•j-'A 8bu "3 75-QOi
                                   Ecological Research  Series
Impact of the  Use  of  Microorganisms
on the Aquatic Environment
                                   National Environmental Research Centei
                                    Office of Research and Development
                                    U.S. Environmental Protection Agency
                                           Corvallis, Oregon 97330

                      RESEARCH REPORTING SERIES .

Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series.  These five broad categories were established to
facilitate further development and application of environmental
technology.  Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields.  The five series are:

          1.   Environmental Health Effects Research
          2.   Environmental Protection Technology
          3.   Ecological Research
          4.   Environmental Monitoring
          5.   Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series.  This series describes research on the effects of pollution
on humans, plant and animal species, and materials.  Problems are
assessed for their long- and short-term influences.  Investigations
include formation, transport, and pathway studies to determine the
fate of pollutants and their effects.  This work provides the technical
basis for setting standards to minimize undesirable changes in living
organisms in the aquatic, terrestrial and atmospheric environments.

                         EPA REVIEW NOTICE

This report has been reviewed by the National Environmental
Research Center—Corvallis, and approved for publication.  Mention
of trade names or commercial products does not constitute endorsement
or recommendation for use.

                                    EPA 660-3-75-001
                                    JANUARY 1975



               Al  W.  Bourquin
    U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
                Sabine Island
            Gulf Breeze,  FL 32561

              Donald  G. Ahearn
            Department of Biology
          Georgia  State University
              Atlanta, GA 30303


              Samuel  P. Meyers
         Department of Food Science
         Louisiana State  University
            Baton  Rouge,  LA 70803
            ROAP 25 AJN9 Task 010
           Program Element 1EA077
         CORVALLIS, OREGON  97330

    This report contains the proceedings of a symposium-workshop spon-
sored by the EPA Gulf Breeze Environmental Research Laboratory to deter-
mine the possible impact of artificially  introducing microbial insect
control agents or oi1-degrading agents into the aquatic environment.  The
efficacy and safety testing, especially against non-target aquatic orga-
nisms, for use of bacteria, viruses, fungi, and protozoa to control
aquatic insect pests is discussed with remarks of panel members repre-
senting government, academia, and industry.  Special attention is given
to persistence of pathogens in aquatic environments as well as control
of aquatic weeds and other non-insect pests.

    The use of microorganisms to clean up oil spills in aquatic environ-
ments is discussed by industrial, academic, and governmental scientists.
Special considerations are given to selection of hydrocarbonoclastic
microorganisms and use of these microorganisms in special  environments —
Arctic regions and Louisiana salt marshes.

    Summary papers are presented for each panel concerned  with microbiai
pesticides and one summary for the session on microbial degradation of
    This symposium-workshop was held in Pensacola Beach, Florida,  in
April, 1974.
                                  i i i


    This symposium-workshop was hosted by the EPA, Gulf Breeze Environ-
mental Research Laboratory, Gulf Breeze, Florida.  The cooperation of
the GBERL personnel was greatly appreciated, especially the efforts and
encouragement of GBERL's Director, Dr. T. W. Duke.  Principal  contribu-
tors to the preparation and operation of the symposium were Ms.  Chiara
Shanika, Ms. Lynda Keifer, Mr. Scott Cassidy, and Ms. Gerta Guernsey,
GBERL, and Ms. Vicki Tayoe, Ms. Glennis Mitchell, Mr. Norm Smith,  and
Dr. Warren Cook, Georgia State University.

    The exceptional efforts of the session chairmen, Dr. Donald  G.
Ahearn, Georgia State University, and Dr. Carlo M. Ignoffo, U.S. Depart-
ment of Agriculture, are most gratefully acknowledged.  Special  thanks
go to all the participants whose remarks helped make the symposium a

    I  wish to acknowledge the rapid and excellent work of Ms.  Mary H.
Alston, Baton Rouge, Louisiana, in transcribing, editing, and  typing
the many tapes and manuscripts for this volume.  The untiring  and
generous efforts of the co-editors, Dr. Donald G. Ahearn, and  Dr.
Samuel  P. Meyers, Louisiana State University, are most gratefully

                                     Al W. Bourquin
                                     Program Chairman


Absfact	   iii
 Introductory Remarks  	     1
    Al W. Bourquin, Program Chairman
    Thomas W. Duke, Director, EPA Gulf Breeze Environmental
      Research Laboratory

                      Carlo M. Ignoffo, Chairman

Use of Bacteria for Control of Aquatic Insect Pests  	     5
    Samuel Singer
    Discussion	    18

Use of Viruses for Control of Aquatic  Insect Pests	    23
    Darrel1 W. Anthony
    Discussion	    40

Fungal Parasites of Mosquitoes 	    49
    Donald W. Roberts
    Discussion	    65

The Use of Microsporida  (Protozoa) for the Control of Aquatic
Insect Pests 	    69
    Edwin I. Hazard
    Discussion	    76

Persistence'Of Pathogens  in the Aquatic Environment  	    83
    Y. Tanada
    Discussion	   100

Use of Microorganisms to Control  Aquatic Pests Other
than  Insects	   105
    Thomas C. Cheng
    Discussion	   123
Use of Plant Pathogens for Control of Aquatic Weeds  	   127
    R. Charudattan

Discussants:   D.  G. Ahearn, George Allen,  John  Briggs,
              H.  C. Chapman,  T.  B.  Clark,  S.  R.  Dutky,
              Reto Engler, A.  M.  Heimpel,  J.  E.  Henry,
              R.  B. Jaques, Marshall  Laird,  E. M.  McCray,
              Joe Maddox, John Paschke, G.  E. Templeton,
              C.  J. Umphlett,  Al  Undeen

                           DEGRADATION OF OIL
                       Donald  G. Ahearn, Chairman

 Bacterial  Growth  and  Dispersion of Crude Oil  in an Oil Tanker
 During  Its Ballast  Voyage   ....................    157
     Eugene Rosenberg,  E. Eng lander, A. Horowitz, and D. Gutnick

 Selective  Enrichment  Processes  in Resolving Hydrocarbon
 Pollution  Problems  ........................    169
     J.  E.  Zajic and A.  J.  Daugulis

 Petroleum  Biodegradation  in the Arctic ..............    183
     R.  M.  Atlas and E.  A.  Schofield

 Effects of Hydrocarbonoclastic Yeasts on Pollutant Oil
 and  the Environment  .......................    199
     N.  H.  Berner, D.  G. Ahearn, and W. L. Cook

 Microbiological Aspects of Oil  Intrusion
 in Southeastern Louisiana   ....................    221
     S.  A.  Crow, M.  A.  Hood, and S. P. Meyers
 Discussants:   W.  L.  Cook,  R.  L.  Huddleston,  P. A.  LaRock,
               A.  I.  Laskin, W. W.  Leathen, S. P. Meyers,
               R.  L.  Raymond,  M.  F.  Terraso,  R. J.  Miget,
               Gerald  Bower

 Concluding  Remarks by Dr.  William  Upholt, U.S. Environmental
 Protection  Agency
                          III.  SUMMARY PAPERS

Bacteria	    239
Viruses	    2kO
Fungi  	    2k]
Protozoa	    2k3
Persistence in Aquatic Habitats  	    2k$
Microorganisms to Control Aquatic Pests Other than Insects  ....    246
Plant Pathogens for Control  of Aquatic Weeds 	    2kJ
Summary Comments by C. M. Ignoffo	    2kS
Microbial Degradation of Oil	    250

Regulatory Aspects of Microbial Pesticides  	    251
     Reto Engler, William Roessler, and William Upholt
       U.S. Environmental Protection Agency, Washington, D.C.
Concluding Remarks by Dr. John Buckley, Office of Research  and
   Development,  Program  Integration, Environmental Protection
   Agency, Washington, D.C	    25/t
Participants  	    255


                    ON  THE  AQUATIC ENVIRONMENT
                         INTRODUCTORY REMARKS
                   Al W. Bourquin, Program Chairman

    Welcome to Gulf Breeze and the symposium-workshop "Impact  of  the  Use
of Microorganisms on the Aquatic Environment."  The purpose of this  sym-
posium is to assess the impact of biological  control  agents on the aqua-
tic environment.  We have divided the symposium into two sessions, the
first dealing with biological control of insects and other  aquatic pests
and the second session considering the impact of introducing hydrocarbon-
degrading microorganisms into the aquatic environment.
    The objectives of this workshop are:  (1) to determine  the status of
current research on the environmental impact  of artificially introduced
microorganisms, (2) to assess future areas of investigation into  the  en-
vironmental impact of artificially introduced microorganisms,  and  (3) to
promote communication and exchange of information.   The workshop will
include short addresses followed by panel discussions.   Dr. Carlo M.
Ignoffo,  chairman of Session I, and Dr.  Donald G. Ahearn, chairman of
Session II, will present a synopsis of the panel discussions on the
final day with recommended guidelines for future research.
                           WELCOMING REMARKS
                       Thomas W. Duke, Director
           EPA Gulf Breeze Environmental  Research Laboratory

    It is my pleasure to welcome you to the Gulf Breeze Environmental
Research Laboratory and to tell you that  we look forward  to having  you
tour our facilities tomorrow afternoon.

    I  would like to take a few minutes to tell  you briefly of  our re-
search activities at the Gulf Breeze Laboratory and  how we became  inter-
ested in this workshop.  We work in a rather emotional  field  in  that we
are developing a scientific data base on  the effects of chemicals and
natural  organics on marine organisms.  This includes, of  course, pesti-
cides.  I  admit to you that our data base falls far  short of what we
would  like it to be.  The development of  new chemicals  and new uses of
old ones have outstripped our capacity to develop data  on their  effects.

    Recently, we have seen new interest developing on the use  of bio-
logical  control agents to control  agricultural  pests and  to degrade hy-
drocarbons.  Thus we have become more interested in  the impact of  these
uses of  microorganisms on the aquatic environment.  Personally,  I am
well pleased to see this movement  toward  biological  control.   It pleases

me to consider using the forces of nature to control  pests and I  think
that the results could be a cleaner environment than we enjoy at  the
present time.  At the same time, I  see a need, based on past experience,
of having information on the impact of these control  agents on non-
target portions of the aquatic environment.

     Interest in this impact was recently stimulated by research con-
ducted by Dr. John Couch of our laboratory.   He was asked to make some
pathological examinations of pink shrimp that were used as test animals
in a study on the effect of Aroclor 125^, a  polychlorinated biphenyl,
on certain estuarine organisms.  He discovered some large triangular
crystalline  inclusion bodies in the hepatopancreas of the shrimp.
Through electron microscopy he found that these inclusion bodies  con-
tained rod-shaped virus particles and that these virus particles  are
similar to those of a nuclear polyhedrosis virus.   What does this oc-
currence mean?  We do not know at present since we have not yet made a
proper evaluation.  Also, we do not know what relation the chemical has
to manifestation of this virus.

     Dr. Bourquin mentioned that the purpose  of this meeting was to pro-
vide a forum for scientific discussion of the impact of the use of mi-
croorganisms on the aquatic environment.  As we listen to the presenta-
tions and the discussions, I am sure that many questions will arise.
For  example, what pathogens are under consideration now and will  be in
the  future?  What kinds of field testing have been accomplished with
which organisms?  How specific are these pathogens?  Have tests been
conducted to evaluate the safety of organisms other than the target
     On Thursday morning, in the preparation  of the summary, I ask that
chairmen of  the different sessions  address these questions in their
summar ies.



