47
3103
2146
1 • 45
1.13
0.956
0.745
3750
2922
1.39
1.31
0.916
0.863
3593
3385
0.99
0.8l
0.652
0.534
2557
2095
1,12
1 • 36
0.738
0.896
2895
3515
0.92
1.0*+
0.606
0.635
2377
2687
0.90
1.14
0.593
0.751
2326
2946
0.83
1.21
0.547
0.797
2146
3126
3011
20
Aquariurn
10 cf
1.62
1.54
1.068
1.015
3955
3759
1.37
1,6*+
0.903
1.081
3344
400 3
'
1.27
1.64
0.837
1.081
3100
400 3
1.90
1.42
1 .252
0.936
4636
3466
t ¦'1 $1
I « Tt
1 .93
0.949
1.272
3514
^7!0
1.75
2.38
1.153
1.568
^270
5807
»
1.70
2.97
1.120
U957
4148
7247
1.32
1 -45
0.870
0.956
3222
3540
1.26
1.68
0.830
1.107
i074
4099
1.36
1.11
0.896
0.731
3313
2707
X
1.
64
1.
079
3996
SO
0.
43
0.
280
1 n
t U
38
H
20
20
20
August 18
Aquarium 9
0
M3
0.78
0.745
0.514
2922
2016
T
1.32
1.34
0.870
0.883
3413
3464
1.47
1.60
0.969
1.054
3801
41 34
0.96
1,14
0.663
A If 1
V - 12 i
2601
2546
1.19
1.26
0.784
0.330
3075
3256
1.45
1.45
0.956
0.956
3750
3750
1.45
0.89
0.956
0.587
3750
2303
1.38
0.93
0.909
0.613
3560
2404
1.27
0.837
3233
X
1.
24
0.815
3202
SO
0.
24
0.
155
600
N
17
17
17
(continued)
71
-------
TABLE l8, continued.
Dry Weight
q
Ash Free
Dry Weight
a
Ash Free
Dry Weight
cal/crayfish
Aquarium 10
Cf
1.60
1-55
1.054
1.021
3903
3781
1.30
i.59
0.857
1 ,048
3174
3881
1.86
1 ,£40
1.226
0.923
4540
3418
1.45
1.86
0.956
1.226
3540
4540
1,67
!. 48
1.101
0.975
4077
361 1
1.70
2.27
1 . 120
1 .496
4143
5540
1.26
1. 64
0.830
1,08l
3074
4003
1.32
2.90
0.870
1.911
3222
707?
>
1 .49
0.982
363 7
I
1.
67
1.
127
4068
qO
0. i+0
0.
285
982
N
17
17
17
August 29
0.82
Aquarium 9
9
1.13
0. 7U5
0. 5^0
2922
2118
1.31
1.06
0.863
0.698
3385
2738
1.50
1.35
0.989
0.890
3879
3491
1.03
1.16
0.679
0.764
2663
299 7
1.31
1.27
0.863
0.837
3385
3283
1.50
1 .45
0,989
0.956
3879
37 50
1 .41
0.92
. 0,929
0.606
3644
2377
0.96
1 .23
0.633
¦ 0,811
2483
3181
1.26
0.830
3256
X
1.
22
0.801
3143
SO
0,
21
0.
135
531
N
17
17
17
Aquarium 10
1.55
1.48
1.021
0.975
3781
361 1
1.29
1.63
0.850
1.074
3148
3977
2.20
1 »44
1.500
0.9^9
5555
3514
1.70
1.92
1.120
1.265
4148
4685
1.73
t hh
I » I'""?
1. ]40
0-949
4222
3514
1.12
1.65
0.738
1.087
2733
4025
1.58
2.63
1 # 041
1.733
3855
6418
X
i.
67
1,
103
4084
SO
0.
38
0.
256
950
N
1 4
14
14
(continued)
72
-------
TABLE 18, continued.
Ory Weight
q
Ash Free
Dry Weight
a
Ash free
Dry Weight
cal /crayf i 5,
Sept. 8
0.811
3181
Aquarium 9
?
1.23
0,90
0.593
2326
ir
1.50
1.17
0.989
0.771
3879
3024
1.69
1 -57
1,114
1.035
4370
4060
1.15
1.29
0.758
0.850
2973
3334
1.40
1 .44
0.923
0.949
3620
3722
1,69
1.53
1.114
1.008
4370
3954
1.51
1.01
0,995
0,666
390 3
2612
1.40
1.08
0.923
0.712
3620
2793
X
1
.35
0.
888
3483
3D
0
.24
0.
157
617
N
16
16
16
Aquarium 10
d*
1.55
1.73
1.021
1.140
3781
4222
1-25
1.71
0.82^
1,127
3051
4173
1.83
1.67
1.206
1 .101
4466
4077
1,70
1.91
1.120
1.259
4l 48
4662
1*36
1.39
0.896
0.916
3318
3392
1.17
1.64
0.771
1.081
2855
4003
1.53
3.07
1.008
2.023
3733
7492
1.52
1.002
3711
X
i.
67
1.
100
4072
30
a.
kh
0.
290
1073
N
15
15
15
Sept. If
Aquarium 9
9
1.10
1.21
0.725
0.797
2844
3126
T
1,26
0.77
0.830
0.507
3256
1989
1.*+2
1.00
0.936
0.659
36?i
2585
0.97
1.00
0.639
0.659
2506
2535
1.24
1.24
0.817
0.817
3205
3205
1.43
1.37
0.942
0.903
3695
3542
1.33
0.87
0.876
0.573
3436
2248
1-33
0,90
0.876
0.593
3436
2326
X
1
.15
0.670
2973
SO
0
.21
0.
138
543
N
16
16
16
(continued)
)
73
-------
TABLE 18, continued.
Dry Weight
Ash Free
Ory Weight
a
Ash Free
Dry Weight
Aquarium 10
cf
1.55
1.53
1 .021
1.008
378]
3733
1.25
1.55
0.824
1 .02?
3051
3781
1.84
1.32
1.213
0.870
4492
3222
1.64
1.85
1.081
1.219
4003
4514
1,66
1.39
1.094
0.916
4051
3392
1.22
1.60
Q1804
1 ,054
2977
3903
1.34
2.33
0.883
1.865
3270
6906
1.46
0.962
3562
X
1.
60
1
.060
3909
SO
0.
39
0
.257
951
N
15
15
15
Sept.'29
Aquarium 9
9
1.10
I 70
1 * £m
-------
TABLE 18, continued.
Oct, 13
Dry Weight
Ash Free
Dry Weight
Ash Free
Dry Weight
ca1/crayfi sh
Aquarium 9
0
1,10
1.33
0.752
0.883
2950 3464
T
1.23
l .34
C.811
0,784
3181 3075
1 ,42
1.19
0.936
0.639
3671 2506
0.97
0.97
0.639
0.883
2506 3464
1 .22
l.3*+
0.804
0.573
3154 2248
! .if3
0.87
0.942
0.567
3695 2224
0.86
0.876
3436
X
1.
18
0.
774
3044
SO
0.
20
0.
133
520
M
13
13
13
Aquarium 10
cf
1.51
1 .*+3
0.995
0.942
3685 3488
1.21
1.49
0.797
0.982
295? 3636
1.80
1-53
1.186
] .008
4392 3733
1.62
1.37
1.068
0.903
3955 3344
1.21
1.57
0.797
1.035
2951 3833
1.31
2.75
0.863
1.812
3196 6710
X
1.
57
1.
032
3823
SO
0.4l
0.
270
1000
N
12
12
12
Oct, 23
Aquarium 9
q
1.07
1.30
0.705
0.837
2765 3283
-r
1.22
1.27
0.804
0.778
3154 3052
1.41
1.18
0.929
0.633
3644 2483
0.95
0.96
O.626
0.363
2455 3385
1.20
1.31
0.791
0.554
3103 2173
1.39
0.84
0.916
0.573
3593 2243
0.8?
0.857
3362
X
1.
15
0.
759
2977
SO
0.
19
0.
128
502
N
13
13
13
Aquarium 10
cf
J .48
1.27
0.975
0.837
3610 3100
1.19
1 .46
0.784
0.962
290 3 3562
1.77
1-33
1.167
0.876
4321 3244
1,60
1.55
1.054
1 .021
3903 3781
1,18
2.73
0-778
1.799
2881 6662
X
1,
56
0
.925
3797
SO
0.45
0. 1 32
1106
N
10
10
10
(continued)
75
-------
TABLE 18. continued.
Dry Weight
a
Ash Free
Dry Weight
a
Ash Free
Dry Weight
cal ,/crayf i si*
Dec. 7
Aquarium 5
o
0.94 1,17
0.619 0.771
2428 3024
i-
1.19 1.32
0.784 0.870
3075 3413
1.36 0.84
0,896 0.554
3515 2173
1.27 0.86
0.837 0.567
3283 2224
X
1,12
0.737
2892
SO
0.21
0.138
540
N
8
8
8
Aquarium 10
1.15
0.753
2807
1.2%
0.817
3025
X
1.20
0.788
2916
SD
0.06
0.042
154
N
2
2
2
Dec. 14
Aquarium 9
9
0.94 1.17
0.619 0.771
2428 3024
-r
1.20 0.84
0-791 0.554
3103 2173
1.36 0.86
0.896 0.567
3515 2224
1,27
a.837
3283
x"
1.09
0.719
2821
SO
0.21
0.138
539
N
7
7
7
Aquarium 10
-------
TABLE 18, continued.
CTadoohora
July 28
Dry Weight
_£L -
Ash Free
Dry Weight
1.90
1.50
1,218
0.962
3959f
1.73
1.49
1 .109
0.955
3604
1.45
1.78
0.929
1.141
3019
1.71
1.38
1.096
0.S72
3562
1.91
1.47
1.224
0.942
3978
0.92
1.15
0.590
0.737
1918
2.14
1.31
1.372
0.840
4459
1.17
1.38
0.750
0.885
2438
1.34
1.44
0.859
0.923
2792
1.46
1.07
0.936
0.699
3042
Ash Free
Dry Weight
cal/crayfish
3127
3104
3708
2834
3062
2395
2730
2876
3000
2272
X
SD
N
1,49
0.30
20
0.952
0.194
20
3074
60S
29
Aquarium 12- 9
f
SD
N
1.16
0.82
0.784
0.554
29496
2084
0.70
0.84
0.473
0.568
1779
2136
1.12
1.45
0.757
0.980
2847
3686
1.67
1.14
1.129
0.771
4246
2900
1.94
0.74
1 .311
0.500
4931
1881
1.01
0.84
0.683
0.568
2569
2136
0.95
1.39
0.642
0.940
2415
3535
0.81
0.80
0.548
0.541
2061
2035
1.29
1.07
0.872
0.723
3280
2719
1.07
0.59
0.723
0.399
2719
1501
1.07
0,34
20
0.723
0.232
20
2720
871
20
August 18
Aquarium 11'
cP
1.58
1.43
1.013
0.917
3292
2980
1.68
1.45
1.077
0.929
3500
3019
1.47
1.80
0.942
1.154
3062
3751
1.82
1.35
1.167
0.865
3793
2811
1.87
1.41
1.199
0.904
3897
2938
0.91
1.22
0.583
0.782
1895
25^2
2.09
1.35
1.340
0.865
4355
2311
1.31
1.41
0.840
0.904
2730
2938
1 .38
1.04
0.885
0.667
2876
0 f r- n
c 1 68
1.38
0.855
2779
1.47
0.23
19
0.943
0.183
19
3060
598
19
(continued)
77
-------
TABLE 18, continued.
Dry Weight
q
August 18
Ash Free
Dry Weight
q
Ash Free
Dry Weight
cal/crayfish
Aquarium 12- g 1.06
0.86
0.717
0.581
2697
1.21
0.86
0.818
0.581
3076
1.28
0,76
0.865
0.514
3253
0.78
0.86
0.527
0.581
1982
1.38
0.89
0.933
0.602
3509
1,45
0.56
0.980
0.379
3886
1.57
1.15
1 .129
0.777
4246
1 .99
1.15
1.345
0.777
5059
1.07
0.723
2719
2185
2264
1425
2922
1.12
0.35
17
0,755
0,244
17
919
August 29
Aquarium 11
cf
Aquarium 12
1.57 1.
80
1.006
1.154
3270
3751
1.46 1.
37
0.936
0.378
3042
2854
1.81 1
40
1.160
0.897
3770
2915
1.83 1
22
1.173
0.782
3812
2542
2.34 1
35
1.500
0.865
4875
2811
1.39 1
44
0.891
0.923
2896
3000
1.46 1
06
0.936
0.679
3042
2207
1.48
0.949
3084
1,53
0.982
3191
0.31
0.
197
643
15
15
'
15
1.14 0.
90
0.771
0.608
2900
2287
1. 2k 0
89
0.838
0.602
3152
2264
1.32 0
77
0.892
0.521
3355
1959
0.33 0
89
0.551
0.602
2110
2264
1.35 0
92
0.913
0.622
3434
2339
1.43 0
c c
0.967
0.372
3637
1 399
1.72 1
20
1.163
0.811
4374
3050
2.08 1.
15
1.406
0.777
5288
2922
1.12
0.751
2825
1.15
0.
775
2915
0.37
0.
251
944
17
17
17
(continued)
78
-------
TABLE tS, continued,
Dry Weight
a
Sept. 3
Ash free
Dry Weight
Ash Free
Dry Weight
cal/crayfi sn
Aquarium
11
cf
K58
1.80
. 1.013
1.154
3292
3751
1.69
1.02
1.083
0.654
3520
2126
1.76
1.37
1.128
0.878
3660
2854
1.85
1.21
1.186
0. / /o
3«55
2522
2.02
1.34
1.295
0.859
4209
2792
1»36
T h
1 m ®T*T
0.872
0.923
2834
3000
1 .41
1. 0^
0.904
0.667
2938
2168
1.43
0.917
2980
X
l .49
1.
000
3100
SO
0.
29
0.
293
609
. N
15
15
¦ 15
Aquarium
12
9
1.26
1.33
0.852
0.899
3204
3381
1.47
0.89
0-994
0.602
3738
2264
1.11
0.45
0.750
0.304
2821
1 143
0.89
1.04
0.602
Q. 703
2264
2644
1.60
0.90
1.082
0.608
4Q69
2287
1.38
0.78
0.933
0.527
3509
1982
1.69
? .34
1.142
0.906
4295
3407
1.76
1.33
1.190
0.899
44/6
3381
1.2.8
0.865
3253
X
1.
21
0.815
3066
SD
0.
35
0.
235
385
N
17
17
17
Sept. 19
-
Aquarium
1]
CT
1.55
1,78
0.994
1. l4i
3231
3708
1.67
1.33 ~
1.070
0-853
3478
2772
1,79
1 -39
1.147
0.891
3728
2896
1.81
1.22
1.160
0.782
3770
2542
2.02
1.34
1.295
0.859
4209
2792
1.3*+
? .40
0.859
0.897
2792
2915
1,40
1.03
0.897
G. 660
2915
2145
1.4 1
0.904
2938
X
1,
50
0.960
3122
SD
0.
27
0.
170
551
N
15
15
15
(continued)
79
-------
TABLE 18, continued. ftsh fras Ash Frge
Dry Weight Ory Weight Dry Weight
G
Aquarium 12
o
1.10
1 .28
0.744
0,865
2798
3253
T
1.22
0.89
0.825
0.602
3103
2264
1,09
0.90
0.737
0.608
2772
2287
0.77
0.90
0.521
0.608
1959
2287
1 .40
0.76
0.946
0-514
3558
5933
1.40
1.20
0.946
0.811
3558
3050
1.67
1.09
1.129
0.737
4246
2772
1.99
1.345
5059
X
1.
18
0.
800
2993
SO
0.
34
0.
230
363
N
15
15
-
!>
Sept. 29
3188
Aquarium 11
e?
1.53
1.77
0.981
1-135
3689
1.66
1.30
1.064
0.833
3458
2707
1.78
1.40
1.141
0.397
3708
2915
1.82
1.34
1.167
0.859
3793
2792
2.01
1.37
1.288
0.878
4l 86
2854
1.35
1.04
0.865
0.667
2811
2168
1.41
0.904
2938
X
1.
52
0.975
3170
SO
0.
27
0.
173
561
N
13
13
13
Aquarium 12
2
1.05
1.93
0.710
1.305
2670
4908
1.18
0.87
0.798
0.588
3001
221 1
! .06
a.89
0.717
0,602
2697 '
2264
0.77
Q.87
0.521
0.588
1959
2211
1.38
0.77
0,933
0.521
3509
1959
1.38
1-13
0.933
0.764
3509
2873
1.64
1.14
1.109
0,771
4171
2900
X 1.15 0.776 2917
SO 0.34 0.228 860
N l4 14 14
Oct 13
Aquarium 11 d*
? .52
1.38
0.974
0.885
3166
2876
1.66
1.73
1.064
1.109
3458
3604
U75
1.30
1.122
0.833
3647
2707
1.79
1.30
1.147
0.833
3728
2707
1.98
1.36
1.269
0.872
4124
2834
1.35
1.01
O.865
0.647
2811
2103
X 1.51 0-968 3147
SO 0.27 0.176 573
N 12 12 12
(continued) gg
-------
TABLE 18, continued.
Dry Weight
-
Ash Free
Ory Weight
_a_
Ash Free
Dry Weight
cal/crayfish
Aquarium 12
9
1.08 1,93
0.717
1.305
2697 4908
1,18 0-89
0.798
0.602
3001 2264
1.07 0,87
0.723
0.588
2719 2211
0.77 0.76
0.521
0.514
1959 1933
1.37 1.15
0.926
0.777
3463 2922
1.37 1.13
0.926
0.764
3483 2873
1. 64
1.109
4171
X
1.17
0.
790
2971
SD
0.3*+
0.
230
868
N
13
13
13
Oct. 28
Aquarium 11
cT
1.48 1.34
0.949
0.359
3084 2792
1.71 1-70
; .096
1.090
3562 3543
1.93 1.27
1.237
0.814
4020 2646
1.30 1.3*+
0.833
0.859
2707 2792
X
1.51
0.967
3143
SD
0.24
0.
156
506
N
O
Q
8
8
Aquarium 12
¥
1.05 1.88
0.710
1.271
2670 4780
1.15 0.86
0.777
0.581
2922 2185
1.03 0.75
O.696
0.507
2618 1907
0.68 1.13
0.460
0.764
1730 2873
1.33 1.09
0.899
0.737
3381 2772
1.33
0.899
3381
X
1.12
0 •
755
2838
SD
0.33
0.
222
835
N
11
11
11
Dec. 7
Aquarium 11
cf
1.46
0.
936
3042
1.30
0.
833
270?
1.33
0.853
2772
X
1.36
0.874
2840
SO
0.09
0,
055
179
N
3
3
3
{continued)
81
-------
TABLE 18, continued.
Dry Weight
Aquarium 12 Q
1,03 0.96
1 .32 0.7*+
1.89 1.10
1.17
o.i+o
\k
Aquarium 11
1«
1.28
U37
0,13
Aquarium 12
1.02
1.69
0.83
0 • 7%
*1.10
1.11
0.45
Ash Free
Dry Weight
Ash Free
Dry Weight
cal/crayfish
0.696 0.6^9
0,892 0.500
1.2/8 Q.]kk
0.793
0.270
2618 2M+I
3355 1881
£+607
101 %
0.936
0.820
0,082
30^2
2665
285^f
267
0.690
1-.278
0.561
0.500
0.7^
0.308
2595
i+807
27 10
1881
2798
2838
1 160
aPer iphy ton ( 2) - V+98.8 ca!/q__{X)
DPeri phy ton (rf) - 3897 ca 1 /g (X) __
cCl adoohora +• Epiphytes (?) - 3922.^9 cal/g (X)
°C1 adoohora + Epiphtyes Jjf) - 3703 • 18 ca!/g (X)
^C1 adoohora (2) - 3761 (X_)
CIadoohora (*) - 3250 (X)
82
-------
TABLE 19. DATA FOR BASIS OF CONVERSIONS FROM DRY WEIGHT TO
CALORIES, 1977 CRAYFISH TISSUE EXPERIMENTS
Ash Free
Ash Free
Dry Weight
Dry Weight
% Ash Free
Dry Weight
.a
3
Dry Wei aht
cal/q
Peri phyton
Aquari um No.