                           Samuel  Singer*


    The choice of bacterial candidates for control  of  aquatic  insect
pests remain the bacilli.   By now you are  all  aware of the  successes ac-
corded Bacillus fhuringiensis against agricultural  crop insect  pests  in
a terrestrial environment.  For the record of  B.  thuringiensis  one  need
only refer to the rash of  publications and recent conferences:   the re-
marks of Bourgerjon at the Atlanta conference  on  the "Safety of Biologi-
cal Agents for Arthropod Control" in Atlanta,  last  spring;  the  Fifth
International Colloquium on Insect Pathology and  Microbial  Control  at
Oxford this past fall; the National  Academy of Sciences report  "Mosquito
Control:  Some Perspectives for Developing Countries"  (2);  a symposium
on Microbial Insecticides  held last summer at  the Society of  Industrial
Microbiologists meeting (4).   It would be  more profitable to discuss the
newer bacilli candidates,  the most outstanding example of which is  Ba-
cillus sphaericus/SS11-1.   This strain,  according to the calculations of
Goldberg (11), will probably require as  little as 56.7 g of dry material
per acre of application.  This should make this bacterial control agent
truly competitive with chemicals.
    The work discussed here includes input from several people.   It has
been perhaps our naive assumption that the best source for  potential
bacterial candidates against specific insects  or  invertebrate targets
would be from natural  epizootics of these  invertebrates. To date we
have been successful in isolating B. thuringiensis  strains  active against
the Indian-meal moth (Plodia interpunatella) from B. thuringiensis  in-
fected Indian-meal moths.   Similarly we have been successful  in isolat-
ing bacilli active against Culex from Culex larvae  infected with these
bacilli.  Given time we feel  we could do the same for  Aedes, Anopheles,
biting flies and schistosome-bearing snails.

    When we propose a new biological control agent  factors  of efficacy,
safety, environmental  Impact and economic  applicability must be consid-
ered  (21).  In terms of microorganisms,  efficacy  includes  isolation,
identification and biological activity;  safety includes human,  mammalian
and phytological toxicity; environmental impact equates with  impact on
target and non-target invertebrates as well as microbiological/ecologi-
cal considerations; while economic applicability  denotes further devel-
opment in terms of economic,  commercial  and socio-political  considera-
    *Dept. of Biological Sciences, Western Illinois University,  Macomb,
111inois  61455.

tions.  Let us consider each of these four aspects of a new bacterial
insecticide, particularly in terms of the definitions expressed.


    Until recently, the choice of bacterial candidates for the control
of aquatic  insect pests has been primarily strains of Bao-illus fhwci-ngi-
ensis such as BA068.  Reeves and Garcia (19, 20), using BA068, reported
its successful use under laboratory conditions against three species of
Aedes, at a level of 106 spores/ml.  We have tested BA068 against our
laboratory culture of Culex pipiens var. quinquefasoiatus and found i t to
be inactive against these larvae.  However, we did find several type va-
rieties of B. thuringiensi-s active against our Culex larvae at a level
of 107 cells/ml.  Goldberg (personal communication) noted that B. thuvin-
giensis strain HD-1 (the commercial B. thuringiensis strain) as well as
its crystal,  is just as active as BA068 against several Aedes species,
but not against Culex.  Several groups, including one commercial group,
presently are investigating use of these and other B. tkur-ingiensis iso-
lates for control of mosquito  larvae,
    Recently, we  isolated two new groups of bacilli, with activity
against mosquito  larvae, from World Health Organization (WHO) material
(22)  (Table  1).  This material originated  in Delhi, India, from field
samples of dead mosquito larvae that succumbed to natural epizootics
caused by the bacilli.  In the past few years similar field material has
been  sent to  the World Health Organization International Reference Cen-
ter  (WHO/IRC) for diagnosis of Diseases of Vectors (under the direction
of Dr. John D. Briggs) for preliminary diagnosis.  These accessions show-
ing Bacillus  infections have been forwarded to us for further develop-
ment.  Table  2 lists many of the accessions that are available and in
process of  further  investigation.  Note that the material originates
from  countries forming a geographical arc from Korea in the western Pa-
cific to Zambia  in  East Africa and Nigeria in West Africa.

    The WHO/IRC material which forms the basis of our recent work origi-
nated in Delhi,  India.  One of these new bacterial candidates, B.
sphaericus/SS\I-1  (morphological group III bacilli, Table 3) has been
found to be  10,000 X more active than previously examined strains of
bacilli; as few as  102-103 cells/ml result in LD5o and LDgg effects
against C. pip-lens and C. tarsalis.  A second group of bacilli  (morpho-
logical  group II  bacilli) form a complex of strains belonging to the
B. alvei-B. ciraulans-B. brevis group and also have shown larvicidal
activity but  not  to the same degree.

    According to modern microbial physiology all of the bacterial insec-
ticidal  "toxins"  that have been found to date have the characteristics
of secondary metabolites (23, 24).  All are generated during early pre-
spore stages of sporulation  (after the cells have stopped dividing) and
all possess no obvious function  in cell growth.  The B. thuringiensis
crystal   is a  stable material  from a cellular point of view.   It persists
and is released at  the time that the spore is released.  The activity

 of the sphaericus toxin on the other hand, while formed during stages
of secondary metabolism, appears to peak in activity prior to mature
spore formation, with an eventual decay of one or two logs of activity.
The heat  labile soluble toxin produced by the group II bacilli also is
•generated during secondary metabolism, but does not usually appear to
decay once the spore  is formed.  These secondary metabolic aspects
bear heavily on the practical considerations involved in eventual field
application as well as in consideration of initial efficacy.
              FROM DELHI,  INDIA, ACCESSIONS #1321 (l-XV)
                      WITH  ORIGINAL KELLEN STRAIN

         Isolate                                         LD5o*

   B. sphaeriaus**/\1-1                           2.3 x 102 cells/ml

   B. sphaerious/IV  8-B                           2.4 x 103 cells/ml

   B. a£uei(aberrant)/lI 1-3                        1   x 106 cells/ml
   B. ci.rau.lans/alvei intermediate/V 1-12           1   x 106 cells/ml

   B. alvei/\l\\-6                                  1   x 106 cells/ml

   Original Kellen strain
     B. sphaeriaus/K.                              1   x 107 cells/ml

      *LDso = number of viable cells in population of original broth
   culture needed to kill $0% of test insect, Culex pipiens var.
     **A11 B. sphaerious cultures listed can be considered related to
   var. fusifown-is.

    The pathology of SSI 1-1 against Culex has been carried out by Dr.
Elizabeth Davidson of Arizona State University (6).  Briefly  (Table A),
there does not appear to be any gross tissue damage to the larvae at
cell concentrations  above the LDso level.  No bacterial cells have been
found outside of the peritrophic membrane.  Supernatants of the final
whole cultures of SSI 1-1 show no insecticidal activity, indicating that
the "toxin" is probably associated with the cell itself.  Evidence
points to the action of a toxin.  Examining gut populations of treated
cells she has found  populations of SSI 1-1 of the order of 105 cells/
larva with no apparent increase in number with time.    In fact there  is
a decrease in numbers if anything.  When bacterial populations were
treated with chloroform only a one log drop in insecticidal activity
occurred even while  the detectable viable cell count was reduced to
zero.  This chloroform treatment opens the possibility of using

chloroform-killed cells as a "chemical" insecticide.  We do not know
what the toxin sensitive site in the insect is, nor how death occurs.
We do know that death can come as quickly as 12 hr.
., ^ „ . , No. of Bacillus, Group
Vector material Accessions Morphological Group
The Phi 1 ippines
Austral ia
Cal ifornia
Culex fatigans
Vermicu) i te
Culex pipiens quinquefasaiatus
Culex pipiens quinquefasaiatus
Culex annulirostria
Culex pip-lens quinquefasaiatus
Culex fatigans
Aedes aegypti
Culex fatigans
Cattle hair scrapings and ticks
Ecological samples:
Anopheles spp.
Culex spp.
Aedes doTsalis
(1 6 II)
II & III, (II & III)
Bacillus), (II)
B. sphaerieus/\ \-\
B. thuringiensis
       *Parentheses denote presumptive.
    Table 3.  EFFICACY OF Bacillus sphaerieus/SSI 1-1:   MICROBIOLOGY
    Biogenesis of
    bio log ically
    act ive
Origin:    WHO/IRC      Access ion #1321/1 I      Delhi, India

Classification:   Bacillus sphaer-ious var.  fusiformis

                 Associated with cell  itself (heat labile).

                 No activity in supernatant of Final Whole Cultures,

                 Appears during secondary  stage of metabolism.

                 Chloroform treatment of  populations results in one
                 log drop in activity.

                 Most active when grown in synthetic merium.

                 Two logs less  activity when grown in non-synthetic
                 med i urn.

                 Active when grown in "fresh frozen" alfalfa
                 i nfus ion.


           Table 4.  EFFICACY OF Bacillus sphaeriaus/SSI 1-1:
                          BIOLOGICAL ACTIVITY

                      Biological Activity (LD50)*

     Culex pipiens var. quinquefasciatus         102-103 cells/ml

     Culex tarsalis                              102-103 cells/ml

     Aedes aegypti                                   106 cells/ml
     Anopheles spp.                                  106 cells/ml

     House fly                                   Inactive

     Plodi-a interpunctella                       Inactive
     Lasioderma serrioome                       Inactive
     Australorbis glabvatus                      Partially active


     No apparent gross tissue damage.

     No Bacillus found outside of peritrophic membrane,
     Gut population 105 cells/larva, no further increase, usually
     a decrease of gut population.

     Death as early as 12 hours,
     Most activity apparent by 2 days.
     For low population numbers, activity continues for 7 days.

     With low spore preparation, instars I-IV equally sensitive.
         *LDso = Number of cells/ml of bacterial population killing
     50% of test animals.
    Of the cultures that we have been working with, B.  sphaeriaus/SS\\-]
 is also closest to field trial.  Last summer this strain was tried at
 the Desplaines Mosquito Abatement District in Lyons, Illinois, as well
as in Kaduna, Nigeria.  It was also tested by Leonard Goldberg of the
University of California, Berkeley Naval Biomedical Research Labora-
tories.  Goldberg found SSI 1-1 to be effective in his laboratory
against Culex pipiens as well as against Culex tarsalis (11), at levels
comparable to those reported previously (22).  Dr. Calvin Alvarez of
the Desplaines Mosquito Abatement District tried SSI 1-1  against field-
collected Culex pipiens var. pipiens and found LDso values in the order
of lO3-^ cells/ml.  In the Kaduna test, SSI 1-1 was active against
Anopheles in the order of 106 cells/ml.  Our own most recent experience

shows SSI 1-1 to be active against Aedes aegypt-i in the order of 106
cells/ml.  SSI 1-1 is as active as our best B. thuringiensis strains
against Aedes and Anopheles and at least 10,000 X more active against
Culex species.
    To illustrate the rate at which B.  sphaerieus work has been going
recently, I  have just recently learned  from Goldberg that he has iso-
lated substrains of SSI 1-1 capable of high percent sporulation while
still retaining high insecticidal activity.  In terms of efficacy and
depending on available funding, we hope to examine our isolates from
WHO/IRC accessions in order to generate potential  field candidates
against Aedes, Anopheles, biting flies  and schistosome-bearing snails.


    Both national and international programs have been developed to
assay, monitor and evaluate the impact  of chemical insecticides.  The
World Health Organization has adopted a seven-stage evaluation of chemi-
cal insecticides necessary for a chemical product to be adopted for use
by WHO (1).   No comparable program exists in the international  area for
biological   (microbiological) agents although one is being prepared,  At
the national level strict federal regulations exist for chemical and
guidelines for new viral  agents (5).   However,  no guidelines exist for
safety testing or environmental impact  of bacterial control  agents (7),
although B.  thuringiensis has been registered for many food crops.
Safety testing of microbial agents, whether their ultimate fate is for
a terrestrial or an aquatic environment, would  obviously be the same.
Basically,  it will have to be shown that the insect pathogen cannot act
as a pathogen for man, other vertebrates or plants.  The differences
between use in a terrestrial or an aquatic environment will  bear on its
impact on the normal, non-target, invertebrate  and plant taxa,  at each
particular trophic level  or niche of the particular environment.  The
basic difference will be in terms of its environmental impact,  rather
than safety to man and his economically important plants and animals.
For instance, B. thuringi ens-is has been used mainly in terrestrial en-
vironments.   Safety information gathered should be equally valid if B.
thuringiensis were to be used in an aquatic habitat.  Information is
needed on its impact on the plant and animal taxa of the aquatic envi-
ronment where it will be used.  Unlike  a new class of chemical  insecti-
cide, where generally little or nothing is known of its toxicity,  a new
class of microbial insecticides usually concerns a known species.   Thus,
there is often some indication of its potential danger, at least its
potential virulence.

    The consequence of renewed interest in bacterial  agents, as well  as
the emergence of new groups of entomogenous bacilli with unsuspected
entomophilic properties,  hastens the need for acquiring information
concerning  both safety and impact of these agents on the aquatic envi-
ronment.   However, little information is available in terms  of  these
newer bacterial  candidates, but what data are available are encouraging.


The morphological group II and III bacilli, when compared with other
bacilli, are relatively inert metabolically and physiologically.  These
groups of bacilli are also very poorly understood from a taxonomic
sense.  In terms of specificity, we have found that with B.  sphaevious/
SSI 1-1, Culex is most sensitive, requiring in the order of 10 -103
cells/ml for an  LD5o-LD9o effect.  In contrast, Aedes and Anopheles are
less sensitive,  requiring on the order of 106 cells/ml for LD5o effects.
Recently we have found that the Indian-meal moth (Plodia interpunetella),
the cigarette beetle  (Lasioderma serriaome)  and the house fly appear to
be insensitive to this strain.  We find some indication that the schis-
tosome-bearing snail, Australorbis glabratus, may be partially sensitive.

    According to Gordon et al. (12), with the exception of the Anthrax
bacillus, members of  the genus Bacillus are generally considered to be
non-pathogenic for man.  A strain of B. sphaericus, however, was re-
ported by Farrar (8) as the causal agent of a fatal disseminated infec-
tion, but close  examination of this report shows that the "infected"
individual was in a highly traumatized state.  This is a case where
specificity becomes important.  There are as many strains of B. sphaeri-
ous which are inactive against mosquito larvae as there are active ones.
Similarly, assuming Farrar's correct identification of the causative
agent in his patient, this does not necessarily preclude the use of B.
sphaericus against aquatic insect pests, especially if adequate and
careful safety tests are done on prospective field test material.

    We have not as yet done any extensive mammalian toxicity studies of
our new bacterial candidates.  This requires a carefully thought-out
program by workers in several disciplines, which at the moment is beyond
our means.  However,  these studies are under consideration and will  be
instituted especially if new field trials are to be developed under WHO

                         ENVIRONMENTAL IMPACT

    In the area of environmental  impact, we have planned:  1) study of
the effect of these bacilli against isolated insect and invertebrate
target and non-target systems; 2) study of the fate of populations of
these bacilli in simulated and natural  aquatic environments.


    Two areas of impact become important when one considers the inverte-
brate taxa.   These are:  a) the effect of these new agents on inverte-
brates that are predators or are able to parasitize these vector and
pest targets; b) the effect of these new agents on invertebrates that
are non-vector, harmless, but important members of the aquatic ecosys-
tem.   One of the benefits of biological control is specificity, which
translates to mean that the natural predators and parasites are allowed
to contribute to the total control of the aquatic pest(s).   It is there-
fore appropriate to include in our examination the effect of these

                                 1 1

 control agents on  several  invertebrates  that may  eventually  become mem-
 bers  of a  biological control complex.
    Table  5  lists  non-pest and  pest organisms  selected as  representatives
 of  their respective  taxa  in fresh-water  systems.  Three  systems  in par-
 ticular are  noted.
    I I
 Parasites and Predators
 of Mosquito Larvae:
     Di ptera
     Di ptera
       Simu1i idae
                                    NON-TARGET AQUATIC ORGANISMS
                                                   Test  Group
                                        Dugesi,a  (Planar ia)
                                        Fresh water 01 igochaetes
crayf i sh
                                                         ^ Cyclops,
                                        Damselfly and dragonfly  numphs
                                        Mayfly  larvae
                                        Toxorynchi, tes
                                        Chironomid midges
                                        Honey bee

                                        Black f 1 ies
    Toxorynahites are  important potential biological control agents
since they are effective predators of a 11 mosquito  larvae, even members
of their own species.  Transmission of disease from prey to predator
was shown by Nolan et al.  (18) with Coelomomyces fungus infection of
Toxorynahites from its prey Aedes.
    Honey bees may be expected to contact B. sphaericus whi 1 e gathering
water from treated areas, especially in areas such as the southwestern
United States, where the supply of open water is limited and likely

includes mosquito habitats.  Testing of microbial agents, principally
B. thupingiensis against honey bees has been reviewed (3) and little
evidence of pathogenic!ty of B. thuringiensis against the bees is noted.
The effects of B. sphaerious against the honey bee should still  be
tested.  Methods are available (15, 16, 17) that have proven satisfac-
tory in testing  herbicides against bees.  An agreement has been reached
with USDA-ARS bee laboratory in Tucson, Arizona, for us to test B.
sphaeriaus at their facility.
    Among those  aquatic organisms  in North America classified as detri-
mental to man, the black fly ranks second only to the mosquito as a
pest.  Though black flies utilize  bacteria as food (19), few bacterial
pathogens have been reported (13,  ]k) and none has been extensively
tested as a control agent.  Therefore, we feel that preliminary testing
of B. sphaevious, as well as other select isolates, against black fly
larvae is worth  pursuing.

(B. spTzaerieus/SSI 1-1) IN SIMULATED

    Consideration of the impact of these bacterial candidates on inver-
tebrate taxa, whether  in isolated  systems or in nature, is obviously im-
portant, yet  insufficient to our needs.  Since we are dealing with a
two-component system,  invertebrate and bacillus, the fate of the micro-
organism, both in the wild and  in simulated ecosystems, bears a strong
influence on  the question of safety and environmental impact.  There-
fore, a well  planned study of the  fate of populations of the bacilli,
both in a natural as well as simulated ecosystem, becomes important.
With chemical there is the question of residue, with microorganism the
question is one of survival of the physiologically active component(s),
either the vegetative cell, the sporulating cell, the spore or some
product of one of these.  The strategy, of improving efficacy of appli-
cation of microbial material, rests on the keystone of controlling a
man-made epizootic.  Control hopefully means a minimum of reapplication.
It is ideally a  feedback situation with the controlling agent persisting
at some minimal   level during stages of low numbers of target inverte-
bates, and increasing sufficiently, due to "reinfection" of the target
host, when the numbers of the target host itself increases.  This ideal
or theoretical ecological balance  is yet to be demonstrated.

    As noted earlier, the microbiological side of the two-component sys-
tem,  invertebrate-bacillus, has in the past been neglected.  Survival
of the Bacillus  insecticide is comparable to residue of the chemical
insecticide and bears heavily on the eventual environmental impact.
Safety data always have been sought along with the effect of the insec-
ticide on non-target invertebrates.  But little has been done to deter-
mine the fate of the important factor—the microorganism itself.   This
does not diminish the  importance of these other considerations.

    Table 6 lists the approaches that we are interested in developing.
The basic data to be gathered in each case are simply bacterial


population counts, both total viable and total spore counts.  We are
basically  interested  in what happens to high-spored and low-spored popu-
lations, and what happens to these  insecticidal populations of SSI 1-1
over periods of time  in the presence of "native water."  By the latter
is meant water from the various natural mosquito habitats.  These could
be marshland, pond (both  intermittent as well as all year type), back
water, and water from urban habitats such as drainage ditches and sewer
lagoons.  For "simulated  standing systems" it would simply be observa-
tions of dosed native water in Pyrex dishes.  For more active water sys-
tems, double spinner flasks are possible.  The latter is a system of two
flasks each magnetically  stirred and separated by a semi permeable mem-
brane.  The wet-dry-wet system is projected as one wherein dosed native
water  is vacuum-dried, stored, and  reconstituted with native water, all
of which mimics a wet-dry-wet series of seasons,  "Factors affecting
population decline" implies study of the effect of bacterial flora,
found  in the native water, on the potential  insecticide.  The natural
ecosystems series is almost self-explanatory.  This is the non-simulated
natural system whereby we a) simply follow the fate of the population of
the  insecticide in natural ponds dosed with the bacillus and b) examine
the  effect of the bacillus on invertebrates  in the natural pond.
       Table 6.  THE FATE OF POPULATIONS OF Baoillus spbaeriousl

           I.   In Simulated Ecosystems (Native Water*)
              A. Standing (Pyrex dishes)
              B. Moving (double spinner flasks)
              C. Wet-dry-wet sequence
              D. Factors affecting population decline
                 1. Anti biosis plates
                 2. Double spinner flasks
          II.   In Natural Ecosystems
              A. Fate of SSI 1-1 populations  in natural ponds
              B. Effect of SSI 1-1 on target and non-target
                 invertebrates in natural ponds.
            "Native water = water from mosquito habitats.
    At this moment ecological studies similar to those just described
are being carried out as a result of the  Kaduna experiments.  We hope
to interface with these studies, thereby  providing  information of
trophic levels  in similar ecological circumstances  to provide baseline
data for supporting ecological  impact statements.

                        ECONOMIC APPLICABILITY

    At this time,  I would like to discuss mainly commercialization.  In
a recent letter from Abbott Laboratories I  have the following figures.
In the United States over 500,000 acres in agriculture are treated with
B. thuringiensis and another 25,000-50,000 acres in forests.  Total In-
dustrial production of B. thuringiensis exceeds one million pounds and
worldwide use increases this figure to perhaps two million pounds.  All
manufactured products are produced primarily by two companies, Abbott
and Sandoz, with Abbott the major portion,  and a third company, Nutri-
lite, producing a minor component.  Usage is expected to increase in
197**, especially since petrochemicals are in short supply.  To quote
Abbott's letter, "In summary, biological insecticides are not laboratory
curiosities but viable alternatives to chemical control agents.  They
can be produced commercially in quantities equivalent to chemicals and
fear of production inadequacies should not be a deterrent to use."

    Two companies, Nutrilite and Abbott, have shown an interest in ex-
amining SSI 1-1.  In a paper delivered before the California Mosquito
Association Meetings this February, Goldberg (11) tells of plans to
field test active spore preparations in Kerne County, California.

    Data presented here deal with preparations of SSI 1-1  that are neat,
that is, with none of the usual additives,  baits, etc., generally found
in final products of this nature.  Thus, much work will have to be done
in this regard.

    In dealing with impact of bacteria on aquatic environment I  have not
dealt extensively with safety in the sense of usual vertebrate toxicity.
This area needs a full treatment in a symposium-workshop concerned with
just this topic.  It requires carefully thought out input from bacteri-
ologists and insect pathologists as well as from mammalian pathologists,
toxicologists,  immunolegists and others.

    Guidelines for bacterial insecticides, whether aquatic or terres-
trial, will have to be developed.  In terms of safety guidelines for a
new product, the manufacturer will  have to bear the main burden of cost.
The same safety tests outlined for the B. thuringiensis registration
should play a role in development of these guidelines.  One interesting
paper with an excellent treatment on this aspect of toxicity, as well
as initial  field testing (10), reviews work with B, moritai as well as
several B.  thuringiensis strains.

    The effect of adjuvants needed for the special aquatic situation
also will undoubtedly influence these "guidelines."  These problems
will require careful  thought and planning,  What  I would like to empha-
size, however, are the problems concerning efficacy and environmental
impact.  We have been spoiled to some extent by the success of B. thu-
r-Lng-iensis in that we have neglected other bacilli as well as other


bacterial genera.  It has been too easy to stress development of very
active fully sporulated materials.  A major need is to develop "delivery"
systems that will permit field use of non-sporulated material.  In spite
of the current feeling, one can more quickly harvest the fruits of la-
boratory efforts such as ours, so that the time lag between isolation
and field use can be reduced.  A time will come when an active material,
such as B. popilliae, may not be able to be developed into a spored
    In terms of environmental impact, decay or fate of the Bacillus com-
ponent in field activity has been sadly neglected and requires further
thought and effort.  Also, fully developed international and national
programs, integrating examination of new microbial  agents in a logical
series of stages, needs further development.   In spite of the energy
shortage we still need to protect man and his environment.
    It is no longer a question of whether there will be bacterial  field
candidates, or even whether we will  be ready for them as they appear in
the next year(s).  If Goldberg's estimate of 56.7 g/acre for SSI 1-1
proves to be correct we have a bacterial agent that is competitive with
chemicals now.
                           LITERATURE CITED

 1. Anonymous.  1971.  "Vector Control."  WHO Chronicle 25(5):227-235.
 2. Anonymous.  1973.  Mosquito Control:  Some perspectives for develop-
        ing countries.  National Academy of Sciences, Washington, D.C,
        63 P.
 3. Bailey, L.  1971.  The safety of pest-insect pathogens for beneficial
        insects.  In H. D. Surges and N. W. Hussey (ed.), Microbial  Con-
        trol of Insects and Mites.  Academic Press, New York,   p, 491-505.
 4, Surges, H. D., and N. W. Hussey.  1971.  Microbial  Control of Insects
        and Mites.  Academic Press, New York.  871  p.