7$
Aug. 15
0.869
0.667
76.8
4104
0.955
0.660
69* 1
4614
Aug, 22
0,932
0.705
75-6
U630
0.934
0.721
77-2
4720
Sept, 27
Q.925
0.707
76. 4
4571
Oct, 10
0.924
0.678
73-4
4354
X
74.8
4498,8
SO
3.1
228.7
N
6
6
Aquarium Mo.
ad*
Aug. IS
0.955
0.669
70.1
M353
0.728
0.517
7!.0
M303
Aug. 22
0.779
0.525
67.4
3961
0.967
0.674
69-7
4a4 1
Sept. 1
0.568
0.361
63.6
3506
Sept. 30
0-954
0.632
66.2
1.017
0.680
66.9
3820
X
67.8
3897
SO
2.6
209
N
7
6
Aquarium Aquari1
9-9 10 - <
Cl adophora +¦ Epiphytes
March 1 March
Aquaria Ncs.
3 & 10 S & ^
Feb. 9, 1978 1.56
1.09
69.9
3472 3550
2.31
1 .Ml
60.6
4040 3855
1.17
0.79
67.5
3941
3986
41/0
f
66.0
3922 3703
SD
4.8
266 216
N
3
5 2
(continued)
83
-------
TABLE 19. continued.
CIadophora
Ash Free Ash Free
Dry Weight Dry Weight % Ash Free Dry Weight
q q Dry Weight cal /g
Aquarium Mo, 11 0
Aug. 22 0.918 0.567 61.8 3208
0.831 0.495 59*6 3358
Sept. 1 0-947 0,626 66.1 3413
Sept, 30 t .230 0.892 72.5
Oct, 10 0,912 0.551 60.4
X 64. 1
SO 5.3 175.1
N 5 4
Aquarium No. 12 2
Aug. 15 0.648 0,452 69.7 3844
0.580 0.400 69.0 3906
Sept. 1 O.947 0.659 69.6 403'
Sept. 30 0.973 0.642 66.0 3664
Oct. 10 0.458 0.290 63.3 3361
X 67.5 3761
84
-------
TABLE 20. RESPIRATION, 1977 CRAYFISH EXPERIMENTS
Dry Weight (gin)
Crayfish Tissue
X SO N
nil 02/
g crayfish/
hr
cal/ *
g crayfish/
hr
cal /
crayfish/
hr
cal /
cray f i si
day
Periphyton
Oct, 3-4
Aquarium
7 I
1.49
0.33
10
0.488
2.29 '
3.41
81.84
Aquarium
8 cf
2.02
0.54
7
0.349
. 1.64
3-31
79.44
Dec. 13-
Aquarium
7 9
1.36
0.32
7
0.152
0.71
0.97
23-17
Aquarium
8 cf
1.99
0.59
3
0.057
0.27
0.54
12.96
Cladophora +
Epiphytes
Oct. 3-4
Aquarium
9
1.30
0.10
10
0.246
1.16
1 51
36.24
Aquar ium
10
1,44
0.40
10
0.227
1.0?
1 54
36.96
Dec. 13-14
Aquarium
9 9*
1.09
0.21
7
0.080
0.38
0.41
9.84
AquarIuffi
10
1.19
0.07
2
0.059
0.28
0,33
7-92
Cladophora
Oct. 3-4
Aquar ium
11 cf
1.51
0.25
10
0.318
J.49
: 25
54.00
Aquarium
12 9
1.21
0.28
10
0.257
1.21
1 *46
35-04
Dec. 13-14
11
Aquarium
1.41
0.13
2
0.077
0.36
0.51
12.24
Aquar ium
12 9
1.15
0.42
6
0.138
0.65
0.75
18.00
* 1 ml 02 - 4.7 calories
-------
TABLE 21. FOOD CONSUMPTION, 1977 CRAYFISH EXPERIMENTS
co
cn
Met Weight Dry Weight
Periphyton
July 19-2?
Aquarium 7
Dry gin
Consumed/
Crayfish/
da*
Ash Free
Dry Weight
gm
No.
Cray-
f i sh
Ash Free
Dry Weight/
Crayfi sh/
day
CaS
Consumed/
Crayfi sh/
jl ~d 'j
X
138.33
58.65
2.93
13.901
20
0.695
2990
so
41.21
17.47
0.87
4.141
0.207
890.7
N
9
9
9
9
9
9
Aquarium 8 -
&
X
100,89
42.78
2.28
10.138
20
0.540
2323
SD
32.09
13.61
0.77
3.225
(7/19-22)
0.182
782.6
N
9
9
9
9
18
9
9
(7/23-27)
July 28-Aug.
17
Aquarium 7 -
Q
X
161.82
68.61
3.74
16.261
19
0.833
3583.6
SD
43.55
18.46
1.04
4.376
(7/28-8/7)
0.312
1061.0
N
19
19
19
19
18
19
19
' (8/8-12)
17
(8/15-17)
Aquarium 8 -
c?
83.84
X
35.55
2.30
8.425
16
0.549
2361.8
SD
27.49
11.65
0.79
2.762
(7/18-8/11)
0.199
056.0
N
19
19
19
19
15
19
19
(8/12)
13
(8/15-17)
{con t i nued)
-------
TABLE 21, continued.
Wet Weight Dry Weight
qm qm
Aug. 18-28
Aquarium 7-9
X 82.14 34.83
N
34.51 14.63
Aquarium 8 - c?
X 43.67 18.51
SD 22,41 9.SO
Aug. 29-Sept. 8
Aquarium 7 - §
X 43.30 10.39
SD 9.37 3.97
N 8 8
Aquarium 8 - ^
X 26.50 11.24
SO 11.37 4.82
Sept. 9-19
Aquarium 7- §
X 67.43 28.59
SD 34.83 14.77
N 10 10
(continued)
Dry gm Ash Free Cal
Consumed/ Ash Free No. Dry We}(jht/ Consumed/
Crayfish/ Dry Weight Cray- Crayfish/ Crayfish/
day gm fish day day
8.254 15 0.595 2559,7
(8/18) 0.237 1019.6
14
(8/19-24)
13
(8/25-28)
13 0.340' 1462.7
0.73 2.252 0.174 745.3
1.47 4.359 13 0,348 1497
0.31 0.942 (8/29-9/1) 0.073 314.7
8 8 12 8 8
(9/4-8)
1.04 2.66 13 0.247 1062.5
0.44 1.14 (8/29) 0.104 447.1
8 8 118 8
(8/30-9/1)
10
(9/4-8)
2.38 6.777 12 0.565 2430.6
0.292 1255.3
10 10 10 10
-------
TABLE 21, continued.
Aquarium 8 - ^
Wet Weight Dry Weight
tiro gin
X 29.30 12.46
SD 10,04 , 7.989
N 10 10
Sept. 20-29
Aquarium 7-9
X 51.68 21.91
SD 20.19 8,56
N 8 8
Aquarium 8 -
X 36.54 12,47
SD 11.59 5.72
N 7 7
Sept. 30-0ct. 4
Aquarium 7-5
X 57.73 24,48
SD 6,62 1.73
N 3 3
Aquarium 8 - cf
X 33.17 14.06
SD 17.64 7.48
N
Oct. 5-6
Aquarium 7 - $
X 39.20 16.62
SD 22.77 9.66
N 2 2
(continued)
Dry gm Ash free Cal
Consumed/ Ash Free No. Dry Weight/ Consumed/
Crayfish/ Dry Weight Cray- Crayfish/ Crayfish/
day gm fish day day
1.25 2.952 10 0.297 1277.7
0.79 1.894 (9/9-18) 0.188 809.2
10 10 9 10 10
(9/19)
1.83 5.193 12 0.433 1062.7
0.71 2.029 0.169 727.4
8 8 8 8
1.76 2.954 9 0.339 1458.4
0.50 1.356 (9/20-26) 0.140 ¦ 637.1
7 7 8 7 7
(9/28-29)
2.04 5.802 12 0.484 2082.1
0.24 0.666 0.055 238.4
3 3 3 3
1.81 2.636 8 0.420 1841.2
0.87 0.162 (9/30-10/1) 0.208 89b.6
7
(10/4)
1.39 3.939 12 0.315 1355 J
0.80 2.290 0.172 821.7
2 2 2 2
-------
TABLE 21, continued.
Wet Weight Dry Weight
qni gm
Aquarium 8 -
X 11.05 5,03
SO 10.54 4.46
N 2 2
Cladophora + Epiphytes
July *20-27
Aquarium 9 - 5
X 4.79 0.503
SD 0.5378 0,0565
N 8 8
Aquarium 10 -tf
I 4.77 0.501
SD 0.8583 0.0902
N 0 8
July 28-Aug. 18
Aquarium 9 - 9
X 5.17 0.543
SD 1.2202 0.1284
N 19 19
Aquarium 10 -cf
X 4.257 0.44?
SD 1.3061 0,1371
N .19 19
(continued)
Dry gin Ash Free Cal
Consumed/ Ash Free No, Dry Weight/ Consumed/
Crayfish/ Dry Weight Cray- Crayfish/ Crayfish/
day gin fish day day
0.72 1.191 7 0.170 731.3
0.64 1.058 0.151 651.2
2 2 2 2
0.025 0.347 20 0.017 74,94
0.0028 0.0388 0.0021 9.1063
8 8 8 8
0.026 0.345 20 0.018 79.34
0.0045 0.0621 (7/20-21) 0.0029 12.7849
S 8 19 8 8
(7/22-27)
0.027 0,374 20 0.019 83.75
0.0063 0.0883 (7/28- 0.0041 18.1150
19 19 8/14) 19 19
(8/15-18)
0.024 0.308 19 0.0165 72.73
0.0073 0.0944 (7/20-8/7) 0.0051 22.4939
19 19 18 19 19
(8/8-17)
-------
TABLE 21, continued,
Met Height Dry Weight
X 2.27 0,238
SO 0.9402 0.0988
N 10 10
Aquarium 10
X 1.964 0,200
SO 1.2376 0.1271
N 10 10
Au(j. 29-Sept. 9
Aquarium 9 - §
X 1,94 0,2037
SD 0.7151 0.0750
0 N 7 7
Aquarium 10 -a1
X 1.85 0,194
SO 0.7190 0.0755
N 7 1
Sept, 9-19
Aquarium 9 -o
X 0,928 0.973
SO 0.7416 0.0781
fi 10 10
Aquarium 10
X 0.745 0,078
SO 0.3899 0.0410
H 10 10
(continued)
10
Dry gm Ash Free Cal
Consumed/ Ash Free No. Dry Ue iijlit/ Consumed/
Crayfish/ Dry Weight Cray- Crayfish/ Crayfish/
day gm fish day day
0.014 0.164 17 0.0096 42.32
0.0058 0.0880 0.0039 17.2074
10 10 10 10
0.012 0.142 18 0.0084 37.027
0.0074 0.0896 (8/18) 0.0050 21,9132
10 10 17 10 10
(8/19-28)
0.0122 0.1401 17 0.0082 36.15
0.0045 0.0515 (8/29-9/6) 0.0031 13,8726
7 7 16 7 7
(9/8)
0.013 0,134 17 0,009 39.871
0,0051 0.0520 {8/29) 0,0036 15.6887
7 7 15 7 7
(8/30- 9/8)
0.0062 0.067 16 0.0042 18.51
0.004/ 0.0540 0.0035 15.5219
10 10 ¦ 10 10
0.005 0.054 15 0,0038 15,87
0.0028 0,0282 0.0021 9.1044
10 10 10 10
-------
TABLE 21, continued.
Wet weight Dry Weight
ffla
Sept, 20-28
Aquarium 9-9
X 0.910 0.095
SO 0.5772 0.0606
N 8 8
Aquarium 10 - ^
X 0,668 0.070
SD 0.2642 0.027?
N 8 8
Sept, 30-0ct. 4
Aquarium 9-9
X 0.51 0.054
SD 0.2009 0.0212
N 5 5
Aqua ri urn 10 - ^
X 0.492 0.052
SO 0.1401 0.0146
N 5 5
Oct. 5-12
Aquarium 9 - $
X 0.503 0.053
SD 0.4156 0.435
N 7 7
Aquarium 10
X 0.55 0.058
SO 0.2489 0.0260
N 7 7
(continued)
Dry gm
Consumed/ Ash Free No.
Crayfish/ Dry Weight Cray
day gm fish
Ash Free Cal
Dry Weight/ Consumed/
Crayfish/ Crayfish/
day day _ _
0.006 0.0656 16 0.004 17.63
0.0039 0.0420 (9/20-26) 0.0027 11.9352
8 8 15 8 3
(9/27-28)
0.0046 0.048 15 0.003 13.22
0.0019 0.0190 0.0013 5.6497
8 8 E8
0.0036 0.037 15 0.0024 10.57
0.0013 0.0147 0.0011 5.0367
5 5 5 5
0.0036 0.035 15 0.0024 10.57
0.0009 0.0102 . 0.0005 2.4100
5 5 5 5
0.0037 0.036 15 0.0026 1 1,46
0.0028 0.0300 (10/5-9) 0,0020 8,7636
7 7 14 ? 7
(10/10-12)
0.004 0.040 15 0.003 13.22
0.0020 0.0179 (10/5-9) 0.0011 4.7105
7 7 14 7 7
(10/14-12)
-------
TABLE 21, continued.
Met Weight Dry Weight
qm qm
Oct. 13-26
Aquarium 9-9
X 0.2485 0.0715
SD 0.3425 0.0097
N 13 13
Aquarium 10 - ^
X 0.295 0.031
SD 0.2188 0.0231
N 13 13
Nov. 1-Dec. 2
Aquarium 9 - $
X 0.333 0.009
SD 0.194 0.006
N 30 30
Aquarium 10 - &
X 0.327 0.348
SD 0.188 0.020
N 30 30
(continued)
Dry gm
Consumed/
Crayfi sh/
_da*
Ash Free
Dry Weight
No.
Cray-
fish
Ash Free CaI
Dry Weight/ Consumed/
Crayfi sh/
day
Crayfi sh/
day
0.0046
0,005
13
0 0
0.0
0.0008
0.0066
-
13
13
13
13
0.0028
0.021
12
0.0018
7.93
0.0020
0.0162
0.0012
5,1369
13
13
13
13
0.001
0.006
11
0.0004
1.76
0.0006
0.004
(11/1-8)
0.0005
2.22
30
30
9
30
30
(11/9-20)
8
(11/21-26)
*7
(11/27-12/2)
0.004
0.024
11
0.003
12.22
0.0016
0.014
(11/1-6)
0.001
4.43
30
30
10
30
30
(11/7-11)
9
(11/12-18)
8
11/19)
7
11/20-24)
6 (11/25)
4
11/26-12/2)
-------
t3 j£
— 1
itt 3 4- na'
<-> "£> >i-C
S ^
O
o o
(43' CM
I—
r- CM
IT)
esj rn
CM CO
CM *3-
ur>
O
« *
"5- o
CM
cm
cm O
CM o
O
o
CTi
m
o o
CM LD
CO
CTI
CM !"—
«sr 23
Ol "C
CO
o
CT JZ
OJ
««— bO
!U
•
LJU
3 ^ "t3
>>T3
JZ
>s ra
c/l
S- U
<
a o
un
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o o
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LO
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o o
o o
. • I
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LO
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d d
cm
cn
o
o o
o d
CM
00 CM
CM i—
o o
d o
CO
CM r—
CM r—
a o
» * ,
a o
CO
>,
. fa 1/1
O S- •—
s: o n-
CM
m cm
*T CM
d d
CO
"O _c
E a> i/j
cn E —
3 h— fa
>> ,yl >,
C fQ
a o 5_
U3 LD
o o
o o
o o
* • .
o o
¦ to
o o
o o
. . U1
o o
o
o o
» *
o o
*3*
o
o o
* *
o o
¦ CM
cm cn
cn t~
O O
o d
cn
O CM
CM i—
O O
o a
as
o»
a
in m
O O
o o
d d
CO LO
foumi f i
o a
d d
un
o o
• a
a o
CM
_ o
d d
¦ to
CM CO
r~~
o
o o
to
o
cn
CM O
o d
CM
r~ O
. - CM
O O
Q*
r- S
I 3
o —
s-
. fa a
U 3 X (/I
<
• to a
u 5lx tn
aj cr
o
-------
TABLE 21, continued.
July 28-Aug, 17
Aquarium 11 -
-------
T3 ^
y —
= V)
— 3 T* >1
fO .y, '¦*- iT3
u c
O
r-«
a m
CO
S3
r-» *3-
Lf)
m ci
CM i—
CO
¦sr cn
— CO
— CO
un
cn cm
CM O
C3
cn
m
J
u. 3s 4- , rg
« U Sw
C a tJ=
¦ 'O l/l
o s, •—
2U^
m
U3
en
CO r™ r~
r-» i
cn —
lo go
a%
o\
cn
U3 CO
CM CM
1 I
WOtis.
¦— c\i i— eg
CTi
cn
W3 GO
CM CM
I 1
UTi O "3-
r—* CM r™- CM
cn
o
cn
aj s.
a> ra
5_ *—
U- 0J
a o
GQ
cn r—
CO
o o
a o
co
"O" r*»
co cm
o o
• * 1
o o
CM
«— cm
F- O
d d
m
£= ^ 7T
ai E 4/1
3 -i™» >»
>* v? *tr .2
a Q
o
ra
S-
o
ud un
o o
O O
O O -
§5
o o
¦ •
O o ¦—
U3
LO
a a
a o
o d
CO
to LO
o o
o o
o d
CO
CO tn
CM CM
o o
o o
* * I
o o
at cm.
o a
O
d o""
•jrs
en
*r—
cr
Of
[
CM
L.
m a
3|X
«c
4J I
0 _
o ¦—
1
o B
t*0 3
• S«k
its ~
a. n!x co
cr
u~>
-------
TABLE 21, continued.
Met Weight Dry Weight
_ 9.1 ,
Oct. 5-11
Aquarium 11
X 0.57 0.106
SO 0.099 0.018
N 7 ?
Aquarium 12 - $
X 0.55 0.101
SO 0.154 0,028
N 7 7
* fJer iphyton - 4302 cal./gin
C1adophora + epiphytes - 4408 cal
C1 atlojihora - 4286 cal ./gm
Dry gm Ash Free Cal
Consumed/ Ash Free NnDry Weight/ Consulted/
Crayfish/ Dry Weight Cray- Crayfish/ Crayfish/
day gin fish day day _
0.0083 0.094 13 0.0074 31.71
0.0015 0.016 (10/5-9) 0.0014 5.99
7 7 12 7 7
(10/10-11}
0.007 0.090 14 0.0066 28.28
0.002 0.025 (10/5-9) 0.002 7.77
7 7 13 7 7
(10/10-11)
/gm
-------
TABLE 22. DATA FOR BASIS OF CONVERSIONS FROM DRY WEIGHT
TO CALORIES, 1977 CRAYFISH FOOD EXPERIMENTS
Ash Free Ash Free
Dry Weight Dry Weight % Ash Free Dry Weight
g q Dry Weight cat /q
Pertphyton
Dec. 9, 1377 1.020 0,287 28.1 5455
1.116 0,302 27.1 4382
1.061 0,296 27-9 ¥+30
1.053 0.196 18,6 3776
1,069 0.207 19.*+ 3832
1.152 0.245 21 .3 393*+
X 23.7 4302
SO 4.5 630
N 6 6
CIadoohora + Epiphytes
Feb. 9, 1978 12.72 9.6*+ 75.3 4480
13-74 10.18 74.1 4358
17.9*+ 10.^+8 58.*+ ' if 167
19.30 10.9*+ 56,7 *+406
12.80 10.*+0 81 .3 4308
14.98 12.06 80.1 4470
20.28 13.14 64.8 U379
19-24 11.50 59.8 *4699
X 68.9 4408
SO 10.1 153.6
N 8 8
CIadoohora
Oec. 9, 1977 0-981 0.870 88.7 *+095
0.960 0.850 88.5 5699
0.983 0.885 90.0 3972
0.988 0.872 88.3 4122
1.004 0.897 89.3 4039
0.989 0.886 89.6 *<038
0.972 0.865 89.0 4037
X 89.1 4286
SO 0.6 624.9
N 7 7
97
-------
TABLE 23. CLADOPHORA BREAKAGE, 1977 EXPERIMENTS3*b>c
_ . d
Tray 1 - o
Added
q
Retrieved
g 2L
Broken
q
%
Consumed
Q
%
July 23
2,0
0.5*4
27
0.315
15.6
1,145
57-2E
24
2.0
0.70
35
0.137
6.9
1.163
58
25
2.0
0.27
13.5
0.28
14
1.45
72.5
26
2.0
0.015
0.75
0.41
20.5
1.575
79
27
2.0
0.29
1*4.5
0.238
11.9
1.472
73-6
X
0-36
18.2
0.28
13.8
1.36
6a. 1
SO
Q - 26
13.2
0.10
5-0
0.19
9.9
N
5
5
5
5
5
5
Tray 2 - cf ^
July 23
2.0
0.23
11.5
0.4l
21
1.36
68
24
2.0
0.64
32
0.27
13-5
1.09
54.5
25
2.0
0.82
4l
0.3*4
17
0.84
42
26
2.0
0.63
31.5
0.165
8.25
1.20$
60.3
27
2.0
0.53
26. S
0.305
15.3
1.165
58
X
0.57
28.5
0.30
15.0
1.13
56.6
SO
0.22
10.9
0.09
4.?