 5. Code of Federal Regulations.  1973.  Nuclear Polyhedrosis  Virus  of
        Hel-iothis zea; Exemption from the Requirement of a Tolerance.
        Vol. 38, No. 103, p. 14169.

 6. Davidson, E. W. , and S. Singer.  1973.  Pathogenesis of Bao-illus
        sphaericus  infections in mosquito larvae.   Fifth International
        Colloquium on  Insect Pathology and Microbial  Control.   Oxford,
        England .  p. 95-

 7. Engler, R.  1973.  Government regulation of microbial insecticides.
       Dev. in Industrial Microbiology, Vol. 15.   In press.

 8. Farrar, W. E., Jr.  1963.  Serious infections  due to "non-pathogenic"
       organisms of the genus Baoillus,  Amer. J.  Med. 34:134-141.

 9. Fredeen, F. J. H.  1964.  Bacteria as food for blackfly larvae in
        laboratory cultures and in natural streams,  Can. J,  Zool.


10. Fujiyoshi, N.  1973.  Studies on the utilization of spore-forming
        bacteria for the control of house flies and mosquitoes.  Re-
        search Report of the Seibu Chemical Industry Co. Ltd., Special
        Issue No. 1.

11. Goldberg, L. J., I. Ford, and S, Singer.  197**.  Bacillus sphaeri-
        aus var. fusiform-is as a potential pathogen against Culex tca>-
        salis and Culex pip-Lens,  AMCA-CMCA meetings, February 1974.
        In press.

12. Gordon, R. E., W. C. Haynes, and C. Hor-Nay Pang.  1973.  The Genus
        Bacillus.  Agriculture Handbook No. 427, Agr. Res.  Service.
        U.S. Dept. of Agriculture, Washington, D.C.

13- Jamnback, H.  1973-  Recent developments in control of  black flies.
        Ann. Rev. Entomol. 18:281-304.

14. Jenkins, D. W.   1964.  Pathogens, parasites and predators of medi-
        cally important arthropods.  Annotated list and bibliography.
        Bull. World  Health Organization, Vol.  30 (suppl.).

15. Moffett, J. 0.,  H. L. Morton, and R. H. Macdonald.  1972.  Toxicity
        of some herbicidal sprays to honey bees.  J. Econ.  Entomol.

16. Morton, H. L., J. 0. Moffett, and R, H. Macdonald.  1972.  Toxicity
        of herbicides to newly emerged honey bees.   Environ, Entomol.

17. Morton, H. L., and J. 0. Moffett.  1973.  Ovicidal and  larvicidal
        effects of certain herbicides on honey bees.  Environ. Entomol.

18. Nolan, R. A., M. Laird, H. C. Chapman, and F.  E. Glenn, Jr.  1973.
        A mosquito parasite from a mosquito predator.  J,  Invertebrate
        Pathol. 21:172-175.
19. Reeves, E. L., and C. Garcia.  1971.  Pathogenicity of  bicrystal-
        liferous Bacillus isolate of Aedes aegypti  and other Aedine
        mosquito larvae.  In Proc. Fourth International Colloquium on
        Insect Pathology.  College Park, Md.  August 1970.   p. 219-228.

20. Reeves, E. L., and C. Garcia.  1971.  Susceptibility of Aedes mos-
        quito larvae to certain crystal 1iferous Bacillus pathogens.
        Proc. Calif. Mosquito Control Assoc. 39:118-120.

21. Schneider, M. J.  1973.  Living witn pests:  New strategies in the
        war between men and insects.  The Sciences  (December), p. 22-28,

22. Singer, S.  1973.  Insecticidal activity of recent bacterial iso-
        lates and their toxins against mosquito larvae.  Nature
23. Singer, S.  1973.  Entomogenous bacilli against mosquito larvae.
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2k. Weinberg, E. D.  1970.  Biosynthesis of secondary metabolites:
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        Advances in Microbial Physiology, Vol. k.  Academic Press, New
        York.  p. 1-W.

C. M. IGNOFFO:   We are dealing with the aquatic  environment,  and  I
think it will  become evident quite soon that the advancements in  this
area are not as great as those that have occurred in the terrestrial
environment.  People have been working  with the  terrestrial  environment
in terms of biological control agents,  more specifically pathogens,  for
the last 25 years.  So they have had an opportunity to build  up a sig-
nificant amount of baseline data.

S. R. DUTKY:  One question, in regard to the numbers.   It sounds  simple
to say you need 103-102 to 106-107 per  mill Miter,  but there are  a  lot
of mill Miters  in an acre-foot of  water.  So the statement that the  or-
ganism does not develop in the host and kills it by magic otherwise  is
no advantage to the inoculation as compared with B.  popilliae in  a
closed environment.  Here you're using  a very small  dosage,  relatively
speaking, at intervals of high concentration, and thereby can use it  at
a level that will kill 50% of the  contact insects in a month.  Then  you
realign the build-up so that 10 years later you  go  back to the treated
areas and find  that the population of remaining  spores in the soil  is
sufficient to give 10% immediate infection.  The whole thing  builds
back up again quickly.  Fields of  that  type have been  under  test  since
1938, and they've been tested considerably since then.

JOHN BRIGGS:   It's appropriate as  we look at the theme of this meeting
to appreciate that we have two individuals who have devoted  an enormous
amount of time in matters of this  type.  One is  Dr.  A. A. Arata,  who  is
representing the World Health Organization and is an observer here,  and
the other is one of his predecessors, Dr.  Marshall  Laird, now at  Memo-
rial University of Newfoundland, who will  appear on the program later
this morning.  Both men contribute significantly in bringing  to bear
the efforts now  identified as consortia in the vector  management  busi-
ness and in the matter of impact of biological and  chemical  agents on
aquatic environments.  The consortia are made up of national  public  in-
stitutions, universities, federal  granting agencies, international  pub-
lic agencies,  like FAO and WHO, and commercial groups  throughout  the
world, frequently  identified as the fermentation industry or  industrial
groups interested  in propagation and formulation of biological  agents.
The consortia are evident, and I think  Dr. Singer's remarks were  perti-
nent in calling our attention to the activities  of  these consortia that
we already see  in work with B. sphaericus.  We will  hear from Dr.  Laird
later concerning some industrial groups and their activities  with inter-
national agencies  in Upper Volta,  Africa,  with respect to work on black


flies, a fine example of cooperative efforts, unprecedented in terms of
public health.

    Dr. Singer commented briefly about the review system for biological
agents.  This is analogous to the review system for chemical agents that
has been observed for a number of years by WHO.  I  trust that Dr. Arata
may have an opportunity to enlarge upon this subject later since it is
quite appropriate to the subject of this entire conference, not only on
the biological agents but on the chemical ones as well.  In brief,  we
are dealing with a 5~step review system that begins with laboratory re-
view, some presumptive safety examinations of biological agents, i.e.,
B. sphaericus, Lagenidium sp., Coelomomyces, and Metarrhisium.

    In conclusion, Dr. Singer, there was one point mentioned which  is
quite pertinent to the whole review system.  How does one positively
identify an agent and distinguish it from other agents?  A case in
point is the  reported pathogenicity of B. sphaeriaus to humans.  When
we attempt to identify or characterize something like B. sphaeriaus,  we
have already  heard this morning that there are some "strains" that  ex-
ceed by 10,000 X the activities of other "strains."  It may be beyond
the capacity of some groups to differentiate this strain difference by
appropriate bioassays.  Thus, we look at the conventional chemical, bio-
chemical characteristics of microorganisms and morphological charactei—
istics to identify species.

SINGER:  I'd  like to answer three things that were brought up,   One,  in
terms of safety, two, in terms of how you measure activity, and three,
the specific article by Farrar.  These groups of organisms, morphologi-
cal group 3 and morphological group 2, are very poorly understood and
characterized.  Now, what this all boils down to is, What is a  species
in procaryots (bacteria and viruses) compared with plants, animals,
fungi, etc., which are, generally speaking, eucaryots?  The concept of
species in procaryots is poor.  In the recent Sergey's Manual  identifi-
cation is to  species, genus, family, period.  What  most microbiologists,
or bacteriologists, consider as a species may be a  closely related
group of strains.  This is a spectrum of organisms  with similar or  com-
mon characteristics.  For example, in first dealing with B.  sphaer-leus,
we went to the American type culture collection and obtained B. sphaeri-
ous, B. sphaeriaus var. fusiformis, which is still  a B. sphaerious  (and
it is B. sphaevieus var. fusiformis that happens to hit the mosquitoes).
The point is  that we don't know too much about differences within this
one species.  This is not a question of species, for there are strains
that hit the mosquito and strains that won't.  Somebody might say,  "It
has a round terminal spore.  It's B. sphaeriaus."  Now, this fellow in
Farrar's paper, who has everything in the world wrong with him, is  said
to be infected with B.  sphaevieus.  This has not been proven,  and I
question whether there had been an adequate amount  of work done.

    The second thing is, how do you measure?  In working with a non-
obi igate parasite, it's easier to set up an in vitro test, in terms of
a huge number of insects per point or a screening type test like we do.
It's easy to measure in terms of numbers per mi 11 Miter.  When  you  go

into the field, with a product you want to use, it's measured  in terms
of amount of powder (or whatever the preparation is) per acre.  B.
sphaericus is only a year and a half old, thus, we have to have a model
with which to measure.  We've just chosen the convenience of organisms
per millilltei—to give it a reference, to measure or compare B. thur-
ingiensis (which we know a lot about) and B.  sphaericus.  Again, in
terms of safety, my clinical friends tell me (in terms of this point)
that bacteria as innocuous as B. subtilis can give galloping infections
in many hospitals.  Does this mean that B. subtilis is dangerous?  It
depends upon the strain we're working with.

IGNOFFO:  I  think the point  is valid.  There are many reports that get
into the literature and become expanded beyond  their original worth.
This may inhibit development of a lot of these microbiological  agents.

MARSHALL LAIRD:  Just a question on the matter  of safety testing.  The
honeybee and silk worm are well entrenched insects which one must look
at.  Bear in mind the kind of target areas that WHO, for example, might
wish to test a Bacillus thuring-Lens-is strain upon.   In Southeast Asia,
not  infrequently the larval habitat has Eiohhovnia cvassipes, or water
hyacinth.  The water hyacinth, in turn, is a harbor for mansonia mos-
quitoes that are filariasis vectors.  A certain amount of work is in
progress on the feasibility of using biological approaches in the inte-
grated control of water hyacinths.  One of the leading candidates is  a
lepidopteran and B.  thiafingiens'Ls is well known to hit a very wide range
of  lepidoptera.  I wonder whether that particular semi-aquatic moth
might be regarded as a worthwhile candidate for safety testing in evalu-
ating B. thuringiens-is strains.

SINGER:  If you recall, we tested B. sphaerieus against the Indian meal
moth, which is a lepidopteran  (and one we could rear).  It lacked ac-
tivity, even against beetles.  It's very obvious that you have to test
against various kinds of non-target hosts, which hasn't been done,  ex-
cept in these few cases.   It depends upon where in the world or in this
country you want to go, which determines the non-target material that  is
needed to test against.  For tests in these rice paddies, you will  have
to do this.   But this is part of the considerations WHO is making in
their scheme for testing biological  agents.

B. W. DAVIDSON:  I'd like to speak to Dr. Dutky's remarks on amounts
necessary for field testing.  Our preliminary work has shown that mos-
quito larvae are incredibly efficient at removing bacteria from the
water.  We found that very brief contacts, i.e., five minutes, with  in-
secticidal B. sphaericus was sufficient for them to pick up enough to
kill in LD98 amounts.  Hopefully, in the field situation this would  al-
low  the insecticide to drift past them, and they might  possibly pick up
enough.  It depends on how actively the  larvae are feeding and  if they
are  filter feeding with the  rapidity and efficiency with which Culex
quinquefasciatus appears to do  in the  laboratory.  The  second thing  is


the question on the honeybees.   It really depends on what part of the
world you're working  in.   In Arizona, for example, almost all open water
is honeybee water.  They visit every open area where they can possibly
find water.  Honeybees  in Arizona are extremely important because of the
citrus  industry.  Thus, any time you treat water in an area of the world
where water  is  in short supply, you're going to have honeybees picking
up the  insecticide.

A. A. ARATA:  For quite a number of years now, we've had the very in-
tensive program in screening of chemical insecticides.  As John Briggs
mentioned, this goes  through several different stages, with the whole
series  collaborating  with  laboratories around the world and a number of
WHO field  research units who conduct actual field trials of the insecti-
cides.  When  I  left Geneva several weeks ago, we had passed the 2000
mark in number of particular compounds that were examined in this period
of time.   Of these, the vast majority were rejected in very early stages.
A much  smaller number have gotten into field operations.  When I  took
over the responsibilities for biological control activities in WHO,  it
became  apparent that  we lacked any sort of coordination of this type in
the biological control field, not only for a particular class of  agents
or a particular class of vector species, but rather across the board.
Therefore, I've proposed an analogous scheme in five stages — it's only
tentative, I've discussed  it with various people.  From our standpoint
we see  that this  is a reasonable approach where, in the first stage, we
are concerned with identification and characterization of the organism,
its efficacy, and some  indication as to its production in reasonable
quantities.  The second stage, of course, would be mammalian infec-
tivity and, following some satisfactory results at this stage, we would
like to review the status of the organism.  Two WHO research units would
probably receive the  materials, the one in Kaduna, Nigeria, and the
other in Jakarta, Indonesia.  Here we have people concerned with  bio-
logical control.  In  addition, we have many entomologists and others
working on the whole  population dynamics of the target species.  So  we
know what's  in the area, we have very good backup for the entomological
assessment.  Following satisfactory results on very small field trials,
rice paddy plots or other situations as the environment demands,  we
would then consider going back into the laboratory, and concerning our-
selves with the non-target species in depth.  As has been brought out,
a satisfactory field  test  in Indonesia or one in Africa would present a
completely different  spectrum of non-target organisms.  Thus, we'd like
to find out what the  efficacy in the field is against the target  before
we concern ourselves  with this huge spectrum that we might have to deal
with.  Otherwise, we've got the whole aquatic biota of the world  with
which to be concerned.

    Following a limited field trial, should this prove satisfactory,
and then testing against non-target organisms with perhaps even more
detailed testing for mammalian and specifically human safety, then we
can consider  using the auspices of our WHO field units, and through
them conduct  larger-scale testing.  This is proposed in the particular
situation for Aedes,  Culicine Anopheles and, hopefully, in the future,


with black flies and even triatoma bugs.  Here we are examining the pos-
sibility of using insect parasites.

GEORGE ALLEN:  We should take into consideration that we have two pests
of the aquatic environment.  One developing rapidly from the interna-
tional standpoint in the last few years is aquatic weeds.  In a recent
meeting in India, UNESCO sponsored a program establishing the most im-
portant aquatic weeds in the world.  As Marshall Laird pointed out,
water hyacinth, Eiahhomia orassi-pes, is foremost.  One of the problems
we are working on in Florida is this tremendous problem of aquatic
weeds, on which we are spending about $5-5 million/yr.  We are looking
for many areas of biological control from two standpoints:  the sub-
merged weeds of what we call hydrilla, water milfoil, areas like this,
then we're talking about the floating, or the type Pistia or water let-
tuce and water hyacinth.  One of the major areas that we're putting a
lot of money into is importation and/or utilization of natural  enemies,
primarily  insects.  Dr. Laird mentioned one lepidoptera against water
hyacinth; we're looking at six, or potentially six, insects from Argen-
tina on water hyacinth alone.  One of the biggest problems that we've
been faced with, from a pathological standpoint, is that they have dis-
ease problems of their own.  One of the most promising insects we have
is a  lepidopteran on a water hyacinth.  But there's no way that Fred
Bennett, in Trinidad, can rear this insect for biological study because
of a microsporidan parasite.  We have found a lepidopteran that attacks
water hyacinth in Florida.   India is interested in bringing it into
their country, but one of our biggest problems is getting the insect
free of disease.  We've got to be careful  with any type of organism we
put into a new environment.

RETO ENGLER:  It has been brought out today that identification is quite
important for safety considerations.  I would like to add two things
about the  identification, namely, that since we are working with toxins,
it would be  important to find out what these toxins are, and their
chemical nature.  For instance, with Bacillus fhur-ingiensis two toxins,
the endotoxin and the exotoxin, have been described and could be used
for pest control.  Since one is proteinaceous and another is a nucleic
acid analog, we have to approach their safety considerations quite
d i fferently.

    Furthermore, if we are working with bacteria,  I believe it would
also be important to describe its antibiotic spectrum.  Thus, we will
have a means of interrupting any infection that should occur.

IGNOFFO:^ Thank you.  We should be careful of another pitfall, and that
is spending so much time trying to  identify a compound.   If that were a
restriction prior to use,  it might take as long as. 25 years to find out
what that protein complex  is.  In the meantime we've neglected a thor-
ough developmental phase which could result in a replacement for a more
toxic materia1.


                        Darrel1  W. Anthony*


    Virus diseases have been observed in  many invertebrate  species  in
aquatic and semi-aquatic habitats.  These include Baauloviicuses of  the
nuclear polyhedrosis (NPV) type,  cytoplasmic polyhedrosis viruses  (CPVs),
entomopoxviruses, Iridovivuses,  and other small  nonoccluded icosohedral
viruses.  This report reviews the literature on  laboratory  and field
studies concerning better known  virus diseases of aquatic  insects with
special emphasis given to viruses pathogenic to  mosquitoes.   Infectivity,
host range, specificity, and cross transmissibi1ity  are  discussed as  im-
portant factors in consideration of an entomopathogen  for potential field

    There are still insufficient research data on any  of the  viruses of
mosquitoes, or other aquatic insects, to  make definitive judgments  re-
garding their use as biocontrol  agents.  A nuclear polyhedrosis virus
affecting Aedes sollicitans shows promise for further  research.  Epizo-
otics of this virus have been observed in natural mosquito  breeding
areas, and laboratory studies indicate that relatively high infection
rates can be achieved.   There are no commercially available preparations
containing mosquito viruses, or  other invertebrate viruses  for control
of aquatic insect pests.  Furthermore, it is doubtful  that  such prepara-
tions can be developed within the next 5  years,


    The usefulness of virus diseases as an alternate method for control-
ling certain insect pests of crops and forests  has been  well  known  for
many years.  However, their effectiveness for management of aquatic  in-
sect pests has not been shown, thus insects such as  mosquitoes and
blackflies have been controlled  primarily by extensive use  of organic
insecticides.  Development of insecticide resistance in  some  insects
and danger of environmental pollution by  persistent  insecticides has re-
sulted in an intensive search for nonchemical methods  of controlling
these pests.  There are a large  number of viruses, protozoa,  fungi,  bac-
teria, and nematodes which may be useful  as biological control agents  in
management of mosquito populations (9).  The present paper  deals gener-
ally with virus diseases known to occur naturally in aquatic  inverte-
brates, with special emphasis to virus diseases  of mosquitoes, and  the
    *lnsects Affecting Man Research Laboratory,  Agr.  Res.  Serv.,  USDA,
Gainesville, Florida 32604.


factors that may affect their possible use as biocontrol  agents.
    Intensive study of insect viruses is a relatively young endeavor.
The earliest reports referring to virus diseases in aquatic insects were
from mosquitoes (U, 15, 18), however, the viral nature of these  appar-
ent infections was not confirmed.  The first virus  from an aquatic in-
sect was described by Xeros  (52)  when he reported an Iridovirus  (Tipula
iridescent virus [TIV]) from larvae of the cranefly, Tipula palidosa,^
Subsequently, a transmissible agent, believed to be a polyhedrosis virus,
was reported from the mosquito, Culex tarsalis (33).  Since that  time,
many virus diseases, including occluded and nonoccluded types,  have been
found  in a wide variety of aquatic invertebrates.  The types of virus^
diseases in aquatic invertebrates and some of the host groups from which
these were recorded are noted below.
       TABLE 1.   Virus  Diseases  of  Aquatic  Invertebrate Organisms

    Hosts          T .,   .     Baaulovirus   Reovirus  Entomopox-  Other
                  I*™™-™*   (NPV  types)  (CVP  types)    virus   viruses3

   Coleoptera                       +
   D i ptera
     Ceratopogonidae    +
     Chaoboridae        +
     Chironomidae      +                       +          +         +
     Culic idae          +            +           +                    +
     S imu1idae          +
     Tipulidae          +
   Tr ichoptera
     Limnephi1idae                 +
   Malacostraca         +            +

       a Includes  small  nonoccluded  icosohedral viruses, densonucleosis
 virus, and  the  tetragonal virus.
           classification and nomenclature used herein is that given by:
Wildy, P.  1971.  Classification and Nomenclature of Viruses.  Mono-
graphs in Virology No. 5-  81 p.  S. Karger, Basel, Munchen, Paris, Lon-
don, New York, Sidney.

In addition to the viruses in this tabulation, we have seen in our
studies viral infections in Ephemeridae, Odonata, and in several addi-
tional groups of Crustacea.   It is believed that further investigation
will show that viruses are, indeed, quite common in many species of
aquatic invertebrates.

    The virus diseases of aquatic  insects can be placed in two general
groups, occluded viruses and nonoccluded viruses.  The occluded viruses,
as  indicated fay the name, have their virions formed within proteinaceous
crystals, commonly referred to as  inclusion bodies or polyhedra.  These
crystals range in size from 0.5 to several micrometers and can be readily
recognized by phase microscopy or by conventional 1ight microscopy from
hematoxylin-stained preparations.  Three types of occluded viruses are
known from aquatic insects:  Baculoviruses (nuclear polyhedrosis viruses
or  NPVs), cytoplasmic polyhedrosis viruses of CPVs (possibly allied to
Reoviruses) and entomopoxviruses.  Only the NPVs and CPVs occur with
regularity in mosquitoes; however, an entomopoxvirus of mosquitoes is
reportedly being studied in France (5).

    The nonoccluded viruses include a large number of virus diseases in
which the virions are not enclosed within proteinaceous crystals.  Of
those occurring in aquatic insects, the Iridoviruses are the best known.
These viruses develop within the cytoplasm of infected cells and form
paracrystal1ine arrays which may, by Bragg reflection, impact a distinc-
tive  iridescence when viewed by reflected light  (35).  The iridescent
viruses (IVs) are the largest symmetrical viruses known, ranging from
125 to more than 200 nm  in diameter.  All that have been studied bio-
chemically have been shown to be DMA viruses.  The morphology, DNA con-
tent and the site of replication of iridescent viruses are similar in
many aspects to other nonoccluded viruses of fungi, reptiles, amphibians,
fish, and mammals.  Therefore, Stoltz (41) has suggested that they all
be  referred to as icosohedral cytoplasmic deoxyriboviruses (iCDVs).
                  IRIDESCENT VIRUSES  (GENUS Iridovirus)

    Mosquito  iridescent viruses  (MIV) were first reported in larvae of
Aedes taeniorhynehus from Florida  (13) and from larvae of A. annulipes
and A. cantons from Czechoslovakia  (48).  MIVs have now been reported
from  13 species of mosquitoes as follows:
Aedes annulipes
A.  aantans
A.  aantans
A,  detritus
A.  detritus
A.  dorsalis
A.  fulvus pattens
A.  stietiaus
A.  stimulans
A.  taeniorhynchus
A.  vexans
Czechoslovakia (48)
Great Britain (46)
Tunisia (47)
France (30)
USA (Nevada) (7)
USA (Louisiana) (7)
USA (Louisiana) (9)
USA (Connecticut)   (1)
USA (Florida) (13)
USA (Louisiana) (7)

                  Psopophora ferox     USA (Louisiana) (7)
                  P. confinnis         USA (Louisiana) (5)
                  P. horrida           USA (Louisiana) (9)
                  P. varipes           USA (Louisiana) (9)
An MIV from A. taeniorhynchus was reported which produced a blue irides-
cence in patently infected specimens (51).  This was designated as TMIV
(T for turquoise) and the original isolate as RMIV (R for regular) (39).
The two isolates were compared by electron microscopy and it was found
that the TMIV was a distinctly smaller virus, averaging 160-175 nm com-
pared to 185-195 nm for RMIV (2).
    Observations on field infections indicate that the incidence of MIV
in larval populations is very low, rarely exceeding one percent (1, 7).
Also, laboratory studies have shown that only a relatively small percent
of larvae exposed to MIV suspensions actually develop patent infections.
Infection rates  in  larvae were demonstrated as high as 33% in some ex-
periments, however, the average was only k,k% (37)-  The average level
of infection for 68 serial passages of RMIV in A.  taeniorhynchus was 16%,
although 60% infection was obtained in one test; for 30 passages of TMIV
the average infection rate was 21% (51).  Hembree (30, with the same
mosquito species, obtained only slightly more than 10% infection in most
of his tes.ts.  Transovarial  transmission has been demonstrated  by a num-
ber of workers (27, 37, 51).  However, since all patently infected larvae
usually die prior to pupation, only female larvae exposed to MIV during
the late instars develop to the adult stage and transmit the virus to
their progeny.  Although the percentage of females that transmit the
virus transovarially has been found to be 15% or less (27, 31), this
mode of transmission does provide a mechanism for perpetuation  of the
virus in nature.  Thus, a tentative cycle of natural  transmission has
been proposed (38).  The latter workers state that transovarial trans-
mission produces infected larvae which die in the fourth instar.  These
larvae provide a source of new infection when healthy larvae feed on
the diseased cadavers prior to pupation.  This in turn leads to the
presence of infected adults which complete the cycle by depositing in-
fected eggs.