0.19
9-5
N
5
5
5
5
5
5
Retrieved: that portion easily removable with forceps,
Broken; that portion of very small filaments found on the bottom of the tray and collected by
fi1 Eration.
Consumed: calculated by subtracting the retrieved and broken portions from the amount added.
1>AJ 1 weights are wet weights.
cFour crayfish were in each tray, except Trays 3 and 6, which were empty.
dfood in for 16 hours each day. (continued)
-------
TABLE 23, continued.
Added
d
Tray 3 - empty
July 23 2.0
2*1 2.0
25 2.0
26 2.0
2? 2.0
X
SD
N
Tray k - q£
July 25 2,0
26 2.0
27 2.0
28 2.0
29 2.0
X
SD
N
Tray 5 - o*e
July 25 2,0
26 2.0
27 2.0
28 2.0
29 2.0
X
SO
N
Retrieved
q
%
2.0
100
1.87
93.5
1.83
91.5
1.75
87.5
2.0
100
1.89
9*4.5
0.11
5.5
5
5
0.95
*~7-5
0.?6
38
0.53
26.5
0.75
37.5
0.78
39
0.75
37.7
0.15
7.5
5
S
1.23
61.5
0.36
^3
l .08
$h
1.06
53
0.90
^5
0.93
51.3
0.3 k
7.5
5
5
eFood in for h hours each day.
Broken Consumed
q
... %
fit..
%
0.015
0.75
0
0
0.02
1.0
0.11
6.5
0.025
1.3
0.1^(5
7-3
0.0*45
2.3
0.205
12.5
0.05
2.5
0
0
0.03
1.57
0.09
5.3
0.02
0.79
0.09
5.3
5
5
5
5
0.276
13.8
0. 77*1
38.7
O.312
15.6
0.928
hS.h
0.320
16.0
1.15
57.5
0. 100
5-0
1.15
57.5
0.275
13-8
0.9^5
T/-3
0,26
12.8
0.99
^9.5
0.09
k.S
0.16
8.0
5
5
5
5
0.262
13-1
O.508
25.
0.338
16.9
0.802
to
0.28
I't.Q
0.6*}
32
0.21
10.5
0.73
36.5
0.285
1'4.3
0.815
'»0.8
0.28
13.8
O./O
3*1-9
0.05
2.5
0.13
6,k
5
5
5
5
{continued)
-------
TABLE 23, continued.
Added
fl
y 6 - empty®
July 25
2.0
26
2.0
27
2.0
28
2.0
29
2.0
X
SD
N
o
o
Broken
Q
%
0.075
3.8
0,021
1.1
0.115
5.8
0.06$
3-3
0,065
3-3
0.070
3.5
0.03
1.7
5
5
Consumed
9 %
0.2
10
0.189
9.5
0,065
3
0.195
9.8
0.105
5
0.15
7.5
0.06
3»2
5
5
-------
. 15
23
25
30
t. 6
16
21
22
23
2.7
. 26
27
28
. 9
11
X
so
N
TABLE 24. CRAYfISH FECES, 1977 EXPERIMENTS
Dry Weight (mg)
No,
Crayfi sh
Aquar. 7 (9)
Aquar. 8 {
-------
TABLE 24» continued.
CIadophora + Epiphytes
Auy. 15
23
25
30
Sept, 6
16
21
22
23
27
Oct. 7
X
SD
N
CIadophora
Aug, 23
25
30
Sept. 6
16
21
22
23
27
_
SO
N
Dry Weight (nig)
Aquar. 9 (p) Aquar. 10 (cf)
32.0
14. 2
23.1
17.1
5.6
4.2
0.4
6.5
3.0
3.9
2.8
3.0
0.7
6.9
Q.5
1.8
0.8
1.3
0.4
3.7
4.6
Q, 60
4. 99
9-25
6.88
11
10
Aquar. 12 (o) Aquar. II (rf)
4.7
if.6
14.8
1.0
4.7
3.9
2.5
0.1
0.5
0.0
0.2
0.2
0.2
3.3
3.8
0.8
3-97
1.9%
5.22
1.77
7
9
Dry Weight of
Ho. Crayfish Fecos/Cruy(?sh
Aquar. 9 (y) Aquar. 10 (cf) Aquar. 9 (y) Aquar. 10 (cf)
18
17
17
17
17
17
15
17
15
16
15
16
15
16
15
16
15
15
15
15
15
1.78
0.84
1.36
1.01
0.33
0.25
0.56
0.38
0.20
0.24
o. iy
0. 19
0.05
0.43
0.03
0.11
0,05
0.09
0.03
0.25
0.31
0.51
0.31
0.51
0.41
11
10
Aquar. 12 (
-------
TABLE 25. UTILIZATION OF CALORIES PER CRAYFISH, 1977 CRAYFISH EXPERIMENT
O
_Ti ssue * Gain Resp. To tal % Wt. Total % Wt. X
X Cal./ or X Cal./ Cal. Gain or % Food Gain or % Ingest.
Per ipliy ton Crayf i sh Loss Cray f i sh Assim. Loss Resp. Ingest, Loss Resp. Assiin.
July lO
(Start of Expt.)
o 4288
o< 5016
Oct. 3-4
o 4639 351 6301.68 6652.68 5-3 94.7 207,314 0.2 3.04 3-2
o' 5686 6?0 6116.88 6786.88 4.9 90.1 136,074 0.5 4.50 5.0
Dec- 13-14
o 4563 - 73 1168 17*
-------
TABLE 25, continued.
C1 atlophora
~~ Jul y 18
Ti ssue
X Cal./
Crayfish
2800
3068
Gain JResp. Total % Wt.
or X Cal./ Cal. Gain or %
Loas Cray f i sh Assim. Loss Resp.
Total
Food
6 nqes t,
% Wt
Gai n
Loss
or
%
Resp ¦
%
Ingest
Ass int.
Oct. 3-'*
?
O*
Occ. 13-l'i
o
•I
O"
2918
3170
2038
285*4
118
10?
- 80
-316
2698
'~158
1289
882
2816
^260
1369
1198
'*.2
2.'»
(5-8)
(26.M
95-8
97-6
9^.2
63.6
M70.5
^286
2.8
2.h
64.7
97.01
67.5
99 Ji
o
•C"
* Periphyton - 4498.8 cal./gm - ?
3897 cal./nig -
-------
TABLE 26. CRAYFISH MOLTING EXPERIMENT, 1977-78
Cray-
Wet
Wet
Dry
%
Dry
% Gain
'X Gain
f i sh
Wt.(g)
Wt.(g)
Wt.(g)
Dry
Wt.(g)
or Loss
or Los:
Oesiq,
Start
End
Calcu.
Wt.(q)..
Molted
End
Me t W L.
Dry Wt
Periphyton
0.65
1*4.*4
Aquarium 7 d
R-B
*<.52
5.10
yes
0.735
+ 11.*4
+ 11.6
B
2.30
3.28
0.*t?
16.1
yes
0.5*il
+ 11,6
+ 13.1
R-R
*1.10
*~.**7
0.68
16.5
yes
0-739
+ 8.3
+ 8.0
Br
*4.56
*1.89
0.69
15.1
yes
0.736
+ 6*7
+ 6.3
Aquarium 8 ?
R-B
6.30
5.52
1.76
27.9
parti al
1.5**
-12.*4
-12.5
Br
7-25
7.0*i
1.99
27.*4
no
1.93
- 2.9
- 3*0
Br-R-fl
3,90
3.81
1.0*4
26.8
no
1.02
- 2.3
- 1.9
L 1 ddijpitoi'cj +
Ep i jjI 1 y Lus
AcjUciC it till 9 J
Br-B
*4.57
kjfy
no
- 2.8
R-B
*~.90
**¦73
no
- 3-5
CIadophora
Aquarium 12 5
Br-R-B
*i.07
*1.08
0.96
23.6
no
0,%3
even
even
B-Br
7.00
7.2*»
!.?*»
2*1,9
no
1.80
+ 3.3
+ 3.3
B
3.76
3.63
0.88
23.5
no
O.85?
- 3-5
- 3-2
No. of
Days
Alive
18
(12/1^-12/31)
25
(12/1^-1/7)
20
(l*/l'i-l/2)
21
(1 2/1*1-1/3
( S 2/1 it-12/27)
15
{ 12/Hi- 12/28)
Mi
{ I 2/Mi- 12/27)
{ 12/1*1-12/28)
20
{1 2/1*4- l/2)
Mj
(12/l%-12/2?)
13
{12/Mm 2/26)
10
{12/Mi-12/31
-------
TABLE 27. INDIVIDUAL TISSUE WEIGHTS, 1977 TADPOLE
EXPERIMENTS
Start of Experiment
April 16
X
SO
N
Wet Weight
Ory Weight
mq/tadoole
ma/tadpole
9-0
10.3
0.79
0.92
10.0
10,4
0.82
0.73
13.5
9.6
1.00
0.73
12.7
9,4
0.10
0.82
15-0
10.6
0.11
0.85
11.0
11,1
0.80
0,39
12.2
11.3
0.95
0.77
12.3
9.9
0.92
0,84
12.1
IT.7
0.89
0.84
10.6
11.2
0. 10
0.86
10,5
13.4
0.75
0.88
12.7
11.1
0.89
0.96
11 ,4
8.0
0.91
0.14
11.6
8.4
0.87
0.29
10,4
13.7
0.91
0.73
11.4
6.3
0.79
0.42
10.6
11.4
0.91
0.85
8.3
12.8
0.81
0.93
10,1
10.9
0.73
0.78
9«9
10.3
0.78
0.85
10
*93
0.748
1
.68
0.
250
40
40
Wet Weight Dry Weight Wet Weight Wet Weight
mq/tadoole mq/tadpole mq/tadooie mq/tadoole
Peri phyton
April 22
351-5
39-48
May 5
1252
130.5
382.1
31.03
915
167-7
234.7
30.96
819
181.5
320.0
20.56
691
123.1
238.1
20.74
907
126.3
273-6
28.17
1072
183.7
202.7
22.59
893
101.5
203-5
1390
79.5
X
275.a
27-65
992
136.7
SO
68.3
6.91
232
37.8
N
8
7
8
8
(continued)
106
-------
TABLE 27, continued.
Wet Weight Dry Weight Wet Weight Dry Weight
ma/tadpole mq/'tadpole ma/tadpol e mq/tadco)e
Peri phy tori
May 18 2570 436 June I 2450 915
2670 459 3690 563
2340 381 ' 5370 91 3
2820 407 5450 692
2950 464 h?30 333
251Q 331 5270 757
2520 358 4960 786
3340 584 5310 764
2470 351 2920 722
2980 432 4840 423
X 2717 420 4437 687
SD 301 7^ 1094 190
N 10 10 10 10
Cladoohora +
Epi phytes
April 22
X
SD
N
302.9
301.5
277-9
400.3
285.4
361.7
447.1
447.5
356.3
77.4
a
31.97
28.74
32.39
21.78
38.30
29.21
47.55
43.55
34.81
9.37
8
May 5
1206
1266
1335
768
944
1547
1104
1144
1164
238
8
113.6
183.3
184.3
215-8
189. l
164.5
138.1
247.8
179.6
42.1
8
May 18 2140 641 June 1 4930 712
3720 303 4690 509
2490 328 3710 731
3920 585 574C 342
3200 525 6510 1009
2730 235 2930 668
3010 434 3960 437
1540 393 6380 899
2910 425 5330 1176
3090 376 7200 1114
X 2875 425 5138 810
SO 705 127 • 1359 245
N 10 10 10 10
(continued)
107
-------
TABLE 27, continued.
CIadophora
ApriI 22
X
SO
N
Wet Weight Dry Weight Wet Weight Dry Weight
mq/tadpole ma/tadpole mq/tadpole mq/tadoo1e
9-3
0.580
May 6
12.0
0.458
9-5
0.40%
9-3
0-700
7-7
0.430
10.6
0.342
10.0
0,484
13.8
0.516
10.7
0.564
19.5
0.382
11.7
0.440
11.2
0.382
10.3
0.596
7.3
0.454
9.9
0.574
7.8
0.328
12.4
0.510
12.5
0.332
9.3
0.510
14.4
0.550
22.8
! .154
31.3
0.826
10.1
0.509
14.4
0,547
1.3
0.069
7.0
0.241
10
10
12
12
May 18
83.5
0.938
102.0
1.472
20.9
0.772
18.7
0.634
18.5
0.666
34.3
0.224
19.7
0.402
29.6
0.290
28.0
1.518
13-7
0.528
27.6
0.820
X
36.0
0.751
SO
29.0
0.428
N
11
11
108
-------
TABLE 28. RESPIRATION, 1977 TADPOLE EXPERIMENTS
Dry Weight (mg)
X SD N
Periphyton
April 5
Apri i 1Q
136.7
*420.0
37.8
/'».0
CIadophora
April 5
ApriI ifl
0.5^7
0,751
8
10
CIadophora + Epiphytes
April 5 179.6 <»2.1 0
April 18 *42*4.0 128.0 10
0.2*41
0.^30
12
11
_ ul Oo/mg/hr
X ' SO N
ca
C 3 1 > C J 1 J
^ tadpole/ tadpole/
i ,/mq/hr hr day
0,89 0.09 k
0,35 0.10 3
0.89 0.36
0,65 0.15 h
2,k\ 0.88 *~
3*h6 1.23 h
Q.00*i2
0.0016
0.00*42
0.0031
0.0114
0.0163
0.574
0.714
0-7 5^
1.300
0.006
0.012
13-78
17.14
18.10
31.09
0. 1 **9
0.293
* 1 ul 02 = 0,00472 calories.
-------
/
TABLE 29. TISSUE WEIGHTS, 197? CATFISH EXPERIMENTS
Tank 1
Wet Heights, mq/fish
Tank 2 Tank 5
TaHFT
June 17
470
420
480 490
660
530
330 490
750
520
330 520
460
450
590 550
460
490
410 480
440
340
510 500
580
510
b5Q 460
370
460
330 540
530
580
510 630
700
540
320 440
510
460
580 540
520
510
320 660
550
400
390 580
530
460
340 590
590
570
400 490
410
460
500 410
400
540
430 310
400
500
580 520
510
450
410 480
560
400
490 370
I
515
474
485
469
SO
80
83
89
105
N
20
20
20
20
Periphyton
Tank
5
Tank 6
Tank 5
Tank 6
July 1
540
690
580 540 July 8 850
780
1580 950
560
550
670 710
530
1140
910 56G
480
660
760 570
1060
860
1180 740
440
540
430 .440
900
710
1280 930
530
450
350 570
990
1320
920 770
440
500
780 740
1490
670
650 1410
470
770 540
810
1390
500 570
480
980
500 610
X
527
596
940
990
SO
78
124
290
310
N
13
18
16
12
Tank
5
Tank 6
July 15
720
470
430 580
460
570
310 590
520
550
360 550
460
390
540 610
470
540
570 710
510
490
810 540
460
41C
530 740
490
570
500 400
460
350
710 330
580
X
490
550
SD
80
140
N
18
19
(continued)
no
-------
TABLE 29, continued
'/Jet Heights, nig/fish
CTadophora
July 1
Tank
5
Tank
6
Tank.
5
Tank
6
36Q
380
330
260 July 8
260
260
200
330
370
370
460
250
450
250
210
250
/f ?A
<+ / u
410
190
490
¦ 230
j 0
190
210
380
460
350
270
230
280
280
280
370
360
390
340
250
260
290
300
320
320
360
350
270
270
280
290
350
370
360
310
190
250
260
330
.370
310
190
320
270
390
310
550
320
390
31f)
390
340
3^3 280 270
78 60 50
19 17 15
111
-------
/
TABLE 30. FATHEAD MINNOW TISSUE WEIGHTS, 1973 EXPERIMENT
Wet wt. Dry wt. a AFDW b " c
cm/fish am/fish cm/fish. Cal../fish AFDW
Start of Experiment
Peri phyton 1,14 1.36 0.301 0,359 0.269 0.321 1716 2047
March 7 0,95 1.07 0.132 0.282 0.118 0.252 Q753 1807
Tray 1 0.85 1.25 0.224 0.330 0.200 0.295 1276 1382
1.22 1.03 0.322 0.272 0.288 0.243 1837 1530
0.92 1.16 0.243 0.306 0.217 0.273 1384 17^1
0.77 0.94 0.203 0.248 0.181 0.221 11 54 H10
1.77 1.44 0.467 0.380 0.417 0.339 2660 2162
1.70 0.95 0.449 0.132 0.401 0.113 2558 0753
1.01 1.10 0.267 0.290 0.238 0.259 1513 1652
1.56 1.29 0.412 0.341 0.368 0.305 2347 1945
X 1,17 0.298 0.266 1700
50 0,28 0.090 0.081 520
N 20 20 20 20
aAll wet to dry conversions for Start of Experiment, March 7,
based on 26.4X
b 39.3* of dry weight, March 3, see Table 21.
c Based on 6378 cal/gm, March 3, see Table 21.
Tray 2
0.73
0.52
0.193
0.164a
0.172 0.146
1097
0931 c
0.92
0.90
0.243
0.238
0.217 0.213 •
1384
1359
0.50
0.S6
0,132
0.148
0,118 0.132
0753
0842
0.80
0.75
0.211
0.199
0.138 0.178
1199
1135
0.95
0.72
0.2S1
0.190
0.224 0.170
1429
1084
0.67
0.84
0.177
0.222
0.158 0.198
1008
1263
0.75
0.72
0.199
0.190
0.173 0.170
1135
1084
0.94
0.96
0.248
Q.253
0.221 0.226
1410
U41
0 .57
0.71
0.177
0.187
0.15S 0.167
1008
1065
0.79
0.75
0.209
0.199
0.187 0.178
1193
1135
X
Q
.75
0
.202
0.180
1148
50
0
.13
0.034
0.030
193
N
20
20
20
20
(csnti nued)
112
-------
Start of Experiment
.-let wt.
--/fisft
Cladoohora +¦ epiphytes
Marcn 7
Tray 3
Dry we.
cm/1", sn
AFDW
cm / r 1 sr
r - *
vc J »/
'-•sn m
•0.33
0.76
0.213
0.201
0.196
3.179
1 26u
1 U2
0.63
0.55
0.166
0,148
0.143
0.132
CS44
0842
0.78
0.74
0.2G6
0.195
0 .13d
0.174
1174
1110
0.65
0.72
0.172
0.130
0.154
0.170
0982
1084
0,90
0,63
0.238
0.166
0.213
0.143
1359
09^
0.71
0.34
0.187
0.222
0.167
0.193
1065
1263
0.95
1.11
0.251
0.293
0.224
0.262
1429
1671
0.71
0.7S
0.187
0.201
0.167
0.179
1065
1H2
0.99
0.73
0.251
0.193
0.233
0.172
1486
1097
0.71
0.77
0.187
0.203
0.167
0.181
1065
1154
0,78
0.140
0,204
0.035
20
0.182
0.031
1153
201
20
i ray
1.15
0.96
0
304
0,253 a
0.271
0.226 b
1728
1441
0.90
1.18
0
238
0.312
0.212
r% "Vf*
\J . Li 3
1352
1779
1 .05
1.07
0
277
0.282
0.247
0,252
1575
1607
1.30
1 .08
0
343
0.285
0.306
0.255
1952
1626
1.07
1.15
0
232
0.304
0.252
0.271
1607
1728
1.09
1.09
Q
238
0.288
0.257
0.257
1639
1639
1.12
1 .00
0
296
0.264
0.264
0.236
1634
1505
1 .06
1.44
0
280
0.380
0.250
0.339
1595
2162
1.14
1 .04
0
301
0.275
0.269
0.246
1716
1569
1.49
1.15
0
393
0.304
0.351
0.271
2239
1728
X
1.
13
0.
.297
0,
,266
1694
SD
0,
14
0.
038
0.
,034
214
N
20
20
20
20
Start of Experiment
H
Cladoohora
0.66
0.74
0.174
0.195a
0,
,155
0.1 74
989
1110
March 9
0.53
0.95
0.166
0.251
0.
. 1 £3
0.22*
9
-------
TABLE 30, continued.
Tray 6
Wet wt.
Dry wt.
a
AFDW
b
qm/fis h
qm/fish
qm/fish
Cal./fish >
0.64 1.04
0.169
0.275
0.151
0.246
963
1569
1.23 0,92
0.325
0.243
0.290
0.217
1850
1384
1.30 0.88
0.343
0.232
0.306
0.207
1952
1320
0.87 1.18
0.230
0.312
0.205
0.279
1307
1779
1.02 1.22
0.263
0.322
0.240
0.238
1531
1837
1.13 1.04
0.298
0.275
0.266
0.246
1697
1569
0.84 1.42
0.222
0.375
0.198
0.335
1263
2137
0.93 0.91
0.246
0.240
0.220
0.214
1403
1365
0.84 0.80
0.222
0.211
0.198
0.188
1263
1199
1.00 1.03
0.264
0.272
0.236
0.243
1505
1550
1.01
0.267
0.239
1522
Periphyton
March 25
Tray 1
0.19
0.050
0.045
287
20
20
20
20
1.34 1.29
0
323
0.311
0.286 0.275®
1735
1668 f
1.05 1.88
0
253
0.453
0.224 0.401
1359
2432
1.57 1.42
0
378
0.342
0.335 0.303
2032
1838
1.44 1.37
0
347
0.330
0.307 0.292
1862
1771
1.92 1.22
0
463
0.294
0,410 0.260
2487
1577
2.37 1.20
0
571
0.289
0.505 0.256
3063
1 553'
1.13 1.17
0
272
0.282
0.241 0.250
1462
1516
1.59 1.44
0
383
0.347
0.339 0.307
2056
1862,
1.46
0
.352
0.312
1892
0.34
0
.083
0.074
446
16
16
16
16
d 24.1* of wet weight based on April 8-11 values.
|38.5X of dry weight, based on April 8-11 values, see Table 21
'6065 cal/gm based on April 8-11 values, see Table 21.
(continued)
114
-------
TABLE 30, continued.
Wet vt.
gm/fish
Dry wt.i
gm/fish
AFDW e
gtn/ Eish
Cal./fish AFDW1
1.04 0.95
Tray 2
,79
03
,19
,19
0.78
1.63
0.71
,35
,05
,27
0.89
1,19
0.251
0.190
0.248
0.287
0,188
0.393
0.171
0.229
0.246
0,326
0.253
0.306
0.214
0.237
0,222
0.168
0.219
0,254
0.254
0.166
0.348
0.151
0.203
0.218
0.289
0.224
0.271
0.189
0.254
1346
1019
1328
1541
1541
1007
2111
916
1231
1322
1753
1359
1644
1146
1541
X 1.07 0.258 0.229 1387
SD 0.24 0.058 0 052 315
N 15 15 15 15
924.2% of wet weight based an April 8-11 values.
April 14
Tray 1
)
1.27 1.27
2.02 1.31
1.63 1.53
1.89
0.306 0.306
0.487 0,316
0.393 0.369
0.455
0.271 0.271e
0.431 0.280
0.348 0.327
0,403
1644 I644f
2614 1698
2111 1983
2444
SD
1.560
0.305
7
0.376
0,073
7
0.333
0.065
2020
393
April 14
Tray 2
0.83 0.80
1.08 1.02
1.20 1.23
0.80 1.20
0.201 0.1943 0.178 0.172e 1080 1043'
0.261 0.247 0.231 0.219 1401 1328
0.290 0.298 0.257 0.264 .1559 1601
0.194 0.290 0.172 0.257 1043 1559
(continued)
1.020
0.187
0.247
0.045
0.219
0,040
115
-------
/
TABLE 30» continued.
Wat vt.
ga / fish
Dry wt.5
ga/£ish
AFDW h
gm/fish
Cal. / fish ArDI^
May 16-23
Tray 1
1.34
0,513 0.352 0.458 0.314
0.539 0.352 0,481 0,314
2886 1979
3031 1979
1.670
0.383
0.439
0.101
0,392
0.090
4
^26.3% of wet weight, based on May 16-20 values.
l"s3,2% of wet weight, based on May 16-20 values.
May 16-23
Tray 2
1.10
1.32
1.11
0.85
0.272
0.274 0.243 0.2441 1531 153?J
0.210 0.291 0.187 1834 1178
1.095
0.192
0.271
0.047
0.242
0.041
J6301 cal/gin, May 16-20, see Table 21.
*24.7% of wet weight, based orv May 16-20 values, Table 21,
Cladophora I
March 29
Tray 3
Epiphytes
U * / J
0,50
0.63
0.55
0.75
0.85
0.58
0.49
0.81
0,58
0.75
0.99
0.67
0.62
0,63
0.151
0.135 '
0.125
0.112 m
736
659
0.104
0.126
0.086
0.105
506
618
0.130
0.120
0.108
0.100
636
589
0.114
0.155
0.095
0.129
559
759
0.155
0.205
0.129
0,170
759
1000
0.176
0.139
0.146
0.115
859
677
0.120
0.128
0.100
0.106
589
624
0.101
0,141
0.084
0.117
494
689
0.66
0.13
16
0.140
16
0.114
0.022
16
130
16
12.0.7% of wet weight, based on April 8-11 values.
111 83% of dry weight, based or. April 8-11 values, see Table 21.
n 5885 cal/gm based on April 8-11 values, sea Table 21.
(continued)
116
-------
TABLE 30, continued.
Wet wt.
Dry wt.°
AFDW m
.23/ fish
gin/fish
ga/fish
Cal./fish Af
March 29
Tray 4
1.00 0,8?
0.199 0.173
0.165 0.144
971
847
0.72 0.87
0.143 0.173
0.119 0.144
700
847
0.99 0,94
0.197 0.187
0.164 0.155
965
912
1.12 0.88
0.223 0.175
0.185 0.145
1039
853
0.91 0.83
0.181 0.165
0,150 0.137
883
806
0.89 1.00
0.177 0.199
0.147 0.165
865
971
1.33 0.37
0,265 0.173
0.220 0.144
1295
847
1.30 0,89
0.259 0.177
0.215 0.147
1265
865
_
0.96
0.192
0.159
936
SB
0.16
0.033
0.027
159
N
16
16
16
16
0
19.92 of wet weight, based on April
8-11 values.
April 14
0,47 0.53
0.097 0.1101
0.081 0.091171
477
536
Tray 3
0.59 0.68
0.122 0.141
0.101 0.119
594
700
0,49 0.57
0'. 101 0. IIS
0.C84 0.098
494
577
0.45 0.59
0.095 0.122
0.079 0.101
465
594
0.58
0.100
0.083
488
X
0.550
0.112
0.093
54?
SD
0.072
0.015
0.013
76
N
9
9
9
9
April 14
0.195 0.171°
0.162 0.142m
Tray 4
0.98 0.86
953
836
0.62 0.82
0.123 0.163
0.102 0.135
600
794
1.01 0.76
0.201 0.151
0.167 0.125
983
736
1.22 0.92
0.243 0.183
0.202 0.152
1189
895
0.83
0.165
0.137
306
X
0.891
0.177
0.147
866
SD
0.170
0.034
0.028
167
N
9
9
9
9
(continued)
117
-------
t
TABLE 30, continued.
May 16-23
Trav 3
Wet wc.
gta/flsh
0.38 0.54
0.66 0,62
Dry we,°
gm/fish
0,051 0,072
0.088 0,082
AFDW ^
gm/fish
0.039 0,055
0.067 0.062
Cal,/fish AfDW
201 284
346 320
X
SD
0.550
0.124
4
,073
0.016
4
0.056
0.012
4
288
63
4
Pl4.9% of wee weight, based on May 18-20 values.
^ 76Z of dry weight, based on Hay 16-20 values, see Table 21,
r5165 cal/gm based on May 16-20 values, see Table 21.
Tray 4
0.88
1.06
0.131
0.158p
0.100
0.120q
517
620
0.62
0,77
0.092
0.115
0.070
0.087
362
449
X
0.
833
0.
124
0.094
487
SD
0.
185
0.
028
0.021
109
M
4
4
4
4
CIadophora
•
March 29
Tray 5
0.49
1.01
0.098
0.203S
0.084
0.175
515
1073
0.71
0.62
0.143
0.125
0.123
0.108
754
662
0.59
0.73
0.119
0.147
0.103
0.127
632
779
0.54
0.92
0.109
0.185
0.094
0.159
576
975
0.78
0.51
0.157
0.103
0.135
0.089
828
546
0.89
0.76
0.179
0.153
0.154
0.132
944
810
0.61
0.65
0.123
0.131
0.106
0.113
650
693
0.78
0.79
0.157
0.159
0,135
0.137
828
840
X 0.71 0.143 0.123 737
SD 0.15 0.030 0.026 159
N 16 16 .16 16
320.1% of wet weight, based on April 8-11 values.
86.2% of dry weight, based on April 8-11 values, see Table 21.
u6133 cal/gm based on April 9-11 values, see Table 21,
(continued)
118
-------
TABLE 30, continued,
Wee we.
Dry we.v
AFDW
t
ga/fish
em/fish
am/fish
Cal./fish/AFDW
March 29
Tray 6
0,58 0,80
0.124 0.170
0.107
0.147
656
902
0,98 1,05
0.209 0.224
0.180
0.193
1104
1184
0.93 1,04
0.198 0.222
0.171
0.191
1 049
I 171
0.93 0.86
0.198 0.183
0.171
0.158
1049
969
0.73 1,16
0.155 0,247
0.134
0.213
822
I 306
0.81 0.78
0.173 0.165
0.149
0.142
914
871
0.70 0.69
0.149 0.147
0.128
0.127
785
779
0.85
0.181
0.156
957
I
0.86
0.183
0.
158
968
SD
0.16
0.034
0.
029
177
N
15
15
15
15
v 21.3% of wet weight, based on April
8-11 values.
April 14
f*
Tray 5
0,52 0.82
0.105 0.165s
0.091
0.142
558
8?lu
0.69 0.45
0.139 0,090
0.120
0.078
736
478
0.72 0.72
0.145 0.145
0.125
0.125
767
767
0.90 0.64
0.181 0.129
0.156
0.111
957
681
0.54
0.109
0.094
577
X
0.667
0.134
0.
116
710
SD
0.146
0.029
0.
025
154
N
9
9
9
9
Tray 6
0.84 0.81
0.179 0.173V
0.154
0 .149 1
944
914 u
0,85 1.06
0.l8l 0.226
0.156
0.195
957
1196
0.73 0.73
0.155 0.155
0.134
0.116
822
711
X
0,837
0.178
924
SD
0.121
0.261
162
M
6
6
6
(continued)
119
-------
TABLE 30, continued.
Wee wt.
gin/ fish-
Dry wt.
gm/fish
AJDW '
zth/ fish
Cal./fish/AFDW
May 16-23
Tray 5
0.55 0.65
0,86 0.32
0.39
0.082
0,097
0.048
0.058
0.062
0.097
0,044
0.074
0.037
325
509
231
194
X 0.554 0,083 0.063 330
SD 0.215 0.032 0.024 126
w14.1% of wet weight, based on May 16-23 values.
x 76% of dry weight, based on May 16-23 values, see Table 21.
^ 5244 cal/gai, based on May 18-23 values, see Table 21.
Tray 6 0,74 0.7S 0,104 0,111W 0.079 0.084x 414 44G-V
0,78 1,02 0.120 0.144 0.091 0.110 477 577
0.53 0.66 0.089 0.093 0.068 0.071 357 /">
X 0.770 0,110 0.084
SD 0.138 0.020 0,153
120
-------
TABLE 31, DATA FOR BASIS OF CONVERSIONS FROM DRY WEIGHT
TO CALORIES, 1978 FATHEAD MINNOW TISSUE
EXPERIMENTS
Dry Weight
Lai
March 3 0.4712
Start of Experiment 0.5355
0.5539
0.4440
0.5114
I
SD
H
AFDW
—Lsi.
AFDW
cal /a
0.4205
0.4842
0.4952
0.3869
0,4614
89.2
90.4
89.4
87-1
90«2
6,404
6,431
6,320
6,297
6,437
89-3
1.31
65
5 5
iril 8-11
0.4420
0.3804
86,0
6,174
CIadophora
0.4410
0.3806
86.4
6,091
X
86,2
6,133
SO
0.28
59
N
2
2
Cladophora +
0.4203
0.3511
83.5
6,103
Epi phytes
0.2847
0.2349
82.5
5,666
X
83.O
5,985
SD
0.71
309
N
2
2
Peri phyton
X
SD
N
1.0148
0,5763
0.9091
0,5038
89.6
87.4
88.5
1.56
2
6,326
5,803
6,065
370
2
Hay 16-20 0.4626 0.3467 74.9 5,234
Cladophora 0.6311 0.4676 77-3 5,254
X 76.1 5,244
SO \,7 14.1
N 2 2
(continued)
1 El
-------
TABLE 31, continued.
Dry Weight AFOW %
(c) (q) AFOW cal /c
May 16-20 0.3773 0,2864 75.9 5,132
CIadophora + 0.^589 0.3U90 76.1 5>198
Epiphytes
X 76,0 5,165
SO 0.14 k7
N 2 2
Periphyton 1.0781 0-9610 89.1 6,l8l
1.1199 0-9997 89.2 6,^21
X 89.2 6,301
SO 0.07 170
N 2 2
122
-------
TABLE 32. RESPIRATION, 1978 FATHEAD MINNOW EXPERIMENT
Peri phyton
May 16
Tray 1
Jar 1
Jar 2
X
SO
N
May 17
Tray 2
Jar 3
Jar 4
)
X
SO
N
May 18
Tray 2
Jar 4A
Jar 5
X
SO
N
CIadophora
Epi phytes
May 20
Tray 3
Jar 22
Jar 26
X
SO
N
Tissue (rog)
(Dry wei ght)
Fish Total
0,4869
0.3831
0.5386
0.3396
0.870
0,878
0.2821
0.2624
0.3127
0.2205
0.5^5
0.533
0.202!
0.2624
0.3127
0.2205
0.545
0.533
0.1263
0.1557
0.126
0.156
ul 02/g/hr
X SO N
650.6
674.3
809,7
276.0
2240
1974
5.1 2
25.1 3
642.1 288.7 4
452.9 123.4 3
34
4i8
2
3
cal/
q/hr
3.058
3-183
3-1 2
0.088
2
3-018
2.129
3-806
1.297
2.563
1.087
4
10.528
9-278
9.903
0.884
2
cal/Pi sb/
day
35.734
28.116
40.966
25.943
32.69
6.93
4
20.433
19.006
15-976
11.265
16.67
4.054
4
25-764
23.965
9-734
6.864
16.532
9.664
1+
31.912
34.670
33.291
1.950
2
1 m! 02= 4,7 calories.
~ontinued)
123
-------
TABLE 32, continued.
Tissue (mg)
(dry weight)
Fish Total
_ul 02/9Ar
ca I /
a/hr
caI/fi sh/
day
Hay 22
Tray k
Jar 10
Jar 11
CIadoohora
May 23
Tray 5
Jar 13
May 2b
Tray 6
Jar 20
X
SD
N
0.0905
0.1102 0.201
0,1305 0.131
0.1305 0.131
0.1012
0.1157
0.217
1705.^
223k.9
1404
?%. 1
21*+.6
231
8.015
10.737
9.U00
6.599
17-409
21 ,198
51.957
30.188
18.95
20.667
1^.326
16.383
15.355
1 .U55
1 ml 0- = h.J calories.
124
-------
)
TABLE 33. FOOD CONSUMPTION, 1978 FATHEAD MINNOW EXPERIMENT
Ash Free a gm AfDW ^
Wet Weight Dry Weight Dry Weight No. consumed/ cal, consumed/
gm qm gin Fi sh fish fish/day
Periphyton
Mar. 11-20
Tray 1
X 55.09 23.36 2.570 20 0.142 544.5
SD 20.4 8.7 0.95 (3/11- 0.553
N 7 7 7 3/16) 7
16
(3/17-
3/20)
Tray 2
X 59.6 25.99 2.782 20 0.158 605-9
SO 18.4 7.80 0.358 (3/11- 0.049
N 9 9 9 3/16) 9
15
(3/17-
3/20)
Mar. 21-31
Tray 1
X 66.0 27.99 3.078 16 0.192 736.2
SO 19.1 8.09 0.390 0.056
N 8 8 8 8
Tray 2
X 41.2 17.49 1.924 15 0.128 490.8
SO 12.0 5.08 0.559 0.037
N 9 9 9 9
Apr. 1-15
Tray 1
X 29.7 13.13 1.444 16 0.104 398.8
SO 16.5 * 6.31 0.694 {4/1- 0.058
N 8 8 8 4/13) 8
7
(4/14-15)
Tray 2
X 17.1 7 . 2 5 0 . 79 8 1 5 0 . 0 6 0 230.1
SD 8.0 3.41 0.374 (4/1- 0.031
N 8 8 8 4/13) 8
(continued)
8
(4/14-15)
125
-------
/
TABLE 33, continued,
Periphyton
Apr. 16-30
Tray 1
X
SD
N
Wet Weight
am
53,8
17.2
11
Ash Free gm AFDW
Dry Weight Dry Weight No. consumed/ cal . consumed,
qm qm Fi sh ti sh fi sh/day
22.81
7.27
11
2.509
0.800
11
0. 31)3
0.114
11
1372.3
Tray 2
X
SD
N
May 1-13
Tray 1
X
SD
N
Tray 2
SD
N
May 14-23
Tray 1
X
SD
N
39.0
15.8
12
47.13
30.18
6
22.7
10.5
6
18.2
6.4
6
Tray 2
X
SD
N
Cladophora +¦ Epiphytes
Mar. 11-20
Tray 3
X
SD
N
Tray 4
X
SD
14.5
6.6
6
0.60
0.40
9
0.84
0.57
9
16.15
6.70
12
19.99
12.79
6
9.63
4.46
6
1.817
0.737
12
2.198
1.407
6
1.060
0.490
6
8
0.227
0.092
12
0.314
0.200
6
0.133
0.
6
870.5
(continued)
6.44 0.847 7 0.220
4.66 0.298 (5/14- 0.141
6 6 5/16} 6
3
(5/17-23)
6.13 0.674 8 0.174
2.81 0.309 (5/14- 0.129
6 6 5/17) 6
3
(5/20-23)
0.063 0.044 20 0.003
0.042 0.030 (3/11- 0.002
9 9 3/15) 9
16
(3/16-20)
0.088 0.062 20 0.003
0.060 0.042 (3/11- 0.002
9 9 3/16) 9
16
(3/17-20)
1204.1
510.0
843.6
667.2
13.2
13.2
126
-------
TABLE 33, continued. Ash Free gm AFDW
) Wet Weight Dry Weight Dry Weight No. consumed/ cal . consumed/
gm qm gm Fish fish fish/day
Mar. 21-31
Tray 3
X 0.60 0.063 0.044 16 0.003 13.2
SO 0.31 0.032 0.023 0.002
N 9 9 9 9
Tray 4
X 0.59 0.063 0.044 16 0.003 13.2
SD 0.42 0.044 0.031 0.002
N 9 9 9 9
Apr, 1-15
Tray 3
X 1.67 0.175 0.123 16 0,010 44.43
SD 0.95 0.100 0.070 (4/1- 0.007
N 9 9 9 4/12) 9
9
(4/13-15)
Tray 4
X 1.44 0.151 0.106 16 0.008 35.58
SO 0.65 0.069 0.048 (4/1- 0.005
) N 9 9 9 4/12) 9
' • 9
(4/13-15)
Apr. 16-30
Tray 3
X 1.11 0.131 0.092 9 0.010 44.48
SD 1.38 0.144 0.101 0.011
N 14 14 14 14
Tray 4
X 1.35 0.142 0.119 9 0.013 57,82
SD 1.69 0.178 0.120 0.013
N 14 14 14 14
Hay 1-13
Tray 3
X 0.41 0.067 0.047 9 0.005 22.24
SD 0.33 0.023 0.017 0,002
N 6 6 6 6
Tray 4
X 0.97 0.102 0.717 9 0.008 35.58
SD 0.27 0.029 0.021 0.002
N 6 6 6 6
(continued)
127
-------
TABLE 33, continued.