    Larvae from MIV-infected eggs of A. taen-iovhynohus and Psorophora
ferox stored for 26 weeks became patently infected (51).   This  time in-
terval is believed sufficient for survival of the disease in nature
during long droughts and periods when the eggs are in diapause  (5).

    It is of interest that the 13 species of mosquitoes that are hosts
of MIV all belong to the floodwater genera Aedes and Psorophora which
have diapausing eggs.  MIV seldom if ever occurs in genera of mosquitoes
which lack diapausing eggs  (9).  All attempts to transmit MIVs  to non-
floodwater mosquitoes have failed.  For additional information  on MIVs
and other virus diseases of mosquitoes the reader is referred to the
excellent review of the subject by Federici (22).

    Since the first description of an  iridescent virus from T.  pal-idosa
(Diptera:  Tipulidae) (52), Iridoviruses now have been reported  from
several  other aquatic insects.  Larvae of Culiaoides ccdooyicula (Diptera:


Ceratopogonidae) in two tree holes were reported infected with an Irido-
virus (9); 50% of the larvae in 17 collections made from one tree hole
in about one year were infected.  Another iridescent virus was described
from larvae of Corethrella brakeleyi (Diptera: Chaoboridae) (6).  The
average infection level noted in field collections during that year was
36.1%, from a low of 0% in August to 70% in November.  A virus disease
was described in Ch-vponornus plwnosus which was obviously a member of the
Iridovirus group but which did not produce a characteristic iridescence
in the infected larvae (44).  We have observed similar virus diseases in
Goeldioh-Lvonomus holoprasinus and in Chironomus attenuatus from material
collected at Gainesville, Florida.  The only record of an Iridovirus in
black flies was reported in 1968 (49).  While the disease produced a
violet iridescence  in Simulitan opnatum (Diptera: Simuliidae),  the author
concluded that the disease in black flies is rare.  In our studies at
Gainesville we have observed iridescent types of viruses in several
groups of Crustacea including daphnids and amphipods.  However, we have
had no opportunity to study these diseases.

    Other nonoccluded viruses occurring in aquatic insects include small
icosohedral viruses, 50-70 nm in diameter, that have been observed in
Chironomids.  One of these viruses has been reported in G.  holoprasinus
(20).  The nerve, muscle, fat body, and gut were infected.

    A densonucleosis virus has been described from a laboratory colony
of Aedes aegypti, (36).  Neither this or other similar viruses  have been
reported in naturally occurring mosquito populations.

    The virus disease first observed in larvae of Culex tarsalis was
thought to be a "possible polyhedrosis virus" because of the tetragonal-
shaped crystals observed in infected cells (33, 3*0.  Ultrastructure
studies at our laboratory and more recent studies (43) indicate that the
tetragonal crystals observed by light microscopy are not proteinaceous
bodies containing virions as is the case with NPVs or CPVs.  Instead,
the electron micrographs show crystals composed of very small  particles
in a rectilinear array, which appear to develop in the nucleus of imagi-
nal disc cells and epidermal cells and then invade their cytoplasm.  Un-
equivocal proof of the viral nature of the particle is lacking (5).
However, the disease is transmissible, and similar types of inclusions
are reported in larvae of Aedes sierrensis and Anopheles freeborni (33).
This disease was also reported in larvae of Culex salinarius from Lou-
isiana, and in larvae of Anopheles crucians from the same area (8).


    The virions of the nuclear polyhedrosis viruses (NPVs)  are rod
shaped, 200-400 nm  in length by 68-80 nm in diameter, and are occluded
in proteinaceous inclusion bodies of varying sizes and shapes.  All
NPVs that have been studied from the biochemical and biophysical stand-
point have been shown to be DNA viruses.   In mosquitoes, infections are
confined to the epithelial  cells of the midgut and gastric caecae.  The
disease can usually be recognized by examination with a dissecting


microscope.  When infected larvae are examined against a black back-
ground, greatly hypertrophied, white nuclei can be observed through the
cuticle of the living larva.  As the infection progresses, the entire
midgut and gastric caecae may become white and hypertrophied.
    Since the first confirmation of an NPV infection in mosquitoes which
was found in Aedes sollioitans in Louisiana (11), NPV infections have
been found in field-collected larvae of several additional species.

       Aedes taeniorhynchus             Louisiana (5)
       A. triseriatus                   Louisiana (5)
       Anopheles crucians               Lou i s i a na (12)
       Culex pipiens quinquefasoiatus   Louisiana (5)
       C. salinapius                    Louisiana (12)
       Psorophara oonfinnis             Louisiana (12)
       P. ferox                         Louisiana (5)
       Uranotaenia sappharina           Florida (Hazard, personal com-
                                                 mun ication)
       Wyeomyia smithii                 Massachusetts (28)
In most  instances, the nature of these NPV infections has been confirmed
by electron microscopy.
    NPV from A. sollioitans could reportedly  be transmitted perorally,
and the disease was shown to be highly lethal   (11).   Laboratory trans-
mission tests yielded infections in only about 5% of the exposed larvae.
A naturally occurring viral epizootic in larvae of A, sollioitans was
recorded  in southwestern Louisiana (12); 70%  of the population was in-
fected.  This epizootic  involved a CPV as well as an NPV virus.  Addi-
tional epizootics approaching 1Q% infection levels were reported (8).
Laboratory studies showed that the NPV from A. sollioitans was transmis-
sible to A. triseriatus  and studies on the pathology of this virus in A.
tpiseriatus have been reported (23, 24).  An  NPV infection larvae of the
pitcher plant mosquito,  Wyeomyia smithii, has recently been reported
(28).  This virus is similar in size and appearance to other Baculo-
viruses of mosquitoes; however, the formation of polymorphic inclusion
bodies appears to be a unique feature.

    An NPV from a caddisfly, Neophylax sp., was described in which the
only external sign of disease was the transparent appearance of the in-
fected larva due to the  atrophied condition of the fat body (29).
Healthy larvae had a wel1-developed fat body  and were milky-white in
color.  Light and electron microscopy showed  the midgut to be the site
of infection, and the authors note that the virions were similar to
other NPVs.

                    (POSSIBLY  ALLIED TO Reoviruses)

    Diseases of mosquitoes caused by cytoplasmic polyhedrosis viruses or
CPVs have been recorded in at least 19 species within 9 genera (22).  In
the diseases that have been studied, these viruses also appear to attack
only the midgut and caecae of mosquitoes.

    The CPVs are occluded viruses that are icosohedral in shape, usually
ranging from 50 to 70 nm  in diameter.  The inclusion bodies may be small
and cuboidal in shape (3, 4,  16), or of varying size and irregularly-
shaped  (11, 12, 25).  The CPVs of mosquitoes are believed to be double-
stranded RNA as is the case with CPVs  in other insects,

    The CPV virus in Aedes sollic-Ltans can be recognized by the yel lowish-
white appearance of the posterior half of the affected midgut and gas-
tric caecae (10),  These workers further indicated that although the gut
cells become so pendulous that they burst under the slightest pressure,
heavily infected larvae usually were able to pupate and emerge as appar-
ently healthy adult mosquitoes.  Transovarial transmission of this virus
was noted in A. sollio-Ltans (12).  CPV  in adult A. taenioTi'hynahus which
survived patent infections as larvae was also observed (25).
    Observations have not shown a high  incidence of CPV infections in
the field; however, most mosquito species are hosts of a CPV (5), and
the infections usually are not lethal  to infected larvae.  An exception
to the observed low incidence of natural CPV infections has been re-
ported  (12).  This study showed a high  incidence of CPV associated with
a NPV infection in A, sollioitans in southwestern Louisiana.  It was
postulated that the CPV may have been a predisposing factor for the ex-
tremely high incidence and lethality of the NPV infection.

    Other aquatic insects known to be hosts of CPVs include several
species of Chironomidae.  A CPV was described from small  numbers of
third instar larvae of Chironomus plumosus during laboratory coloniza-
tion of this species  (43).  Two distinctly different CPVs have been de-
scribed from GoeldiohiTonomus holoprasinus (23) and from a Chivanomus
sp. collected from greenhouse basins at the USDA Insect Attractant, Be-
havior and Basic Biology Research Laboratory at Gainesville, Florida.
Recently another CPV, different from those described earlier, has been
seen in a Chironomus sp.  In all of these infections, the midguts of
the larvae appeared to be the primary area of infection.   In an "added
note of proof," the disease was reported found in a field-collected
fourth  instar larva from Lake Winnebago, Wisconsin (42).   These workers
also reported the existence of another CPV which was restricted to the
fat body.
    Patently infected larvae of G. toloprasinus with a CPV collected
from greenhouse basins usually died within 2k to 48 hr after collection
(23).   Laboratory studies showed that the virus could be transmitted
perorally, and infection rates as high as 40% were achieved.


    Entomopoxviruses have been recorded from several species of Lepidop-
tera and Coleoptera and from a single orthopteran (*»5).  However, the
only aquatic insects from which these viruses have been reported are
chironomids.  The following species are known to be hosts:

    Camptochironorms (chironomus)   Czechoslovakia (50)
    Chironomus luridus              Germany  (26)
    C. luridus                      Germany  (32)
    C. attenuatus                   USA (Texas) (k5)
    C. plumosus                     (A 5)
    Goeldichironomus holoprasinus   Florida  (22)
    Procladius sp.                  Florida  (Anthony, unpublished)

    The disease was studied in C. luridus and the fat body was reported
to be the primary site of infection (32).   The disease was described in
C.attenuatus as a hemocytic poxvirus (A5) .   At Gainesville, we have ex-
amined many infected specimens of G. holoprasinus, and it is also our
opinion that the hemocytes are the probable site of infection in this
species.  The virus is highly lethal to G.  holoprasinus.   The disease
becomes apparent in late instar larvae and  all patently infected speci-
mens  die before pupation.  The virus has been transmitted back to G.
holoprasinus perorally in the laboratory,  but in our tests, infectivity
level was low and quite variable.


    MIV has never been found in natural populations of A, sollioitans,
although larvae of this species are frequently found breeding with pat-
ently infected larvae of A. taeniorhynchus.   However, in the laboratory,
RMIV has been transmitted from A.  taeniorhynchus to larvae of A. sollioi-
tans and A. vexans (51).  All rates of transmission were very low (less
than \%) and much lower when the MIV was transmitted back to its normal
host.  These workers also transmitted the MIV from P. ferox to A. taeni-
orhynchus and A.  vexans and, as before, transmission rates were very low.
Additional  cross transmissions include:

             MIV from A. taeniorhynchus to A. nigromaculus
             MIV from P. ferox to P. varipes
             MIV from P. ferox to P. horrida
             MIV from P. horrida to P. confinnis
             MIV from P. aonfinnis to P. horrida

All rates of transmission were low  (Chapman, personal communication),
and all  attempts to transmit MIVs to non-floodwater mosquito species
have proven futile.