May 14-23
Tray 3
Tray 4
X
SD
CI adophora
Mar. 11-20
Tray 5
-mm*
X
Tra
¥
SO
Mar. 21-31
Tray 5
SD
Met Weight
am
0.62
0.07
0.67
0.26
0.07
0.1
0.05
0.07
9
0.06
0.08
Ash Free
Dry Weight Dry Weight
-9-H m
0.065
0.008
0.071
0.028
fi
0.013
0.019
0.009
0,013
0.011
0.014
0.046
0.005
0.050
0.020
0.010
0.016
0.008
0.011
0.009
0.012
gm AFDW
No. consumed/ Cal. consumed
Fish fish fi sh./day
9 0.005
{5/14- 0.005
5/19) 6
5
{5/20-23}
9 0.007
(5/14- 0.002
5/21) 6
5
{5/22-23)
20 0.0005
(3/11- 0.0008
3/15) 8
16
(3/17-20)
20
(3/11-
3/16) !
16
(3/17-20)
0.0004
16
0.0005
0.0006
22.24
31.12
2.0
1.60
2 .0
Tray 6
0.10
0.11
0.018
0.021
0.015
0.017
16
0.0009
0.0011
3 59
Apr. 1-15
Tray 5
X 0.06 0.010 0.009 16 0.0005 2.0
SD 0.10 0.018 0.015 (4/1- 0.0007
N 11 11 11 4/13) 11
(continued)
(4/14-15)
128
-------
TABLE 33, continued.
Tray 6
Tray 6
7
Ash Free gm AFDW
Wet Weight Dry Weight Dry Weight No, consumed/ cal. consumed/
am am gm Fish fish fi sh/day
0,18 0.033 0.028 15 0.0020 7.98
SD 0.19 0.035 0.023 (4/1- 0.0013
N 11 11 11 4/13) 11
6
Cladophora (V14"15>
Apr, 16-30
Tray 5
X 0.12 0.021 0.017 5 0.0019 7,58
SD 0.17 0.031 0.026 0.0029
N 14 14 14 14
0.0013 5.19
SD 0,07 0.012 0-010 0,0017
N 14 14 14 14
May 1-23
Tray 5
X 0.019 0.003 0.002 9 0.0003 1.2
SO 0.037 0.006 0.005 0.0006
N 9 9 9 9
0.02 0.004 0.003 6 0.005 20.0
0.04 0.007 0.005 0.009
8q q q
o o o
® % ash free dry weight, Table 34
Based on Table 34
Periphyton - 3834.6 cal./gm
CIadophora + epiphytes - 4408 cal./gm
Cladophora - 3991.4 cal./gm
129
-------
TABLE 34, DATA FOR BASIS OF CONVERSIONS FROM DRY WEIGHT TO CALORIES,
1978 FATHEAD MINNOW FOOD EXPERIMENTS
Ash Free
Ash Free Dry % Ash Free Dry Dry Weight
Drv Weight (q) Weight (q) Weight , cal/g
Periphyton I. *465 0,830 0,130 0. 108 8.9 13-0 3690.9 '<003.6
1,319 1.027 0. 123 0. 13% 9.3 13-0 *4205.9 3l8«».2
1.109 0.908 0.10 2 0. 116 9.2 12.7 3'*86.1 *40/*4.5
3955-5 ^ 0 7 2.2
X 11.02 383^-6
SD 2.07 350.7
U 6 8
C i adophora +
oZ Epiphytes 0.'»52 0.526 0.321 0.3**9 7K0 66. k *t*i80.0 4308.0
° 0.*<08 0.566 0.30? 0.375 75-3 66.3 <058.0 Mr/0.0
0.*^ 0.513 0. 373 0.3^5 75.5 67.2 M67.O *1379.0
M1O6.0 '1699.0
x 70.30 kkm.k
SO *4.32 153.6
N 6 8
C i adophora
X
SO
N
0,1*47
0.182
0. 150
0.385
0.*4Q0
0.251
82.1
80.2
86.0
82.77
2.96
3
'lOMt. /
>'it90.y
36*18,9
M I5.*t
3977.8
*1036. 3
3835-0
*402?. 1
*(05 i .9
yjcj 1 - *1
s 60. ]
9
-------
TABLE 35- UTILIZATION Of CALORIES PER FATHEAD MINNOW
% Wt. % Wt.
Ti ssue
Gain
Resp.
Total
Gain
Total
Gain
%
X cal/
or
X cal/
cal .
or
%
Food
or
%
1nges t
Fish
Loss
F i sh
Assim.
Loss
Resp.
Inqest.
Loss
Resp.
Ass im.
Periphyton
Tray 1
March 7
1700
April 14
2020
320
1 242.1
1562.1
20.5
79-5
20,760
1.5
6.0
7-5
May 23
2*469
2517.1
3286.1
23.4
76.6
45,080
1.7
5.6
7.3
Tray 2
March 7
1148
April 14
1327
179
631.9
810.9
22.0
78.0
16,497
l.l
3-8
4.9
Hay 23
1520
372
12.80.5
1652.5
22.5
77-5
26,590
1.4
4.8
6. 2
Cladophora +
Epiphytes
Tray 3
Mui ch 7
1163
April 14
5^6
(-617)
1265.0
1882.0 '
32.8
67.2
940
65.6
134.6
200. 2
May 23
288
(-0/5)
2563.0
3438.0
25»5
74.5
1223
71*5
209.6
281 .1
Tray 4
March 7
1694
April I1!
866
(-826)
11%7-1
1973.1
41.9
58.1
815
101.4
140.8
242.1
May 23
'187
(-1207)
2324.5
3531.5
34.2
65-8
1677
72-0
138.6
210.6
Cladophora
Tray 5
March 7
1253
April 14
710
(-543)
785-4
1328.4
40.9
59.1
76
714.5
1033.0
17^7.8
May 13
330
(-923)
1591
251*1.4
36.7
63.3
131
704.6
1214.8
1919.4
Tray 6
March 7
1522
April 14
92'*
(-598)
583-5
1181.5
50.6
49.4
172
347 .7
339.2
686 ,9
May 1 3
440
(-1078)
1182.3
2260.3
47.7
52.3
S0[
1067.3
11 70 .6
2237 .9
-------
TABLE 36. RADIOACTIVITY IN BODY PARTS AND CALCULATED ASSIMILATION RATIOS
ON SUCCESSIVE DAYS FOR FATHEAD MINNOWS FED THREE DIETS
5/25
Diet DFH/iiig AFOW in:
Fish Muscu-
No. 1 a t u re* Viscera
Gut
Assimilation Ratio
Muscu- Musculature
latore & viscera Fish
Gut Gut No.
5/26
PPM/nig AFPU in:
Muscu-
lature* Viscera
Gut Gut
Assimi idi fjiii Rat.io
Muscu- MusculaLure
lature & v i_s c c rvi _
" Gut
Cladophora
1
2
3
4,61
0.03
1.72
Cladophora + Epiphytes
1 161.0
f\3
2
3
4
0.0
45.7
51.4
Periphyton
1 384.5
2 87.1
-** 5.48 0.84***
1.33 0,02
1.26 1.37
61.9
15.0
732.7
701.4
249.3
299.4
0.22
0.0
0.10
0.17
0.30
0.22
0.14+0.10 0.26+0.06
384.5 1.00
132.8 0.66
0.83+0.24
1
2
3
4
1
2
3
4
1
2
3
1.19
88.8
192.0
169.0
165.3
243.5
213.5
180.2
1167.3
512.3
463.9
1,10 2.58 0.46***
— 553.7 0.16
— 50.0 3.84
9.60 80.2 2.10
— 388.0 0.43
133.3 2376.7 0.10
91.6 1810.6 0.12
251.2 1519.9 0.12
0.96
2.22
0.16
0.17
0.28
***
0.19+0.16 0.20+0.07
2096.0 1989.2 0.59
46.8 1533.2 0.33
589.3 1385.9 0.34
1.64
0.37
0.76
0.42+0.15 0.92+0.65
* Head with gills not included
** Missing data
*** Range on ratios too great to warrant calculation of mean.
X®sMpsee'
-------
TABLE 37. PARTIAL RADIOACTIVITY BUDGETS FOR DIET STUDIES OF FATHEAD MINNOWS
5/24
t added
% added
DPM
5/25
d-pm #
%
DPM
DPM
5/26
DPM ^
% OPM
Diet
Introduced
DPM Removed
No. fish
Ingested
Introduced
OPM Removed
No. fish
Ingested
Cladophora
5,151,067
Feces
103
0.0003
4,849,455
Feces
3,662
0 01 I
Fish (7)
1,531
0.0042
Fish (4)
13,752
0 071
Muscle
397
Muscle
3,043
.
Heads
985
Heads
1 ,557
Guts
149
Guts
9,104
Viscera
(0)
Vi scera
(48)
I*
; 0.0049
I 0.066
A**0.0077
A 0.016
Cladophora
+ Epiphytes
5,062,182
Feces
39,124
0.097
4,817,058
Feces
24,592
0.128
Fish (8)
31,213
0.077
Fish (4)
58,626
0.305
Muscle
8,406
Muscle
11,399
Heads
3,936
Meads
7,475
Guts
18,242
Guts
38,692
V i scera
629
Viscera
] ,060
I
0.142
I 0.328
A
0.022
A 0.065
Periphyton
3,973,239
Feces
11,087
0.056
3,666,873
Feces
8,696
0.080
Fish (5)109,512
0,551
Fish (3)190,323
1.000
Muscle
50,005
Muscle
87,696
Heads
18,616
Heads
33,978
Guts
40,891
Guts
62,686
Viscera
(-)
Vi scera
13,963
1
0.262
I 0.649
A
0.252
A 0.924
* I = feces and guts; ** A = Muscle and Viscera
-------
LITERATURE CITED
;
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50-63.
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V. Fretter & J. Peake. Academic Press, New York, pp. 53-104.
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14
15
16
17
18
19
20
21
22
23
24
25
Budd, T, W,, J, C. Lewis, and M. L. Tracey. 1978. The filter-feeding
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26. Ambrose, J.,Jr. and B. E. Brown. 1971, food of the channel catfish
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before and during metamorphosis. Copeia, No. 1, "pp. 214-273"
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136
.)
-------
II. CHEMICAL COMPOSITION OF CLADOPHORA GLOMERATA (L.) Kutz. CELLS
INTRODUCTION
Experiments in which many possible consumer organisms were fed Cladoohora
glomerata showed that the alga was either not eaten or caused deleterious
physiological changes.
The nutritive quality of a food depends on many factors, including not
only its biochemical composition but also the nutritional requirements of
the consumer species. Algae of similar ultimate energy content (caloric
value) may differ widely as food sources because of variations in protein,
lipid, or vitamin concentration, or differences in content of nutritionally
important mineral elements. In addition, textural , gustatory,ana toxic
factors may be important in governing feeding behavior.
The first line of defense for an organism which may be eaten is its
exterior layer; in the case of algae, this means its cell wall. The cell
walls of Cladophora spp. and those of other members of the Cladophorales
consist largely of crystalline cellulose microfibri1s laid down in a mat-
like structure in two directions, lying at somewhat less than a right angle
(1). This orientation is unusual and may be restricted to this and closely
related orders of the Chlorophyceae. The sole hydrolysis product of this
cellulose is glucose, unlike many other algal celluloses which afford large
quantities of pentoses or other sugars upon hydrolysis. The walls of
Cladoohora contain other constituents also, including proteins (2) and
polysaccharides containing sulfate esters (3).
Some of the proteins in Cladophora walls are structurally important,
as shown by studies which showed significant weakening of wall strength after
treatment witn proteolytic enzymes or disulfide reducing agents (4).
It is not known whether the unusual structure of Cladoohora vial Is con-
¦ tributes to its resistance to predation.
The question of the existence of feeding inhibitors or toxins in C,
glomerata has not been fully studied. However, it has been reported that
several green algae, including Cladoohora species (5; LaLonde, personal
communication) contain substances toxic to mosquito larvae, and a marine
CIadophora produces acrylic acid ( 6 ), an irritating substance with anti-
biotic activity. Several other bloom-forming algae, particularly blue-
greens (7), produce toxic compounds; Chara and Mitel la appear to contain
mosquito larvicides (8); and several planktonic algae are rejected by
137
-------
fi1 tar-feeding grazers, notably Daohnia, presumably on the basis of taste
(9).
In this section of the report we summarize analyses of the chemical
composition of CIadophora, with emphasis on possible nutritionally important
factors.
METHODS AND PROCEDURES
Nitrogen Compounds
Proteins--Tota1 protein was determined by a modified Lowry (10) pro-
cedure, Freeze-dried algal samples (100-500 mg} were ultrasonically homogen-
ized with ice cooling in 10 ml of 0,02 M phosphate buffer (pH 7.0). After
centrifugation (500 rpm) of the homogenate, protein was precipitated from
the supernatant liquid by using an equal volume of 10% trichloroacetic acid.
After further centrifugation, the supernatant was removed for soluble carbo-
hydrate and amino acid determinations, and the pellet was redissolved in
1.0 ml of phosphate buffer. A 1.0-ml portion of the Lowry Cu50tartrate
reagent was added, f011 owed after 10 minutes by 3.0 ml of 2N pnosphomolybdate-
phosphotungstate ("Phenol reagent solution," Fisher Scientific Co.), The
color was allowed to develop for 45 minutes and absorbance was read at 540
nm. Bovine serum albumin was used as a standard protein.
Amino acids—Free amino acid and peptide content of algal material was de-
termined fluorimetrically using the method of Udenfn'end, et al. (11). An ali-
quot (0.1 ml) of the supernatant from the protein determination was mixed wiin
2.0 ml 0,2 M phosphate buffer (pH 8.0), 0.2 ml of an 0.05 M aqueous ninhydrin
solution and 0.1 ml of an 0.01 M solution of ohenylacetaldehyde in ethanol.
After mixing, the tubes were covered with aluminum foi 1 and "heated for 15
minutes in a water bath at 60°C. After cooling in cold water for 15 minutes,
the fluorescence intensity (excitation 390 nm, emission 490 nm) was read
using a Turner model 430 spectrofluorometer. Solutions of 0-20 mg/1 glycine
in 10% trichloroacetic acid were used as standards.
Amino acid composition of algal proteins was determined by acid hydroly-
sis. In a typical experiment, 309 mg of Cladoohora (dried at 60°C) was
suspended in 6 ml of 502 (8 M) HC1 , purged with "I2, and heated in a Pyrex
tube sealed with a Teflon-lined screw cap for 20 hours at 11Q°C. The hy-
drolysis mixture was fi1tered (glass fiber filter) and the filtrate was
passed over a 2 cm x 12.5 cm column of Bio-Rad AG50 (H+ form). The column
was rinsed successively with three column volumes (4-0 ml) of deionizad water
and three column volumes of 3M NH4OH. The ammonia eluate was freeze-dried
and portions were converted to amino acid N-trifluoroacetyl-n_-butyl esters
using the procedure of Roach and Gehrke (12). The ester mixture was in-
jected onto a gas chromatograph fitted with a glass (183 x 0.2 cm) 3* 0V-210
column, The areas and retention times of the emerging peaks were determined
electronically and compared to that of an internal standard (n-butyl stearate).
As a check on the method, a protein of known amino acid composition (bovine
serum albumin) was hydrolyzsd and derivati zed by the same procedure.
138
)
-------
Lipids
Total 1ipids-"Tota1 lipid was determined gravimetrically by Soxhlet
extraction of freeze-dried algae {usually 2-3 g) in a pre-extracted paper
thimble. Chloroform-cnethanol (2:1 v;v) was used as solvent; a blank thimble
containing no algae was used as a control,
Free fatty acids and hydrocarbons--Freeze-dried algae (3-6g) were ex-
tracted in a Soxhlet apparatus with pesticide-grade pentane. The pentane
was evaporated and the total pentane-solubla lipid was determined gravi-
metrically, with correction for simultaneously extracted blank thimble.
Fatty acids were removed by shaking the total extract three times with 25-ml
portions of 1% Na?C03 (pH 11), The combined Na^CQ-? extract was back-extracted
three times with pentane, acidified to pH 3 with 5% HC1, and re-extracted
with pentane and evaporated to obtain the total free fatty acids. The weight
of this fraction was determined; a portion was removed for yeast toxicity
studies, and the remainder was esterified using absolute methanol containing
12% redistilled (13). For each sample, 3,0 ml of the reagent was added
and the solution was boiled for 3 minutes; after cooling 6 ml water and 3 ml
pesticide-grade petroleum ether was added. The top {petroleum ether) layer
was removed and concentrated to about half its volume under a stream of
nitrogen. After accurate volume determination {calibrated microsyringe)» the
solution was transferred to a GC vial and a 4.5-ul portion of the sample was
injected onto a 1 OS diethylene glycol succinate column {glass, 200 x 0.2 cm)»
The GC oven temperature was programmed from 30° to 190°C at 6°/min. The
areas of peaks emerging from the column were electronically integrated. Each
sample was injected three times for determination of peak areas and relative
retention times. A mixture containing known amounts of Cg-C^g fatty acid
methyl esters (Sigma Chemical Co.} was used to identify and quantirate the
individual fatty acids, Pentane-soluble (nan-fatty acid) material remaining
after carbonate extraction was weighed, used for yeast toxicity studies, and
also partly characterized by ultraviolet and infrared spectra.
Sterols—Sterols were isolated from the total lipid fraction by sapon-
ification (25% methanolic KQH: reflux 45 min.), extraction with ether, and
precipitation from the nonsaponifiable fraction with dig;tonin. Recovery
of free sterols from the digitonide precipitate was done by the method of
Issidorides, at al. (14); the crude precipitate was heated with dimethyl-
sulfoxide and extracted with hexane. The hexana extract was dried (.NC2SO4),
filtered ana evaporated to dryness. An aliquot of the sterol fraction^was
injected into a 3% OV-210 glass GC column held isothermally at 250°C. Under
these conditions, authentic cholesterol emerged at 9.5 minutes.
The total lipid fraction of CIadoohora and its nonsaponifiable portion
were also examined by thin layer chromatography on silica gel. The solvent
used was 1/1/0.02 hexane/ether/acetic acid,and the spots were visualized by
spraying with 51 H2S04 in methanol followed by charring in a 120°C oven.
Authentic cholesterol gave a red spot at Rf 0.45 when run under these
conditions.
139
-------
Soluble Carbohydrates
Soluble carbohydrate in algal samples was determined using the super-
natant material from the protein determination. The phenol-sulfuric acid
colorimetric method of Dubois, at al. {15} was used. Standard solutions
used consisted of glucose in 51 trichloroacetic acid.
Total Phosphorus
A persulfate digestion method was used. Determinations were performed
on accurately weighed freeze-dried samples of about 1 mg dry weight. Finely
snipped algal fragments were suspended in 50 ml deionized water, 5 ml of 5,4
f4 H0SO4 and 7.5 ml of 0.18 M KgSgOo were added, and the mixture was digested
in an autoclave (1,4 kg/cm2"]" for 30 minutes. After cooling, the samples
were brought just to the phenolphthalein end point and diluted with deionized
water to exactly 100 ml. The phosphate concentration in a 50-ml subsample
was determined by the ascorbic acid method (16).
Toxicity Studies
Preliminary feeding experiments with snails presented problems which
made the results difficult to interpret. We therefore decided to carry out
a series of experiments on yeasts.
Yeast screening experiments—In preliminary studies designed to identify
the chemical nature of toxic materials Cladophora might contain, a yeast
growth inhibition test was used. Yeast, rather than some other microorganism,
was chosen because of its eukaryotic nature; it is similar in its cellular
organization to higher animals, and most materials toxic to yeast are also
toxic to higher forms. The method has been briefly outlined (17). Freeze-
dried yeast (90" viable Saccharomyces cerevisiae Sigma) was grown in a de-
fined basal medium containing 1% glucose as sole carbon source (13) to the
midexponential growth phase. A portion of this suspension containing (1+ 0,1)
x 10° cells was removed and added to fresh medium containing the test mater-
ial. Six replicate cultures were incubated at 25°C using a reciprocating
water-bath shaker for 48 hours. Growth was measured turbid metrically and
expressed as a percentage relative to six control cultures. Differences in
growth were assessed for significance using Student's t-test.
Nutrient Content
Proteins--It has been suggested that the amounts of protein and com-
ponent amino acids are the primary determinants of the food quality of plant
materials (19). The protein content of algae is extremely variable, with
levels from 55 to 60S being reported in different species. Within an individ-
ual species there can also be great variations in protein composition depend-
ing on light, temperature, phase of growth, and other factors. Hare and
Schmidt (20) showed a fourfold variance in protein content in synchronized
-------
Ch1 ore 11 a cultures, with young and rapidly growing calls having the highest
levels of protein.