    Apparently  there  have  been  few attempts  to  transmit  the Iridoviruses
from mosquitoes  to other hosts.  Attempts at  the Gainesville  laboratory
to  transmit RMIV  to Eeliothis zea, Triehoplusia ni, and  Galleria mello-
nella  by feeding  hea'vy virus suspensions were unsuccessful.   Intrahemo-
celic  inoculation of  purified virus  into G. mellonella resulted  in death
to  all  larvae within  72 hr after  injection.   Electron microscope studies
of  the specimens  failed to show evidence of virus  replication.

    The successful transmission of the Chilo  iridescent  virus  (originally
from the lepidopteran, Chilo supressalis but  maintained  in G. mellonella)
to  13  species of  mosquitoes has been  reported  (25).  Even though trans-
mission levels  were generally low, it  is highly significant that species
of  Anopheles, Culex and Culiseta  (which were  refractive  to infection by
all MIVs)  became  infected  with  this virus.
    An iridescent virus from the grub, Serioesthis pruinosa (SIV) was
transmitted to Aedes  aegypti (17).  Two larvae  developed a blue irides-
cence  two  weeks after exposing  early  instar  larvae to media to which SIV
had been added.   It was concluded that the larvae were infected with SIV.
    Tipula  iridescent virus  (TIV) apparently has a wide host range
This virus  has  been experimentally  transmitted  (mostly by the  inocula-
tion of virus material)  to  12  species of Lepidoptera, 3 of Coleoptera,
and 7 species of Diptera.   The susceptible Diptera include:  4 species
of Tipula,  Bibio marci,  Calliphova  vomitora and a Myoetophila sp.  No
attempts to transmit this virus  to  mosquitoes are noted.

    The iridescent virus from  the chaoborid Covethrella brakeleyi is
cross-transmissible to C. appendiaulata; however, early instar mosquito
larvae of A, sollioitans , A. taeniorhynohus , and P. ferox were not sus-
ceptible to this virus  (6).
     In tests with the iridescent virus from the chironomid, G. holo-
prasinus , Hazard  (personal  communication) found that the virus was
transmissible back to larvae of G.  holoprasinus but not to ChLronomus
attenuates,  or to the mosquitoes, A. aegypti and Anopheles quadrimaeu-
    Transmission studies with  the tetragonal virus isolated from C.
salinarius  were reported  (10).   It  was found that this virus was trans-
missible to larvae of C. tarsalis,  but transmission trials with C.
pipiens quinquefasoiatus, C. peooator, Culiseta inomata, and A. taeni-
orhynohus were  negative.

    Because of  the limited amount of virus material available from the
isolated collections of  infected larvae, only a few transmission studies
have been conducted with the NPVs from mosquitoes.  The NPV from A.  sol-
lioitans has been transmitted  to larvae of A. triseriatus, A. aegypti,
A. nigromaoulus , A. tormentor, P. ferox, and P. vavipes (5).  Species
of Culex and Anopheles were not susceptible to this virus.  Hazard (per-
sonal communication) was unable to  transmit the NPV from Uranotaenia


sappharina to either A. aegypti or Anopheles quadrimaaulatus.  There
have been no tests with mosquito NPVs and nontarget organisms.


    Although CPVs have been recorded from at least 19 species of mosqui-
toes, there is a great lack of knowledge in regard to their infectivity
to other species of mosquitoes.  Nothing is known regarding their ability
to infect nontarget organisms.  The CPV from larvae of C, salinarius was
transmitted to larvae of C. territans and Culiseta inornata but not to A.
sollioitans (5).  A CPV from the larvae of A. sollioitans was transmitted
to A. taeniorhynchus and P. ferox, but this virus failed to infect larvae
of C. salinarius (12).  This may indicate that the two viruses are dis-
tinct.  Hazard (personal communication) found a dual  infection of NPV
and CPV in U. sappharina and successfully transmitted the CPV to A.
aegypti but not to Anopheles quadrimaaulatus.  As noted earlier, the NPV
did not infect either of the test species.
     Infection trials with CPVs from chironomids have shown that the CPV
from G. holoprasinus was not infective to larvae of C, attenuatus; simi-
 larly, the CPV from C, attenuatus was not infective to larvae of G, holo-
prasinus  (Hazard, personal communication),


    During 1972 and early 1973, we observed a mixed population of C. at-
 tenuatus and G. holoprasinus breeding in an aeration pond at the Univer-
 sity of Florida Sewage Treatment Plant at Gainesville, Florida.  C. at-
 tenuatus was by far the predominant species in this population, but the
 entomopoxvirus was found only  in G, holoprasinus.   Infectious levels in
 G. holoprasinus were usually quite low (\% or less), but the virus was
 found  in nearly every collection made over a period of several months.
The virus from G. holoprasinus was very similar in size, morphology, and
 tissue specificity  (20) when compared with the virus reported by Stoltz
and Summers  (45) in C. attenuatus.  However, it may be a different virus
as attempts to transmit it to C. attenuatus in the laboratory have never
been successful.  We also have recorded an entomopoxvirus from the larvae
of a Procladius sp. collected from the Waccassassa River, about 30 miles
west of Gainesville.  From the ultrastructural  standpoint, this virus
appears to be identical to that from G. holoprasinus.


    Laboratory experiments (37) showed that cadavers of A. taeniorhynchus
patently infected with MIV were a good source of infection to susceptible
larvae.  Field related studies (38) were then conducted to discover how
the virus was acquired and perpetuated in nature.   These workers con-
ducted several  types of experiments to (l) demonstrate infection rates
in natural  habitats after addition of infected  larvae or cadavers,


(2) study the infectivity of the virus after storage in artesian water
and 10% sea water, and (3) determine the persistence of the virus on sod
after varying periods of time.  The field studies were conducted by
placing the experimental  larvae (infected and noninfected) in large gal-
vanized cylinders sunk into the side of swales.  In some tests,  the ef-
fects of the virus were evaluated against both first and second  genera-
tion mosquito larvae.  The first generation larvae were those that de-
veloped patent infections after exposure to the virus.   Second genera-
tion larvae were the progeny of individuals exposed to the virus, but
did not become patently infected.

    Tests showed that live patently infected larvae (which would die
within 2k hr) or macerated larvae provided a source of  infection in the
field.  Infection levels ranged from a low of 1,k%, using an  inoculum
of 5 live infected larvae, to a high of 9-9%, using 100 macerated in-
fected larvae.  Comparisons were made of the infectivity of virus sus-
pensions stored in artesian water and 10% sea water to  first  and second
generation A. taeniorhynchus,  In all  cases infectivity rates in the
first generation decreased with the storage period of the virus.  This
was especially obvious with virus stored in artesian water, where infec-
tivity rates fell  from 15,6% to 2.5% in the first 2 days.  The virus re-
mained active longer in sea water, producing infection  after  20  days of
storage, whereas there was no infection in artesian water after  10 days.
In the second generation, infection rates were generally lower than in
the first, and again, the loss of infectivity occurred  sooner after ar-
tesian water storage.

    Studies on the persistence of the virus were conducted with  small
pieces of sod which had been sprinkled with a suspension of 5 macerated
infected larval cadavers in artesian water.  When the pieces  of  sod were
flooded and susceptible larvae added,  all  infectivity (to both first and
second generation mosquitoes) was lost after 2 days of  storage.   It was
indicated that the chance of larvae becoming infected in nature  by virus
liberated from the soil on flooding was very small.  Under natural  con-
ditions, the virus liberated into floodwater probably would become unin-
fective before the breeding area became dry.  The likelihood  of  its sur-
viving even a few days between successive flooding seems remote,

    Linley and Nielson (38) concluded  that in nature the greatest number
of individuals become infected through direct feeding of larvae  on ca-
davers or fragments thereof.  Larvae acquiring the virus during  their
early instars develop patent infections and die, thereby serving as a
source of virus for older larvae which may acquire the  disease in the
late instars and transmit the virus to their progeny, thus perpetuating
the cycle.

                          POLYHEDROSIS VIRUSES

    A viral  epizootic  involving  both a nuclear polyhedrosis virus and a
 cytoplasmic  polyhedrosis  virus was  studied  in natural  populations of A.
 sollicitans  in  southwestern  Louisiana  (12).  The affected population was
 followed  through  three periods of flooding, and  laboratory studies were
 conducted  to aid  in understanding of events that occurred in  the field.
 Infective  material also was  introduced into an area having a  very low
 infection  rate  to  determine  whether the rate of  infection could be in-
 creased .
     In  the study  it was  noted that a series of overlapping broods of A.
 sollieitans  may lead to a  significant buildup of infective material
 within  the habitat.  A rise  in the  infection level from 8.6%  to 70,8%
 was observed during a  10-day period when two successive broods of mos-
 quitoes occurred.  During  the following 9 days there was no rain and the
 soil  became  parched and cracked  because of  the intense heat and sunlight.
 Rain  brought forth another brood of mosquitoes, but at this time the in-
 fection  level never exceeded k%,

    Another  area was studied where  these virus diseases occurred at fre-
 quencies  of  less  than  0.1% of the population sampled.  The authors se-
 lected  a  pond (10  x 20 ft x  8 in.)  in this area and "seeded"  it with 5 g
 of freeze-dried infected  larvae  collected at the site of the  viral epi-
 zootic  described earlier.  After treatment, collections from  this pond
 showed  a  significant increase in infection  levels from less than 0,1% to
 16.3%.  The  virus  persisted  in the area through at least two  more broods
 at infection levels of about 5%, after which it dropped to its original
 incidence  of less  than 0.1%.

    An  evaluation  of the  separate roles of the two viruses was precluded
 by the  presence of an unknown number of double infections.  Laboratory
 tests showed that  the CPV was not usually fatal; however, patent NPV in-
 fections were invariably  fatal.  It was suggested that the CPV may pre-
 dispose the  larvae to NPV  infection and that the dual  infection may
 bring about  a synergistic effect.   It was also pointed out that oil pol-
 lution at  the site of the viral   epizootic may have acted as a stressing

     It is apparent from this review that we know very little about the
viruses that cause disease  in aquatic insects or in other aquatic in-
vertebrates.  Occurrence and host records have been compiled, and some
information has been accumulated on the pathology of a few of the
viruses.   In the case of the better known iridescent viruses, there is
data on their biochemical and biophysical characterization.  Although
host range data are available for some viruses, they are not complete,
and essentially nothing is  known in regard to total  ecology or the


possible effect of these viruses on nontarget organisms.  The studies
by Linley and Nielson (38), using sod that had been contaminated with
MIV, indicated no infective residue after a one-day storage period.
Similarly, the NPV/CPV epizootic in A. sollioitans diminished rapidly
after the habitat became dry (12).   Both studies suggest that the low
incidence of virus infection seen in nature is maintained by a cycle of
lateral (larva to larva) and transovarial transmission.  Other than  the
tests and observations cited previously, there have been no experiments
with viruses of aquatic insects to show their persistence in soil,  run-
off water, or in different types of water and habitats.
    More basic research is  needed  before definitive conclusions can be
made on the potential use of these viruses for control  of aquatic insect
pests.  For instance, production of sufficient quantities of virus ma-
terial for field experimentation, nontarget organism, and persistence
studies has been a major problem.  The science of insect virology is
still very young, and although we have found a few viruses in aquatic
insects, there probably are many more yet to be discovered.   It seems
possible, and quite probable, that a future undiscovered virus may  have
far greater potential for field use than any of those discussed in  this

    There is no commercially available preparation containing a  mosquito
virus, or any other invertebrate virus for use in control  of aquatic  in-
sect pests, and it is extremely doubtful that any preparations can be
developed within the next 5 years.  Research has not yet progressed to
the point where a particular virus can be selected as a promising  candi-
date for further development.  Of the virus diseases discussed here,  the
NPV from A. sollioitans appears to offer the greatest potential  for
future research.  This virus (1) can be propagated in the  laboratory
with relatively high infection rates, (2) is lethal  to all  patently in-
fected larvae, and (3) has been observed in epizootic proportions  in
field populations of mosquitoes.  However, a concentrated  effort of ex-
panded research is needed before this virus can be developed for use  as
an effective agent for control  of mosquitoes.