Our results on C. glomerata indicate that this alga contains only about
8;j protein (Table 1). It is likely that this is partly due to its fila-
mentous growth habit and to the presence of a thick, largely cellulosic cell
wall. The data of others, as well as our own determinations on Vaucheria
and Stigeoclonium spp. show that filamentous algal forms are low in protein
relative to other types. Literature data for diatoms, and our analysis of a
diatom-dominated mixed periphyton community readily acceptable to a variety
of consumer organisms, indicate that a much higher level (over 40% protein)
is characteristic of these algae.
The possibility was considered that Cladophora protein might also be
deficient in some essential amino acid constituents. Although the amino
acid requirernents of most aquatic invertebrates are not very well under-
stock, most insects are known to require branched-chain (iso leucine, leucine,
valine), basic (lysine, arginine, histidine), and aromatic (phenylalanine,
tyrosine, tryptophan) amino acids. CIadophora was nvdrolyzea in strong acid
and its amino acid constituents were concentrated and analyzed by GC. As a
check on the applicability of the method, a standard protein (bovine serum
albumin) whose amino acid composition has been determined by traditional ion-
exchange cnronatographic procedures was hydrolyzed, derivatized, and analyzed.
The individual amino acid percentages computed using the GC method were com-
pared with those from the literature by linear regression; a hign degree of
correlation (r=0.94Q, P
-------
Lipids—-The lipids of algae make up a heterogeneous group of substances,
having as common characteristics the ability to dissolve in rtonpolar solvents
and a high ratio of carbon to hydrogen. Their reduced character rakes them
a useful source of metabolic energy. Some lipids are also essential nutri-
ents for consumer organisms; thus some invertebrates require a dietary source
of polyunsaturated fatty acids, such as 1inoleic or 1inolenic acid. In
addition, all insects require dietary sterols. The requirement for sterols
by other invertebrates is not as well established but it may be rather
general. Many blue-green algae either contain no sterol or very low con-
centrations of sterols; this may explain, to some extent, why they are such
poor dietary constituents for invertebrates. For green algae, the reported
range of sterol content is 0.01 - 0,381 (25, 26). The only member of the
genus Cladophora previous studied, C_. flexuosa, contained a rather low 0.06%
sterol, with the major constituents being cholesterol, 24-methylene-choles~
terol, and 28-isofucosterol (26). Other members of the Cladophora!as also
have been reported to produce sterols; for example, Chaetomorpha crassa
contains sitosterol, cholesterol, 24-methylenecholesterol, and two methylated
sterols (27).
The combined fatty acids of algal lipids have been extensively studied.
Although there is considerable variation among individual genera, larger
groups of algae are characterized by more or less typical fatty acid patterns
For example, the usual fatty acid composition of diatoms is dominated by un-
saturated Cig and C20 fatty acids. Unsaturated C-j3 fatty acids, particularly
1inolenic acid, are normally present at much lower levels (21). Green algae,
on the other hand, resemble the higher plants in their fatty acid composition
their unsaturated fatty acids are usually C|g types, with unsaturated C]q or
C20 acids present in low concentration (28),
Much less work has been reported on the free fatty acids of algae, al-
though there are a few interesting observations on their toxicity to other
algae (29), protozoa (30), and bacteria (31),
There are a considerable number of other reports of lipid toxicity.
Polar lipids, in particular, may interact with cell or organelle membranes
and cause disorganization of systems involved in transport, photosynthesis,
or respiration. Surfactants, such as linear alky!sulfonates, have similar
toxic effects on aquatic organisms for related reasons. Restricting con-
sideration only to simple fatty acids and their esters, there are reports of
toxicity to bacteria (32), fungi (33), and algae (34) in addition to those
mentioned previously. Although toxicity of C]g - C20 fatty acids and un-
saturated fatty acid peroxides (35, 36} has been mentioned by a few investi-
gators, there are many more reports of activity in the Cg - C]4 range of
saturated acids with a peak at about Ci2-
The total lipid content of Cladophora (about 9.2%; Table 1) was some-
what less than that of mixed periphyton (10.4"), other diatoms (about 13%).
or Vaucheria (12.2%). Examination of the total lipid extract by thin-layer
chromatography revealed two closely spaced bands, running at practically the
142
-------
same Rf as an authentic sample of cholesterol. The lipid fraction was
fractionated by means of digitonin to obtain the sterols; decomposition of
the digitonide complex gave a crude sterol fraction amounting to 0.13" of
the total weight of the CIadophora. This sterol content is in the range
reported for other green algae, and is about twice the amount found in
Cladophora flexuosa (25). the crude sterol fraction, when examined by GC,
gave only a single broad peak, eluting at the same time as cholesterol, which
was previously reported as a constituent of algae of this order, further
study will be required to confirm this tentative identification.
The free fatty acid content of CIadoonora was investigated because of
the several reports concerning potentially toxic free fatty acids in other
algae. Similar studies were performed on a mixed periphyton community
(dominated by diatoms) which was readily eaten by the snail Physa. The algae
were extracted with refluxing pentane and the total extract was partitioned
into carbonate soluble (free fatty acids) and insoluble (hydrocarbon, etc.)
portions. The free fatty acid fraction was examined by GC and the results
are shown in Table 3, Periph.yton contained more free fatty acids, with the
majority consisting of C74 and C]g isomers. CIadophora contained mostly
C]g - unsaturated fatty acids. These data are consi stent with other reports
of the relative abundances of fatty acids in the triglycerides of diatoms
and green algae, respectively.
Short-chain free fatty acids were also found in these algae. 80th con-
tained some Cg, but Cladophora contained 170 ppm Ct2 Cauric) acid, which was
absent in the periphyton. Conversely, C]q (capric) acid occurred in peri-
phyton but not in Cladophora. LaLonde (personal communication) has indepen-
dently shown that Cladophora contains 1 auric but not capric acid. The occur-
rence of lauric acid in Cladophora is interesting from an ecological stand-
point since there are many reports of its toxici ty; these wi 11 be discussed
under toxicity experiments.
The pentane-soluble, carbonate-insoluble fraction of Cladophora was
examined by infrared and ultraviolet spectra; it included hydrocarbons and
long-chain alcohols as well as soma carbonyl-containing material.
Carbohydrates--The bulk of the carbohydrate in most algal cells is
polymeric, with a majority in many forms (green, brown, red, and some yellow-
green algae) being cellulose. Because cellulose is not digestible by most
animals, requiring a specialized gut microflora such as that found in ruminant
animals, it may contribute little or nothing to the nutritional value of a
given alga although it may contribute a significant number of calories as
measured by combustion. Since Cladophora is known to have a eellulosic call
wall (1), we chose to measure only soluble carbohydrates, assuming these to
be more available to digestion by a wide range of predators. Although little
quantitative data on the soluble carbohydrate concentrations of algae have
been reported, a great variety of mono- and oligosaccharides have been re-
ported to occur, along with starches, agar-!ike gels, and sugar sulfonates.
Several species of the CIadophorales, including Cladophora spp. , produce
143
-------
unusual fructose-containing oligosaccharides (37). 4hen used as storage
products, as in the brown algae, soluble carbohydrate may comprise as much
as SCR of algal dry weight (38),
C. qlomerata contained about 4.5;i soluble carbohydrate (Table 1), more
than was found in the filamentous xanthophyte Vaucheria, but less than was
present in a mixed periphyton (diatom-dominated) community. Because both
Vaucheria and periphyton were readily eaten, it is unlikely that Cladophora
was not consumed because of an insufficiency of carbohydrate. Further studies
of Cladophora carbohydrates may be necessary to establish their roles in
nutrition and feeding behavior. Although toxic carbohydrates are unusual,
they do vary widely in palatabi1ity and susceptibility to enzymatic degrada-
tion. Starches are also feeding stimulants for a slug (39).
Total phosphorus-~the phosphorus content of Cladophora (Table 1} is
about 0.752, a value intermediate between that"for Spiroqyra (0.19%} and
periphyton (1,85/0, The extent of availability of the phosphorus as a
nutrient mineral for consumer organisms was not assessed.
Toxicity Experiments
CIadoohora extracts (and some corresponding extracts of the readily
eaten periphyton) were screened for possible toxic substances. The effect
of the extract on growth of a yeast (Saccharomyces cerevisiae) in a defined
medium was used for the analysis (17), Solvents used for the extraction
were pentane, acetone, methanol, and cold and hot water. Results of the
studies are shown in Table 4,
Water and acetone extracts of Cladophora significantly promoted the
growth of Saccharomyces, probably due to the provision of respirable sub-
strates , co-enzymes or vitamins. Methanol extracts had a limited growth-
inhibiting effect; at 800 ppm growth of the treated cultures was signifi-
cantly less than that of controls. Pentane extracts of CIadoohora had little
effect at 100 or ZOO ppm, but significantly suppressed growth at 400 ppm,
Pentane extracts of periphyton promoted growth significantly.
In a preliminary experiment, it was determined that the observed tox-
icity of the Cladophora extract was not due to its pentane-soluble hydro-
carbon constituents. Yeasts exposed to this fraction (200-400 ppm) grew at
37-1052 of the control growth. Because insufficient material was available,
it was not possible to test the free fatty acid fraction of Cladophora for
toxicity. However, LaLonde (personal communication) has shown using pure
compounds that some of the free fatty acids of Cladophora are toxic to mos-
quito larvae, with the C72 (1 auric), C]4 (myristic) and Ci5.1 (palmitoleic)
acids being most active. Our work shows that the latter two acids are more
abundant in the readily eaten mixed periphyton than in CIadoohora (Table 3),
and that lauric acid, absent from periphyton, is present in CIadoohora. Our
tentative conclusion is that lauric acid is responsible for the toxicity of
Cladophora extracts to yeast, and may also contribute to Physa toxicity.
144
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Further study will be required to confirm this hypothesis. It is interesting
that 1 auric acid, extracted from the seaweed LHva lactuca, was shewn to be
toxic to protozoa, causing cell lysis at 250 pprn and inhibiting mobility at
lower concentrations (less than 100 pot; 30). There are also many reports of
antimicrobial activity of 1 auric acid (40, 34, 41, 42, 32, 43).
The results of these experiments show that CIadoohora glomerata is lower
in overall nutritive value (less protein, lipid, and phosphorus per unit of
dry weight) than many other readily eaten algae. CIadophora contains low con-
centrations of steroids and free polyunsaturated fatty acids. Although Clad-
ophora is also low in sulfur-containing amino acids, it is not unique in this
respect, and appears to contain most of the other essential amino acids in
adequate quantities. Cladophora has a thick and resistant cell wall, which
affords mechanical protection and may also be resistant to enzymatic attack.
The evidence suggests that Cladophora dome rata may contain substances
which are toxic based on yeast growth inhibition. One of these substances
may be free lauric acid, which is present in the organism at a concentration
of about 170 pprn.
-------
TABLE i
NUTRIENT CONTENT OF FILAMENTOUS ALGAE, PERIPHYTON, AND1 DIATOMS3
Cl^il'Sjthora
«jlM»ar."»t a
Vauchcr t a
SM iji.oc 1 on i urn 5r i rnqyra
Mixed
peri pliyinn
l> 1.1 tiua-.'1
I'roLc i n
n.2il.6 |9|
5.4(2)
9.67(1) 15.93C
44.111)
4 ti. 4 i 1 (1.1( II!
Free run i no acid
0.17(2)
0,65 (1S
Sol . carbohydrate*'
4 , t,6(5)
1.6(11
0,112)
?.i.r.l'K l id)''
Total lipid
2ll.O(5>
1.2.2(1)
10.4(1)
l 3. 0l 11. -1 III)
Tentano-solubi«
0.06(2)
1.11(11
Free f.i 11 y «c id
0,1512}
n.9r. ti)
Plnrol
0.11 (2 i
t« ii-*) §>
0.74*ft.35(41
1.9012)
1.12(2) 0,1912)
1,#3(2|
Mil vjIul-s in [!crcc!il of ash-five dry weight, f tgures in parentheses are (winter of dnterwiiutlons. Stamlaril
ifi;v u t iuns art; i)lv<>n where this figure Is greater than 3.
'•Iln .1 Iculdtoil f rum Jit a In l.itiie 7.1 of liar ley {21).
< ilnldoii I! f Idl speck's {S4).
') jii Iul) I e tn tot
-------
TABLE 2
AMINO ACID COMPOSITION OF PROTEINS OF SOME FILAMENTOUS ALGAE AND DIATOMS
Ami m> Acid
d
Clailoplinra
Stiqeocloniu«b
b
Vauolier irt
Spi r: i i 1 J iju -1 h Ic
2 0 . 0 4 10 . 6 7
ia„l
20. U
14.79
2 0. 1
Serine
4 . 9811.4 0
7.2
fi, 3
4.49
9.0
llya I L- i tie
(o.no)
0.5
1.0
0.22
0.5
S'rol uia
7.72*0.35
7.6
7. i
3.97
6.5
Hiitii i on i ue
a.3sio.7i
1.1
3.0
1.50
1. f]
I'licjiy 1.» r t u: Acid
H . 5 U 10 .
-------
TABLE 3
FREE FATTY ACIDS OF CLADCPHQRA AND NIXED PERIPHYTON
Acid
Cladoohora
Periphvton
Cg (caprylic)
C^q (capric)
C12 (lauric)
C14 (myristic)
C^g {palmitic)
C - . (palir.itoleic)
ID . 1
C (stearic)
18
^"18 1 (oleic)
c18 2 (linoleic)
C^8 3 {linolenic)
30
o1
170
130
70
40
75
105
200
520
420
170
G1
1560
3020
1370
200
470
1280
' 230
All values in micrograms per gram ash-free dry weight (ppm)
None detectable bv GC.
148
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TABLE 4
GROWTH OF SACCHARGMYCES CEREVISIAE
EXPOSED TO ALGAL EXTRACTS
3. hC
Alca Solvent Concn, (ppm) Growth S ig.
c
Pentane
100
98
_
200
115
400
3
+
p
Pentane
100
152
+
200
154
+
400
157
+
c
Acetone
200
203
400
151
+
c
Methanol
200
86
—
400
85
_
800
53
+
c
Water (hot)
200
. 200
+
a oo
^Oc;
+
c
Water (cold)
200
151
+
400
16 5
+
800
183
4-
a
C = Cladophora cyloroerata ; P = mixed periphyton.
o
Growth relative to a control {only solvent added).
Control growth = 100, Mo growth = 0. Usually mean of 6 replicates,
except for pentane extracts where limited quantity of material allowed
only duplicates.
c+ = Significant at P+0,05 (t-Test) relative to control.
149
-------
1
2
3
4
f
5
6
7
8
9
0
1
)
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31. Harder, R. and A. Qppermann, 1953. Uber antibiotische Stoffe bei den
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41. :
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III. A FUNGAL PATHOGEN FOR POSSIBLE BIOLOGICAL CONTROL
OF CLADOPHORA GLOMERATA
INTRODUCTION
These experiments were performed to explore the potential of fungal
pathogens for biological control of Cladophora glomerata, Considerable re-
search has been conducted concerning the biological control of aquatic nacro-
phytes, such as A1ternanthera philoxeroides (alligator weed) and Eichornia
crassipes (water hyacinth), using both insect and microbial pathogens. In
contrast, the potential for biological control of algal growths has been
only superficia1ly explored. Because Cladophora growths are problematical
in the Great Lakes, other lakes and ponds, streams arid brackish water en-
vironments we initiated a search for a microbial pathogen of the alga. We
began with the fungi because of the demonstrated utility of some for the
control of macrophytes and because several fungi were reported associated
with Cladophora in the early literature.
LITERATURE REVIEW )
Historically, control of algal growths has been accomplished using copper
sulfate ( 1 )» but effects on other organisms and copper accumulation in sedi-
ments are undesirable complications. Herbicides have been used in some in-
stances { 2. 3» 4} but in addition to lack of specificity, resistant algal
strains may develop, and the herbicides may be expensive. Control of nutri-
ent inputs will reduce algal biomass (5, 6). However, depending on the
degree of reduction and prior storage in the system, biomass accumulation
may still occur, albeit to. a lesser extent, if the dominant algaa are species
not readily consumed by herbivores. Manipulation of micronutrient metal con-
centrations has been suggested as a possible means of altering algal com-
munity structure to favor species desired by herbivores, resulting in more
efficient transfer of energy in the food web ( 7 , 8 ). This innovative idea
needs further testing before its utility can be assessed.
Biological control measures also offer the potential of control of se-
lected algal species or groups. A number of observations have been made of
viruses, bacteria, and fungi that have antagonistic activity against algae
of many types (Table 1). Viral and bacterial agents have been most often
associated with blue-green algae (Cyanobacteria), Most fungal-algal associ-
ations have involved Phycomycete fungi, particularly the chytrid group.
However, biological control programs using these agents have yet to be
developed.
154
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Seine associations between fungi and Cladophora species reported in the
early literature were saprophytic (Diplophylyctis iasvis, 9); Chvtridium
"J aggrsoatum, 10), but some were parasitic (Phlyctidium soinulosum and
Phi '/ctochvtrium zyqnemati s, 11; Myzocvti um pro! i farum, and Acntyoqeton
entoohvtum, 12), or unclassified 1 ?h 1 ytoehyzriua qua aricorne11K~Cancer
(13) and Sparrow (14) later identified the Myzocytium species noted by Martin
cn Cladophora as H, ffleqastonnum and Dogma (15} recently found H, megastomum
on CTadophora from a stream in the Philippines.
It is noteworthy that some fungi show considerable promise in programs
to control the development of some aquatic macropnytes. Acremonium zonaturn
(16), Cercospora rodmani i (17), and Rhizoctonia sp. (18, 1~9) in particular
have demonstrated utility for the control of water hyacinth (Eichornia
crassipes) in the greenhouse and in some instances in field tests (20).
Many toxins produced by fungi are host-specific (21), which makes them par-
ticularly promising biological control agents.
METHODS AND PROCEDURES
Isolation, Culture, and Identification of Fungi
CTadophora was collected from three streams where large populations were
present for several months of the year (Plum Run, Chester County, Pennsyl-
vania; Lititz Creek and Cocalico Creek, Lancaster County, Pennsylvania).
Collections were examined microscopicaTly for the presence of fungi associated
with the Cladophora-^ A fungal-infested CTadophora stock culture was also
) sampled for fungal isolates.
Small pieces of Cladophora with associated fungi were transferred to
plates of autoclaved CIaaopnora agar consisting of 6 g C. glomerata in T9C ml
water {macerated in a blender) and 1,5 g Prepurified agar (Oifco, Detroit,
Michigan), Cultures were incubated at room temperature for 7-21 days to
permit fungal development. Isolates were purified using malt extract broth
and agar as well as chitin and cellulose (both identified in early studies
of the Cladophora cell wall (22), although the presence of chitin has not
been substantiated) in mineral salts solution (23). Stock cultures were
maintained on male extract agar and on CTadophora agar at 10°C.
Identification of one isolate as Acremonium kiliense Grutz (Fungi
Imperfacti) was achieved using slide culture techniques and microscopic
observations. The keys of Barnett (24), von Arx (25), and Gams (26) were
used. We gratefully acknowledge Dr. Walter Gams, Institut fur Schimmel-
spilze, Baarn, the Netherlands, who confirmed our generic identification
and affixed the species epithet for us.
Throughout all phases of this work any material exposed to any fungal
agent was sterilized by autoclaving or by the addition of formaldehyde
(where large volumes might preclude autoelaving) before discarding.
)
155
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Culture of C, glomerata
Unialgal C. glomerata was grown In the defined Cladophora Medium II of
Fitzgerald and Gerloff (27) at 20°C using | 16-hour photoperiod with average
illumination intensity of 55 u Einsteins/m^/sec from Vita-Litss (Duro-Test
Corp., North Bergen, New Jersey). These lamps possess a spectrum approxi-
mating solar radiation. Cultures were bubbled continuously with filtered
air.
Laboratory Assay of Fungi for Anti-Cladophora Activity
Fungi were grown on malt extract, chit in, or CIadophora substrates,
usually for 7-15 days at 18-20°C. Conidia were collected from solid substrates
by rinsing with 5 ml sterile water and centrifuging the suspension. Conidia
were retrieved from liquid cultures containing chitin or Cladophora by centri-
fugation of the liquid portion of the medium; the mycelium remained largely
with the solid added substrate. Numbers for addition were estimated by direct
microscopic counts using a- Petroff-Hausser eel 1. 'Viable numbers reported here
were determined from serial dilutions spread on plates of malt extract agar in
triplicate. Growth foci were enumerated after 5 and 7 days' incubation at 2Q°C.
The C. glomerata used in assays was either subcultured in CIadophora
Medium Ii for 5-7 days or was collected from a field site just prior to use.
Material from the field was dominated heavily by Cladophora, but diatoms,
other green, and blue-green algae were present in small amounts.
Fungal-algal interaction was studied in 125 ml Erlenmeyer flasks by )
adding serial dilutions of A. kiiiense conidia suspended in water to 25 mg
(wet weight) £, glomerata in 50 ml filter sterilized (0.45 urn pore size)
Lititz Creek water. Controls received no addition. Cultures were incubated
for 10-14 days with continuous agitation under Vita-Lites (72 u Einsteins/m^/
sec intensity at the flask surface; 16-hour photoperiod) at 2C°C.
Three measures were used to assess fungal effects on Cladophora: cell
condition, chlorophyll a_ content, and dry weight. The algae were examined
microscopically for cell condition. At least 1000 cells from each flask
counted and scored as healthy (packed with green chloroplasts), partially
bleached (chloroplasts a pale green or fewer in number than in the control),
chlorotic (chloroplasts a pale yellow-grey) or empty (cell contents absent).
Counts were made across each microscopic field, rather than along Cladophora
filaments. The algae from each flask were blotted dry, weighed, macerated,
and extracted overnight in 10 ml acetone at 4°C for determination of chloro-
phyll a^ (28). The algae were then dried at 60°C and weighed,
Supernatants from fungal cultures or assay flasks in which effects on
Cladophora were obtained were also tested for effects on Cladoohora. The
supernatant fluids were filter-sterilized (0.45 urn pore size J and a iiquots
(5 ml) were added to the 45-ml filter-sterilized Lititz Creek water, Other
5-ml aliquots were heated to 80°C or 100°C for 10 minutes before addition.
Cladoohora {25 mg) was added,and assays were conducted as described,
156
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Microcosm Experiments with A. ki1iense
Experiments with Cladophora-dominated algal ccmmuni ties were conducted
in flowing water (stream) and static (pond} microcosms in a greenhouse labor
atory. Temperature was monitored using maximum-minimum thermometers (Brook-
lyn Thermometer Co., Farraingdale, New York), Radiation was monitored at a
site outside the greenhouse using a pyranometer (Eppley Laboratories, New-
port, Rhode Island), Water chemical parameters (NO3-N, POd-P, and total
alkalinity) were measured using Standard Methods {29), HNn-N by the phenol -
hypochlorite method (30), and metals by atomic absorptionJspectrophotcmetry.
Flowing water microcosm experiments were conducted by placing rocks with
attached algal communities from Lititz Creek in Plexiglas cylinders (10 cm
inside diameter x 61 cm long, fitted with end caps). The microcosms con-
tained 2 liters Lititz Creek water and were connected to reservoirs contain-
ing 10 liters Lititz Creek water. The water in the systems was recirculated
continuously using submersible pumps. The microcosms were submerged partially
and the reservoirs completely in water from White Clay Creek, which flows
near the laboratory. This water was continuously replenished to keep the
systems at near ambient water temperatures, Every three days tne water in
all microcosms was renewed with Lititz Creek water and the experimental sys-
tems were reinoculated with fungal conidia (collected by cantrifugation from
1-1 iter cultures grown on Cladoohora in mineral salts solution) or conidia
and mycelium (collected by cantrifugation from cultures in malt extract broth).
CIadophora condition was determined at intervals from microscopic counts of
1000 cells done on a sample taken from each rock. Additional counts of
another 1000-1500 cells, which included both Cladophora and Ulothrix, were
done to assess dominance and condition.
Static microcosm experiments were conducted by adding 3 grams wet weight
of Clariophora-dominated algal communities from Lititz Creek to 6 liters of
Li ti tz Creek water in plastic trays. The trays were kept at near-ambient
stream temperatures as were the flowing water systems. The water in the
trays was changed and the experimental system was reinoculated every three
days. Counts of 1000 cells each were performed on duplicate samples at inter-
vals to assess Cladophora condition. Another count of 1000 Ulothrix and
Cladoohora cells was done to assess relative dominance and condition.
RESULTS
Laboratory Assays with Isolates P-l, PR-5, and Acremonium kiliense
Fungi were observed in some Cladoohora cells in material collected from
Plum Run and isolates, designated PR-1 and PR-5, were obtained. Isolate PR-5
possessed no virulence against Cladoohora.
In our first experiment with isolate PR-1 anti-bacterial antibiotics
(Streptomycin sulfate and Penicillin G, Sigma Chemical, St. Louis, Missouri)
were added to suppress bacterial numbers since axenic cultures of C. clomerata
were unavailable. A tuft of PR-1 mycelium was added {rather than spores) to
cultures in Cladophora Medium II, White Clay Creek water, or a Z57, dilution
-------
of medium in water, to examine effects when nutrient concentrations were
altered.
Isolate PR-1 had a slight detrimental effect on CIadophora, but pro-
nounced development of another fungus occurred in the control with anti-
biotics (Table 2). This funqus, which was apparently associated with the
Cladophora stock culture, was isolated and subsequently identified as Acra-
moniurn kiliense Grutz. Two other experiments were conducted with isolate
PR-1, but it appeared less antagonistic to Cladoohora than A. ki1iense. A.
ki1iense (Acremonium was formerly known as Cephalosporium) therefore became
the focus of further experimentation.
The results of one experiment with A. ki1iense are shown in Figure 1.
Exposure to A. ki1iense caused chlorosis of many CIadophora cells. CIadophora
collected from lititz Creek was more susceptible to the fungus than CIadophora
from the stock culture, perhaps because the fungus was isolated from the
stock culture or because the Lititz material was a mixed algal community with
other interactions. Assays were repeated several times with variable results
(Table 3). Direct microscopic examination for cell condition tended to be
the most sensitive measure of effect, although in experiments 5 and 6 chloro-
phyll _a concentration showed the effects of fungal pathogenicity. In experi-
ments 1-4 the number of healthy cells was reduced to less than 50" of the
number in the control. (In experiments 1 and 2 50% or more of the cells at
some treatment level were completely chlorotic, but in the other experiments
the effect was less dramatic and the chloroplasts were partially bleached.)
Lack of correlation between eel 1 condition and chlorophyll a_ concentration when
the Lititz Creek community was used may have resulted from the presence of
diatoms, Spi rogyra, Ulothrix, or other algae that were unaffected by A. kiliense
and which contributed chlorophyll a_. Sometimes microscopic observation showed
ruptured cells with external accumulation of intact chloroplasts and other de-
bris along the Cladophora filaments. In such instances, some chlorophyll a_
might still be measured even though the cells were debilitated.
Although the effect produced by A. ki 1 iense was observed repeatedly,
we have had experiments in which effects on Cladophora were not evidenced
under the experimental conditions. Inoculum size, fungal culture conditions
and age of the fungal inoculum, extent of germination of conidia, and incuba-
tion water varied between experiments, but positive or negative results can-
not be traced to a single factor. Since negative results were more common
in later work it is possible that loss of A. ki1iense virulence may also be
involved. Extent of conidial germination may also have affected our results
so that similar inoculum levels did not produce the same result.
At the end of the experiment shown in Figure 1 , supernatants from the
assay flasks in which Cladoohora was damaged (those receiving undiluted
condia 1 inocula) were assayed for effects on Cladoohora. Only one super-
natant (Lititz Cladoohora, assay flask 1) affected the stock culture CIad-
ophora strain in this experiment (Figure 2). Heating at S0aC did not
eliminate the toxic effect, although exposure to 100°C may have lowered it
158
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somewhat. Variability in results may be due to differing concentrations of
the toxic substance(s) in the supernatant fluids added, Furthermore, we
tested only 5 ml volumes so that nutrient concentrations would not be severely
altered by supernatant addition, and positive results may have been obtained
with larger volumes,
In another experiment A. ki1iense was grown in Czapek-Oox broth for five
days after which the conidia and fungal culture supernatant were assayed for
effects, No effect was noted from addition of conidia in this trial, but the
addition of sterilized supernatant damaged Cladophora {Figure 3 )f whether
the supernatant was heated or not. The control f5 ml Czapek-Dox broth) had
no effect on the Cladophora, Another identical experiment was conducted ex-
cept that the fungi were cultured for 9 days prior to addition. Only mild
effects were noted, but the concentration of toxicant probably differed be-
tween experiments. Production and destruction of toxicants may be proceeding
simultaneously in the fungal cultures.
These experimental results demonstrated, however» that contact between
the alga and fungus was not necessary for damage but that this A, ki1iense
isolate produced an antagonistic agent against CIadoohora that was water sol-
uble and not destroyed by heating. This indicates that the deleterious effect
of the fungus on CIadoohora was caused by an antagonistic effect and not to
competition for nutrients.
Microcosm experiments with A, kiliense.
Experiments were conducted with A. ki1iense and Cladophora-dominated
communities in microcosms held under natural light and water temperature con-
ditions in a greenhouse laboratory. Both flowing water microcosms (designed
to simulate stream conditions) and static microcosms (designed to simulate
pond or lake environments) were used.
The first experiment was conducted from July 17-August 1, 1978. Light
input ranged from 134-586 langleys/da^ (x + s.d. = 438 + 126, n = 15) and
temperature averaged 21.2 + 0.98 °C (x + s.d.- n = 15), Duplicate static
systems were inoculated with 2.07 - 18.5 x 10 conidia/ml but effects on
Cladophora were not evident. However, in the stream systems which were
inoculated identically, the CIadophora was in poor condition after one week.
White patches of Cladophora were visible to the naked eye on the surface of
rocks. Direct microscopic examination indicated many cells were chiorotic in
the treated microcosms, but in the controls s>70« of the CIadophora cells were
healthy (Table 4), Diatoms and Ulothrix which were present in the micro-
cosms were unaffected by A. kil iense and protozoa and other meiofauna were
healthy. Extensive 111othrix develooment occurred in both the inoculated and
control microcosms, particularly at the water surface where skeins of Clad-
ophora provided sites for attachment and growth. At two weeks, counts of
1000 cells each were made of attached and surface growths. These counts in-
cluded both CIadophora and Ulothrix cells, CIadophora cells were most often
encountered in the growth scraped from rocks in all microcosms and Ulothrix
159
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at the water surface. CTadophora exposed to A. ki1ieose had many more chlor-
otic cells (60 arid 70S of attached cells) than CIadopnora in the controls (8
and 23%). Ulothrix cells were in good condition in both sets of microcosms.
The experiment was repeated between August 11-31, 1978, with striking
results. Conidia added during the first week were collected from cultures
grown on CIadopnora in mineral salts solution used for chitin-salts medium
(23), Both mycelium and conidia that were harvested from malt extract broth
were added after that.
After one week, Cladophora in the inoculated streams was largely chlor-
otic (Figure 4} • and Ulothrix was replacing Cladoohora as the dominant
alga (Table 5). Diatoms, Scenedesmus, and Spirogyra that were present to
a smaller extent in the communities were in good condition as were protozoans
and rotifers. At the end of the experiment, Cladophora was absent on some
of the rocks in the inoculated microcosms. Althougn the condition of Clad-
ophora in the control microcosms deteriorated through the experiment, on any
date many more healthy cells were encountered in the controls than in the
treated microcosms.
Treatment with A. ki1iense produced similar results in the static micro-
cosms. In one week the fungal-exposed Cladoohora was largely chlorotic
(Figure 5 )» In the controls, however, Cladoohora remained healthy through-
out the experiment. As in the flowing water systems, Ulothrix replaced Clad-
ophora as the dominant alga in the treated microcosms (Table 5}.
Water temperature (measured in the early morning and late afternoon)
ranged between 19.5-2_3-0°C in the microcosms. Light inputs ranged from 169
to 584 langleys/day (x + s.d. = 394 + 126, N = 21). Concentrations of selected
chemical parameters in Lititz Creek water are shown in Table 6 and their con-
centrations in water removed from the microcosms are shown in Table 7. The
lower concentrations of NO3-N in the water removed from the treated microcosms
may reflect the rapid growth of Ulothrix and diatoms in these microcosms, but
PO^-P and total alkalinity concentrations do not show the same pattern.
Negative results were obtained in trials performed in the flowing water
systems between September 18-Gctober 4 and in the static systems between
September 18-0ctober 20. Lower water temperatures (13.0 - 20.CC in the
stream systems and 15.5-20.0°C in the lake microcosms) may have prevented
sufficient fungal development between changes of water and reinoculation.
A final trial was conducted between October 10-20 in the flowing water
microcosms. Temperature during the experiment ranged from 9-17°C Jx + s.d. =
13.6 + 1.67, n=22) and light inputs from 61 to 353 1angleys/day (x + s.d. =
252 101, n=ll). Water chemistry data is shown in Table 8. Conidia and
mycelium were added at three inoculum levels (x, 2x, and 1 Ox) to increase
inocula over those which produced no effect in the preceeding experiments.
In this experiment results were not as dramatic as during the sunnier experiment,
perhaps because of the lower water temperature, but factors such as fungal
160
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virulence may be involved. Nevertheless, within 10 days the Cladophora in
the treated microcosms was in poorer condition than that in the control
.nicrocosms (Figure 6). Ulothrix again developed larger populations in
the treated microcosms, but Cladophora remained dominant in the control
(Table 9}.
The results presented here suggest that microbial pathogens may provide
a useful tool for controlling extensive growths of C. glomerata in nature.
The results of our laboratory experiments, although striking in some instances,
have been variable. Nevertheless Cladophora from both stock cultures and
field sites were adversely affected in laboratory experiments. Redhead and
Wright ( 31) noted that effects of fungi on blue-green algae were least
pronounced in agitatad cultures when compared to effects in static cultures
or in assays using discs impregnated with culture supernatant placed on algal
lawns. Since all our laboratory experiments were conducted with agitation,
effects have been observed under conditions considered by others as least
favorable for detecting antagonism.
Effects were less pronounced in later experiments which may mean some
loss of fungal virulence or increased resistence of our Cladophora stock.
Redhead and Wright (31 ) reported A_. kiliense was one of several fungi and
bacteria possessing lytic activity against blue-green algae but noted their
fungal isolates lost virulence after six transfers on laboratory media.
The identity of the toxic agent is unknown as yet, It is apparently
Ron-proteinaceous because heating (which would denature proteins) did not
eliminate effects. Some A. ki1iense (formerly Caohalosporium acremonium)
strains produce the antibiotic cephalosporin. An antibiotic-producing
strain (ATCC 11550) was obtained from the American Type Culture Collection,
Rockville, Maryland. Laboratory assays with 1.20 x 1CP conidia/ml showed
slightly lowered chlorophyll _a concentrations and dry weight compared to con-
trol cultures and a reduction of the number of healthy cells to approximately
50* of the controls. Supernatants of 2-day cultures a? this isolate cultured
on chitin in Cladophora II medium reduced chlorophyll a concentrations to 50%
of control cultures as did A. ki1iense supernatants in the same experiment
but had no effect on yield or microscopic observations of ceil condition.
Supernatants from 5-and 9-day cultures had no effect on any of these indicators
of cell condition. Additions of 100 and <100 mg/1 cephalosporin C (K salt,
Sigma Chemical, St. Louis, Missouri) produced some chlorosis, but 10 and 50
mg/1 had no effect. It is possible that the toxicant affecting Cladophora
IS a known compound, but additional studies to determine the identify of the
toxic agent must be done.
Many physiological and ecological aspects of the A. ki 1 iense-Cladoohora
interaction will affect the outcome of any attempt at biological control
using the fungus. Similarly use of the toxic agent will require studies of
its production and effective concentration on a range of algal species and
other organisms. The resistance of various Cladophora strains, or the vari-
able resistance of a given population at different times, as well as the
virulence of the fungus culture conditions promoting maximum virulence or
181
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highest titer of the toxic agent, host range of the fungus or toxicant and
effect of environmental conditions require careful study before any control
program could be attempted.
When an endemic pathogen (as opposed to an exotic-introduced pathogen)
with narrow host range is used, biological control will result in control of
the problem-causing organism. Acremonium species are widely distributed in
aquatic environments (32, 33, 34, 35). Where a water supply with multiple
use is involved, use of a natural agent is preferred over the introduction
of a foreign chemical agent.
The positive results in several microcosm experiments with mixed algal
communities conducted at different times of the year lend particularly strong
support to the reasonableness of further investigating the biological control
of CIadophora using A. ki1iense or another fungus. At the end of the August
10-31 microcosm experiment, fish (fathead minnow, Pimephales promelas, five
per microcosm) were added to one inoculated and one control static microcosm,
Every day algae were removed and the water was sieved through a 250 urn sieve
to collect particles. Algal filaments were removed and fecal pellets were
recovered and their dry weight obtained. The weight of fecal matter from
animals fed the A. ki1iense altered algal diet were from 3-13 times (x + s.d.,
6.3+5.1, n = 4j greater than those fed the CIadophora-dominated original
diet. Thus selective control conceivably would increase fish production by
fostering development of algal species more desired as food by herbivores
than CIadophora, thereby promoting more efficient transfer of energy in the
food web.
162
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TABLE 1. Viruses, bacteria, and fungi with antagonistic
activity against algae (other than Cladochora).
Pirchoeeft
Ho at
Hafarencs
VI3QSES'
A-I virus
Anibaen* vari&blis
XosysJcov, 5,, B. Croinov,
and I. Xhud'/aJcov.
.'liJcrabiol. 41:355 (1372S
A-4cv. virus
Anaiaena -ariabli
Khud'/akav, I. and 3.
Stsasv, Itikrobiot. 42:
904 11973).
AP-1 virus
Aonanisomnon flos-aq\ia.«
Geanfealt, u. ?h«/siol.
Plane. 2S«332 (1972) .
Aa-I virus
C-i wir:s
AS-L virus
A.-iabaenco3i3 racilaorskil
JtRtaaaar.aosxs circular's
ftaoSI^laosis' inalca
Cyilr.drosoerr?
An.acysc.i3 .-lidulan*
Svr.«cnococcu3 c8dror*J3»
Singh, H.S., and »,X.
si.-.on. Sacuca 215; 13 20
{1967! .
Saiiecman, 3.S., es *1.
Virol. 4 7: 105 (13721 .
UP-L virus
Lvnqbv>
Piecxortetaa
?hor7aiciiua
Saifemian, a.S., and
M.£. Harris. Sci. 140:
S79 (1963! ,
3-i virus*
LyngSva
?lac~or.ena
?5acm3iua
Oaf-t, M.J. , J, 3*99,
and w. 3 . ? . Ssewacs.
:t«w ?hyt3l. S9:i039
(1970) ,
G-ttl virus1
Lyngbya
PtTeTarTgna bcrvanua
?ho r^idiur.
?idan, £. , M. Shilo ,
and ."f. Ris Lev . Viral,
32: 334 (1967).
lpp-:
Safferman, R.S., at »1.
Virol. 39:773 (1369).
LPP-1G virus
Plectonosia borvinua
Padan . E,, et al.
ViroL. - 5:773 (1971)
M-L virus
Nostoc muscorm
Ado Inn, KM., and a.
Hasaikarn, Virol, 16;
200 (1971),
SM-l
Synechacacsus Ion gnus
Saifsraan, !¦$. tt al
Virol. 57:336 fl969)
163
-------
TABLE 1. (Continued).
)
? it ho :-n Hqs t Re fsrencs
•v.inaaed
Oedoeoniura
Pickett-Heaos, J.3.
J. Phycal. 3:14 CIS?:).
Unnamed
Qvlorslla ovrenoidosi
Tikhonanko, A.S. , and
N.3. Zavar:ina. Mikro-
biol. 55:350 (1968).
Unnamed
Sirodo tia tenuissima
Lee, R.E. J, Csll Sci.
3:623 £ 19T1) ,
BACTERIA1
—
3dalls%'ibrio jjacreriovorus
?ho rniiiurn
Burnhan, J.C. N.T.I.5.
.'•fvcrocvstis
PI-ITS 569/:.
-------
TABLE 1. (Continued).
Host
Re serenes
S&isoaii idi.ua androdiocres Oictvosnhaeriua saldislitm
Canter, H -M , Trans.
3n:. co 1. Soc . So;
US (1371) .
-ygofhiiidiun verms turn
CUrysophyws
Canter, H..M. Nova Hed-
wig. 21:5 77 (1973) ,
Acreacmiua
raer* cgrTobsi s
TTrT; c £173 "
Slue-green (3) and
green (4) algal, species
Redhead, S, , and S.J. I.,
'.'.'rtghc. Aool. Environ.
Microbiol, 33:96: ri9"S)
ZTjaritiiidiua aalosiraa
Melosin iraaulata
Felix, E.A. Trans.
3rit. Mvcol. Soc. 69 r
315 f 19 7?) .
Aquatic PhvcsmycsMS
Blue greens, jreens,
diatoms, desnids
Sparrow, F. K., Jr.
Aquatic Phycanycstes.
*J. Mich.. Press. Ann
Arbor. 1137 pp. (1560)
Rhicosistion. anabaenae
Anabaens alaaktonica
Patterson, ft,A. Ecol.
H:41S (I960).
jthisophidium sphserocafamn
Sntio phiai urn SrtiagIT'"*
Soiragvra
OTocHrtx™*
Barr , D.J.S., and C.J.
Hickman. Can. J. Soz.
4S:423 £196?},
Py.thiua jorahyrae
Porgayra spp.
Takahashi, M.» T, Ichi-
tani, >1. SasuM. Trans.
Mycol. Sac. Jan. 13:2 79
(1977) .
ArasaJti, S. 3ull. Jap.
Soc- Sci. Fish. 12i74 {1947}
Fuller, J!, S., 3, Lewis,
and P. Caok. Mvco1. £3:313
11366 J.
lAcer.-d-uj sp
Cascir.oducui cantraiis
Johnson, ?. '¦!. , Jr.
58:131 ilSSfii.
Mycol.
«a.ce»isr.a. soscir.odisci
Cascirtodisc-is -iranni.
5aI3eria~Har2safiia
Dcebea, G. H«lcr «iss, Saaras-
unter*. 13 t 4 2«"(19661.
Crsnaaa. S. 3xri. PHvcol.
11:57 (19761.
165
-------
TABLE 1. (Continued).
Pwiiocen Host 3acera.nct
SctrocaHa Jerforans
,1-9 are
•rtrsatiis
Swnedra
Johnson, t. ~i.,
33:3T3 i13 661 .
J\j- Myeal.
Phlvciid^aia acg.nedesroL
For:
?hlyctidi.ma seaneaesax
var. acvisx '/»r. nova
Scanedasaws agnates
Scermdasrous acucus
Seeder, a. A, ar.d a.
•Uiw«q. Axch. Hydrciuoi.
66:48 (136ft.
for reviews sa«: Sarferr-ar., 3.3. 19 *3. Phycoviryses, p. 2U-33", In N.G.
Cirr and 3.A. Whittoit i.Jds .! , The biology of the Slue -jrten algae, 07 Calif.
Press, Davis. Padua, E, and M. Shi la. 1973, Cyanophajes - viruses at-icxir-s
blue-green algae. Bac.eriol. S«v. 37:343-370.
I, May be she sane virus as LPP-i
3. For review see Whittan, 3.A. 1973. Interactions with 0titer orjsnisrj ,
p, 413-433, la N.C. Carr and 3.A. Whicton (eds .). The biology of blue-green
alga#. 'J, Calif. Press, Davis.
166
-------
TABLE 2
ASSAY OF EFFECTS OF ISOLATE PR-1 ON C. GLOMERATA IN VARIOUS i NCUBATI ON MEDIA
AND IN THE PRESENCE OF ANTIBIOTICS (STREPTOMYCIN SULFATE, FINAL CONCENTRATION
0.06 MG/ML, AND PENICILLIN G, 0,10 MO/ML, 1625 UNITS/MG).
Culture Conditions MacroacopI?
Control
UToroscopId"
Inoculated with PR-l
""Macroscopic ——— HTcroa copTc
White Clay water,
autoclaved
White Clay water
+ antibiotics
White Clay water
Cladophora modi i
"12
CI adoi»ho ra med I urn
~~IT125*7 + white
Clay Water (751)
Healthy, no
apparen t
con taminatlon
Em t.£iiiai ve
bleaching,
ex tunaive
fuiujal develop-
ment »
Healthy
Healthy, no
apparent
contamination
Healthy
Some chloroplaat
breakdown, much
new growth, no
con tain In an ta
Chloroplas ta
brown, lit J is
hyphae develop-
ment in and on
Cladophora
Ceils-filled
wllli ch!oropl as
greun
Cells heal thy,
much new yrowt h,
no con Lamination
Heal.thy, much
budding - new
yrowth
Cladophora healthy,
fungi
-------
TABLE 3
EFFECT OF A. KILIENSE ON C. GLOMERATA. RESULTS OF ASSAYS WITH
POSITIVE RESULTS.
CI adophiH 4
Source.
Cul lured,
i iit tz
Cut lured
Cut turctl
Cut turetl
Cullured
A,. kiM efjse Inoculum
Rdmjc (CoriidSd/inJ in
fi^y flJik)
8,32 « lo2 - 8.32 * JO5
8.32 * 102 - 8.J2 * I05
2.08 x 105 - 1.08 x Id'
3,92 x |Q5 - 3.92 x »07
l 6
2.70 x 10 - 2.70 x 10
5.12 X SO1 - 5.12 x 103
Lowest inoculum Reduciikj £xp«rimcnidl
Treatments lo 50^ <»> Control 1
* ¦-!'
1 QtiJi L j oiij < h I m 0|iiiy i I ti 0ry Height
8.32 x I0S , 8.32 * I05 8,32 * 10S
8.32 * 10
2.0fl x 105
3.92 x 10&
2.70 x JQ?
5*12 x 10
8.32 * 10
A. k i I 1 civ^c
Cii3 Sure
Guild i t U'l!^
£1 tfduphoi .a dm I s,
I 3 dayi
C I ddophur d iinj I s ,
II tJu/i
Ground CIadopliura in
liliii water, 9 il«y>
Whole CI a(h)|:lloi a in
Lili U water, 9 days
Mai l extract hi uili,
S days
CI ailoilhot a HclJ l mu I I
pi us dii I III, y Lluys
• Conitlia aJiieil In tenfold serial dilution.
"S>Nfc—i**"-"
-------
TABLE 4
OCCURRENCE AND CONDITION Of CtADQPHQRA AND ULQTHRIX FOLLOWING
EXPOSURE TO A. KILIENSE IN FLOWING WATER MICROCOSMS, JULY 17-31,
1978. INOCULATED MICROCOSMS RECEIVED ADDITIONS AS IN FIGURE 4,
noculatea Microcosms
No. Cells
% Cells
in Indicated Condition
Encoun-
Hea1 thy
31eached
Emp ty
Mi cro-
tered/10
Parti -
Ch lo-
Time
cosm
A1 oa
Cells
aJ.br
re tic
1 wk.
1
CIadoohora
1000
<; 1
3
86
12
3
1000
50
17
23
10
2 wks
t
CIadoohora
1
Atnacned
600
22
3
60
15
Surface
20
< 1
0
100
0
J
Attached
630
21
3
70
6
Surface
170
65.
2*+
12
<1
Uiothrix
1
Attached
kQG
88
12
0
0
Surface
980
$k
5
0
1
3
Attached
370
57
0
0
Surface
830
89
8
Q
2
Control
Microcosms
1 wk.
2
CIadoohora
1000
77
9
8
8
k
1000
71
17
9
1+
2 wks
.
CIadoohora
2
Attached
Q7n
7 / u
66
23
8
3
Surface
170
100
0
0
0
k
Attached
880
56
19
23
2
Surface
190
89
< I
6
6
Uiothri x
2
Attached
30
67
0
0
33
Surface
830
89
1
8
J
k
Attached
120
82
0
<1
18
Surface
810
78
9
5
3
169
-------
<1
£
w a
IS
£ .
-3JT5 .
31 ZJ 1
— J C
•si— '
X '
— 1
3
« 3 "J
£ SI
as s> e
¦M C .NN O 3>
33 ** £3 Q
^ry .ar n* cc-ss ^ -c co — o a
¦offl OCS *V ^ SH in S3 rt «5" s3 «M O OO
^ ft SSI^ f^<0 35® **»C3 CO S3
£, o »A-a sS jr w-v o
'3 « 3
cm — 3 -*M —
=3 23 ^ 33 ft "n a ^ ^—
ft ft -*3 C3 ft ft 33 ft ft s» 03
3 2 If, ?S S?S :?S 5
**+¦ S3 fM— kiis *»* — r>% *r* ;**
1
2 =
is
—i 3
sSi
;s
¦ a
; =c
' u
j-<
:i
S3
P
3
^ »
£ 4
so o a — ¦>« o <
o o jr o\
2.3
p* #n oo p*i o® *#¦* o <03 ***> ©a a?
s
ss gg
s;
i J 22
ft ft
S . ££ fU 3a S£ S
p** Of*" t^jr — a
*?* O
¦ «JT <0 ws
— ^ «*"»
a © O 55 «M —
O Q O O -5* 1
— ^ © ^ ft ft f^a ¦s'*r
0* «0* O vj-i
ft SO
it
*P. 0
*- a
* s
2
i
«
5-
170
-------
~ w -T » f w • **J \ WV* t t 'MWI * VWH v~ss wuaiiiiw / *
Counts"were"made across each microscopic field, rattier than along Cladopnora
filaSnt! The algae from each flask were blotted dry, weighed, macerated,
" ' ' ¦ * ~ * 1 MOLL 0
;
CONCENTRATION OF SELECTED CHEMICAL PARAMETERS IN
LITITZ CREEK WATER ADDED TO MICROCOSMS,
Concentration (mg/l)
N
X
SO
Total Alkalinity
6
169.0
16.6
N03-N
6
8.64
0.47
NQ_-N
2
6
0.200
0.105
NH. -M
4
5
0.083
>r®*»
0.116"
P\-P
6
0-295
0.038
PH
6
7.92
0.34
) "One Sample was 0.29 mg/l , others ranged from 0.02-0.04 mg/l.
171
-------
TABLE 7
CONCENTRATIONS OF SELECTED CHEMICAL PARAMETERS IN WATER REMOVED FROM MICROCOSMS, AUGUST 11-31 ,
1978. MICROCOSMS 1 AND 3 WERE INOCULATED WITH A. Kill ENSE: 2 ANU 4 WERE CONTROLS.
Con c e n 11 ¦ a t Vort I»wn71~l in lndicAlc'tnJarow;,iwj ~
flowing Walur Static
i
z
1
<<
1
2
. 3 .
h
pll
a/ia
10.50
IO.'iO
10.'(5
10.'12
10.00
10. to
10.65
10.90
0/2''
10./S
10-72
! 1.00
10.80
0/28
9.75
9-75
iO. 20
9,65
9.90
10,60
11.00
10.60
9/1
IO.JO
10.''lO
10.'(5
10. kd
10,2©
10.20
10.80
10.'|0
Plicnol -
a/ia
Vi
27
20
35
35
28
l>hihalcin
8/2'.
)6
20
'~2
28
Alkaili.lly
a/28
}'¦
22
'i 3
22
27
• 25
'|6
21
9/1
35
17
31
20
36
23
52
29
lolal
a/tu
65
'.8
45
hi
93
6*i
11%
66
Al kalIn 1ly
8/i'i
65
62
n
66
8/20
131
63
ii'<
73
102
'•5
03
0
y/i
n
1)0
56
l»j
Hi
61
112
lib
a/i a
0.015
0.015
o.tOJ
0.021
0.9(3
0.0'l 2
0. 11'»
o.osi
¦>
ll/2h
0. 150
U. J'i2
o J19&
1.020
8/211
0-365
0.023
0,096
0.022
0-033
0.010
O.O'lJ
O.O'i)
v/»
0.020
0.060
0.0 10
0.010
0.265
O.OJ'i
0-305
0. 170
no ,-ii
i/ia
1.02
2.17
2,57
2.27
5.10
'l.63
'(.'(2
'..0'(
/
a/?'-
o. 16
o.s'i
O.JO
1.02
0/20
0. 16
3.79
0.30
<1.0?
0,20
l .15
1.00
1.86
9/1
0.2)
O.l'l
0.23
1.70
0.77
3.16
0.2)
3-15
fi/sa
o.oij
0.0 111
0.015
0.018
0.100
0-075
0, i'lO
0.099
o. 150
0.5'i2
0.'i96
1.02
0/28
0.006 -
O.O06
a.a'ji
0.07'|
0,00'f
0.020
o.osi
0.025
y/i
O.OOJ
0.0 XI
0.003
0.029
0.006
0.062
0.018
0.115
Mi -ti
u/in
0.016
0.UI5
0.020
0.005
0.0115
0-200
o.oi?
.0.023
3
B/2'i
(J.021
0,015
11.021
0.021
O.UuS
U/2U
11.022
0.0'i2
0.060
0.0.13
0.01 I
o.ois
0.0/6
9/1 ,
O.Ol'l
u.oot
0.021
0.022
0
0.0 2'i
O.OU'i
0
-------
TABLE 8
Total Alkalinity
ho3-h
no2-n
Nttj.-N
w P0/(-p
CONCENTRATION OF SELECTED CHEMICAL PARAMETERS IN LITITZ CREEK MATER
AND WATER REMOVED FROM MICROCOSM, OCTOBER 10-20, 1978.
pH
1
2
2
2
1
2
2
Lit Hz Creek
155.0, 60.1
6.93, 0.10
0.62, 0.02
0.649, 0.152
8,10, 0.49
Concentration (inti/nix
. .d.
Mlcrocosm
1
113.0, kl.O
5.40, 2.26
87.0, 32.5 80.0, 0 102.0, 59.4
5.61, 2,22 4,59, 2.08 5.01 , 3-30
0,070, 0.083 0.016, 0.008 0.00?, 0.001 0,035, 0.035
0.024 0.024 0.014 0.163
0.190, 0.221 0.059, 0.039 0.022, 0.001 0.088, 0.091
9.25, 0.21 9-75, 1.41 9.48, 0.8i 9.53, 1.73
-------
TABLE 9
OCCURRENCE AND CONDITION Of CLADOPBQRA AND ULOTHRIX
FOLLOWING EXPOSURE TO A. KILIENSE IN FLOWING WATER
MICROCOSMS, OCTOBER 10-20, 1973.
noculated Microcosms
/O
Cells in Indicated Condition
No, Cells
Heal thy
01eached
Empty
Mi cro-
of
Parti -
Chlo-
Time
cosm
A1 qa
Soecies"
ally
rotic
10/10
1
CIadoohora
1000
91
0
1
9
3
1000
98
1
0
2
990
99
0
0
1
1
Ulothrix
0
««
_
BBB
3
0
-
-
-
-
i*
10
100
0
0
0
10/20
1
CIadoohora
790
72
k
13
1 I
3
840
58
12
11
13
k
1000
65
7
13
15
1
Ulothrix
210
100
0
0
0
3
160
97
0
3
0
k
0
—
*•
r-
Control
Microcosms
10/10
2
CIadophora
990
96
0
1
k
2
Ulothrix
10
100
0
0
0
2
CIadophora
980
95
2
0
3
2
U1 otiir i x
20
100
0
0
0
'1000 cells counted
174
-------
Cultured
m ,oa*
1
af 06*
a
a
-e
u
,04-
.02-
rfl
T1 r fl
a
£
at
•
5
s*.
a
60'
40-
20-
u
-1 - 2. - 3
n
U -1 -2 -3
FIGURE 1, Effects of A. kiliense (8.32 x TO5 conidia/ml U) on
cell condlcion, chlorophyll a_ content and dry weiaht
of £. glomerata from stock culture and Lititz Creek.
Com'dia added in serial dilutions.* U=undiluted; -1,
-2, -3=tenfold serial dilutions; Ocontrol, Cell
condition; 3lack=nealthy, heavy dcts=partially bleached,
light dots=chlorotic, white=empty.
175
-------
100
^4
m
e
i»
M
so
fe.
#
a*
Q
Ui
.Oll-i
6
oi
.06-
>.
.04-
X
&
a
im
.uu-
Q
u
a-
*->»
m-
u»
B
0
0
1]
jc
m
si
m
¦ m
Mi
SO-
IO-
o-
Contr,
;n
ii
&
$
c-i
1] TO
f
!?£
.103
&
_t<4
w
Mhl -lfaii/1-
i
.H
I
11
ftv-5
"S f'
*j# «
' 111
r>*
iifi
y\
80
C-2
100
_ p
i
H
w
r—t n
iLmili
u bo too
Ll
r
i
Ki
m
BO
1-2
iou
FIGURE 2. Effects of filter-sterilized supernatant fluids from assay flasks in which
Cladophora was damaged on C. glomerata from stock cul lure. Contr .=c:ontro 1;
C-l=supernatant from assay with cultured CIadophora receiving undiluted conidia
(U), flask 1, in Figure 1 ; C-2-cu 1 tured Q a doctor a, flask 2; 1-1=1 ititz
flask U L-2=Lititz Cladophora, flask 2. U^unheated, 80 and lf)0=
heated to 80 C or 100°C for 10 min. before addition. Cell condition: desiyna-
tions as in Figure 1.
-------
S3
S
.06-
Of
^ .04-
a
o
«2
u
.02-
0-
f****1 "1
I ! I
OS
30-
<0
>
la
Q
20-
10-
0-
f'"1[—I
S S/80 S/100
FIGURE 3, Effect of supernatant from A. '
-------
I
100
c
w
>»
a
IOCh
t %
HPT™ _"M"*1
jf' fv - " .. V
f : :
\-r, f>:;
w." . •-
v . ""+<**'¦
:V-"^
t
EZ
5Stf
3/14
8/11 f 8/17
8/T4
FIGURE 4. Cond ition of Cladophora cells in flowing water microcosms
inoculated with A. ki1iense conidia; 113, 6.25, and 29.2 x
104/rnl on 8/14, 8/17, and 3/21 respectively, and with
mycelium and conidia after that (0.32 and 0.39 g wet weight/1
to stream I, 0.02 and 0.39 g/1 to stream III on 8/24 and 8/28
respectively}. Microcosms II and IV=controls. Designations
for cell condition as in Figure 1•
178
-------
100
•"Sri
€»
„X*\- -|
u
50-^'
w
m
&
—¦friaTSg
V.'fj/
' \ *
O-^Si
c
9
u
8/11 t 8/22
8/14
8/29
8/1 if 8/22
8/14
8/29
FIGURE 5, Conditicn of Cladophora cells in static microcosms inoculated
with A. kil iense coriidia; (113, 6.25, and 28.3 x !Q4/m! on
8/14, 3/17, and 8/21 respectively) and with mycslium and
conidia after that (0.64 and 0.73 g wet weight/1 to lake I,
0,02 and 0.73 g/1 to lake III on 8/24 and 3/23 respectively).
Microcosms II and IV=controls. Designations for cell con-
dition as in Figure 1.
179
-------
C(3E)
10 0:
c
01
u
L.
01
On
03
O
10/10 10/20
lo/io lty^o
10/10 10/20
EZ
10/10 10/20
FIGURL 6. Condition of Cladophora cells in flowing water microcosms inoculated with
A- kil iense mycelium and conidia; 0.07-0.21 g wet weight/1 added to microcosm
I, 0.03-0.10 g/1 to microcosm III, 0,01-0.02 g/1 to microcosm IV, C (II) =
control. 10/10 = sample before exposure, 10/20 = samples after exposure for
10 days. Designation for cell condition as in Figure 1,
-------
1
2
3
4
5
6
7
3
9
10
11
12
13
LITERATURE CITED
Bartsch, A. F, 1354, Practical methods for control of algae and water
weeds. Pub. Health Rep., 69:749-757.
Newbold, C. 1975. Herbicides in aquatic systems, Biol. Conserv.. 7:
97-118.
Kirby, G. £. and E. W. Shell. 1975. Karmax herbicide with ferti1ization
and aeration to control filamentous algae in hatchery ponds. Proc. 29th
Ann. Conf., Assoc. Game and Fish Comm., 279-281.
Jordan, I. S., 8. E. Day, arid R. T, Hendrixson. 1962. Chemical control
of filamentous green algae. Hilgardia, 32:433-441.
Edmondson, W. T. 1970. Phosphorus, nitrogen, and algae in lake Washington
after diversion of sewage. Science, 169:690-691.
Schindler, 0. W. and E. J. Fee. 1974. Experimental lakes area: Whole-
lake experiments in eutrophication. J. Fish. Res. Bd. Canada, 31:937-953,
Patrick, R.» T. t. Bott, and R. A. Larson. 1 975. The role of trace
elements in management of nuisance growths. EPA 660/2-75-008. 250 pp.
Patrick, R, 1978. The effects of trace metals in the aquatic ecosystem.
American Sci., 66:185-191.
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