                           LITERATURE CITED

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T. B. CLARK:  First of all, with respect to the tetragonal  virus in mos-
quitoes, Culex tarsalis and Culex salinarius, I  recently found a similar
virus in Culiaoides.  This adds another family to your list of aquatic
viruses, and the syndrome in Culiooides is almost identical to that in
mosquitoes.  I  originally thought it was the same virus and tried unsuc-
cessfully to transmit it to Culex tarsalis, which is a susceptible host
to the tetragonal virus, and to Culex quinquefasaiatus and  Aedes dor-
salis.  I  also inoculated a group of flies and larvae of the wax moth
and navel  orange worm without success.
    The electron micrographs of that virus, however, indicate that the
viral particle is just about twice the diameter of the one  in mosquito.
There is no doubt in my mind that the one in the mosquito is a virus of
the same type.  Secondly, I would like to mention the reference to a
rectilinear array in the Culex tarsalis tetragonal virus crystal.  I
have some of Dr. Anthony's electron micrographs that show both hexagonal
array and rectilinear array and both also appear in Culiaoides virus.

    Our original observations on the nuclear polyhedrosis of A.  sollici-
tans were made in a Louisiana pasture situation in which the flooding
was dependent on natural phenomena such as rains, winds, and high tides.
Often, periods of several weeks would occur between rains,  the habitats
would get very dry, and the virus would apparently disappear.  The pas-
ture mosquito, Aedes nigvomaoulus, found to be susceptible  to that
virus,  is found  in  the San Joaquin valley of California which is almost
entirely cultivated by regulated irrigation.  This mosquito is undoubtedly
the most important  pest species in California and it is also, unfortu-
nately, the most resistant to insecticides.  We conducted a simulated
field study in k one-thousandth/acre ponds on the campus of Fresno State
University during the last half of July and through early August.  At
that time of year,  the normal daily maximum is about 101 F, and it
tapers off, dropping to 99 F through the first part of August.  We noted
a decline  in infection levels over three successive floodings of these
small ponds, each one separated by three days.  Mosquito abatement per-
sonnel wouldn't  let us allow any mosquitoes to emerge so we would fill
the ponds to about  k inches of water, add about 2,000 newly hatched
Aedes nigromaculus  larvae, and then add some virus material.  At that
temperature in the  San Joaquin valley we had pupae and emergence by k
days, thus there was very little time for a virus to infect.  On the
fourth day we pumped out the water and screened it to recover any re-
maining larvae or pupae.  We then crushed these, returned them to the
pond and allowed the pond to dry for 3 days.  We did this three times,
of which the typical example is that on the last day, the fourth day of
the first test, k2.2% of the larvae showed patent infection.  In the
next series of tests, the infection rate dropped 20%, that  is, without
adding any viral  material.  At the end of the second flooding, the in-
fection rate was 22% and at the end of the third flooding it was 23%.
The studies were terminated in late August, and the ponds not reflooded


until the following May.  At this time we conducted the same tests, ex-
cept that fresh viral material was not added.  The ponds were filled
with water and about 3,000 larvae were placed in each.  No viral infec-
tions were detected  in examination of most of those 3,000 larvae.  It
was concluded that survival of the virus over that length of time and in
that situation was very poor.

    We have also done some feeding experiments of v5rus-infected mos-
quito larvae to non-target species (none of this is in the literature);
some of these non-target organisms, such as dytiscid  and hydrophylid
beetles, mature Odonata, and dragonfly and damsel fly larvae, were par-
ticularly good predators.  We also tested Gambusia, our important mos-
quito control predator  in California.  To date,  we have found no evi-
dence of any infections or mortality resulting from these exposures.
The Gambusia were tested in 10-gallon aquaria, about  a dozen in each,
and fed infected mosquito larvae over several months  without evidence
that disease could be transmitted in that way.

    The real barrier to use of this virus in mosquitoes is the dosage
required.  The amount of virus required is really quite fantastic,  at
least with our present techniques of application.  Quite possibly,  this
could be improved significantly using other application methods or  pro-
tectants on the inclusion bodies.  There is a fairly  typical  dose re-
sponse of Aedes nigTomaoulus in terms of milligrams of polyhedral inclu-
sion bodies per liter of water.  Infection tests show that almost 10 mg
of inclusion bodies per liter of water are needed to  achieve close  to
100% mortality.  Let me break this down for you:  About 2% of the dry
weight of a patently infected or moribund fourth instar larva is com-
posed of inclusion bodies.  Thus, it takes 28 patently infected larvae
or moribund fourth instar larvae to produce one milligram of virus.   At
this figure, taking  10 mg--of course, this is infecting the larvae  as
third instars and harvesting the virus from the fourth instars (if  you
expose larvae in earlier instars, you can get much better results)--at
this dosage rate, according to my figures, it takes something like  12
pounds per acre to kill the larvae.  That's not exactly practical.

    This virus is one of the few organisms that I have worked with  over
a rather disappointing 13 years that has really done  anything to mos-
quitoes in measurable quantities.  Since the virus develops in the
field, we have produced, in ponds, a 100% mortality within a 2-day  pe-
riod, so the virus is lethal, if you could get it there in the right
quantities.  Perhaps with protectants, or some way of extending the life
of the viral particle and a better method of applying it so you don't
have to worry about environmental saturation, it's quite possible that
it could be useful  under some circumstances.

JOHN PASCHKE:  As far as our work with MIV is concerned, we're not
overly enthusiastic about its use as a biological control agent, and I
think that's been pointed out by Dr.  Chapman a number of times.  In con-
nection with what Dr. Anthony said regarding the propagation and produc-
tion, we have had success in getting infection in a number of tissue
culture cell lines using both R and T type MIV.   However, I'm not


encouraged about the production of the virus in the cells because it
does not appear to be an economical means of propagation.  We haven't
really scaled this up so I  don't have any concrete data — it does appear
that the tissue culture, at this point anyway, would not produce enough
virus to be practical.  Furthermore, if we allow these cell lines to go
for 2-1/2 days, the virus elutes from the cells and picks up a portion
of the plasma membrane of the cells in which the virus was produced,
and as nearly as we can tell, renders it uninfective to surrounding
cells.  Bioassays of the virus conducted on early instar mosquito lar-
vae also appear to give negative results.   I would assume that what we
are looking at  is a virion wrapped in plasma membrane from the host
cell, the adjacent cells recognize that as host cell and viral infection
doesn't take place.  We stripped the outer coat from the virus and tried
to  inoculate virus cores into these cells.  Apparently the chemical pro-
cess of removing the outer coat eliminates the infectivity of the core,
so  that method  doesn't  look like a practical approach to infecting tis-
sue culture eel Is.
     Insect virology has been aimed mainly at biological control of pest
species and  I  think many of us have felt that the insect viruses are
rather unique  in that they have this inclusion body that Dr.  Ignoffo
touched pn.   Incidentally, Dr. Ignoffo, you didn't talk about the nu-
cleic acid being different.  This uniqueness seems to be disappearing
rapidly, as  Dr. Couch has found a NPV in shrimp.  Earlier we thought
NPV's were quite unique, and that the inclusion body viruses  in general
were very unique.  Obviously they're not as unique as we used to think
they were.   The icosohedral deoxyriboviruses  (which include the irides-
cent viruses)  have been shown to infect a number of animals,  including
vertebrates  and plants.  We also have representatives from the Picorna
virus group, the Parvo virus group, and the pox viruses which have been
mentioned; it  seems these viruses are found in insects as well as in
other animals  and plants.  This dictates that we have a very thorough
biochemical/biophysical characterization, to substantiate species, or,
if  you prefer,  to separate one virus from another.  We need to care-
fully conduct  quantified studies in cross-transmission to other insect
species, but  in this  instance we are talking about the aquatic environ-
ment where there are many other species of animals which need to be
carefully scrutinized.  In some situations we may want to conduct com-
parative studies with other animal viruses.

C. M. IGNOFFO:  One comment:  I'm sure there  is probably no living or-
ganism, if enough search is made, that would not harbor a viral entity.
And as in many  of the bacteria, the morphological distinguishing charac-
teristics are  not that distinct.  Further clarification will probably
have to be done on a much lower level.   I'm sure you are aware of what's
going on in an attempt to make the nomenclature and classification or
taxonomy of viruses universal.  There are many different types of vi-
ruses placed  in the same groups—for instance, plant viruses,  amphibian
viruses,  avian viruses, mammalian viruses, and insect viruses.  These
are done initially on the basis of morphological  characteristics of the

virion and then further differentiation into its biochemistry.

H. C. CHAPMAN:  Just about everything has been said on the aquatic  vi-
ruses that we know.  But I  think we've got a long way to go,  for  we
know very little.  Hopefully, as Dr. Anthony has said, there  are  some
better viruses than the NPV's that we found and worked with.

    The virus in A. soll-io-itans seems to be about the best one  we have
and it's not too good.  We have seen at least three or four epizootics
in the field that have reached around 60-70%, which is pretty fair.  Of
course, in Louisiana that doesn't affect too many mosquitoes--the re-
maining ^0% will kill you.    In the last ten years we've learned a lot,
but with the few people we have and the little money that we  have work-
ing in this field, I  agree that probably it'll  be 5, 10,  or 15  years be-
fore we can come up with a good candidate, not necessarily in a virus,
but in biological control in general.

J. N. COUCH:  I am mainly concerned, being a member of EPA, with  aspects
of possible effects on non-target species.  However, as we've heard,
very little is known about impact on non-target species.   There are no
publications available of reports of testing.  I've searched  the  litera-
ture several times and spoken with many people in this area and very
little has been published about testing these viral agents.  I  hope that
data in people's files on tests that they've carried out on non-target
species would be made available.  These data, either positive or  nega-
tive, will be of great value to people in EPA.  Dr. Anthony's list  in
his first slide showed some of the viruses which have been found  in
aquatic organisms, particularly the insects.  I would like to add some
other published works that  may be of interest to people here.  In 1963
Dworky et al. published a paper in Nature describing a rod-shaped virus
isolated from a microanalid.  This was a very interesting relationship
in that the microanalid, when cultured in an aqueous phase, produced
tumor-like lesions.  Virus-like, rod-shaped particles were isolated
from these lesions only when the microanalid was cultured  in  an aqueous
phase.  These workers did not comment on the affinities of the  virus ex-
cept to say that in some respects it was similar to certain members of
the Baculoviruses, ordinarily known only as nuclear polyhedrosis  viruses.
Also, in 1972, Viriton in France reported a nonoccluded but rod-shaped
virus from the whirligig beetle.  This virus had many characteristics
of what we currently call Baculoviruses, but did not appear to  produce
mortality in infected specimens--at least none were observed.  His  con-
clusion was that perhaps this was an enzootic, or  latent  infection  that
rarely produced natural epizootics.  Recently, at  the Gulf Breeze labo-
ratory, we have found an apparent NPV  in pink shrimp, one of  the animals
we use in toxicity testing.  Occluded and free virus occur in the he-
patopancreatic cells of the  shrimp, and it was found during the course
of toxicity studies of chemicals using shrimp as a test animal.  You
will be able to visit my laboratory if you wish, and we have a  display
there of micrographs available to people who are interested  in  examin-
ing this  in greater detail.

    One future area that may pose some concern is the possibility of
interactions between pollutant chemicals or chemicals in the environment
and viruses.  We have found in preliminary tests that when pink shrimp
have been exposed to polychlorinated biphenyl Aroclor, apparently this
chemical stress may elevate the prevalence of the virus in sample test
populations.  Rockendorfer and Denton (1973). in their work on inverte-
brate pathology, reported that they had achieved a nine-fold increase in
virulence of a NPV by exposure of the virus to treatment with 3-methyl-
colanthrine, a carcinogenic drug.  We have environmental factors here
that have not been considered in great detail in the past but will  be of
concern in the future.  Two other factors on which there are data pub-
lished are the effects of temperature and salinity of the environment on
 IGNOFFO:  I  might direct one question to Dr.  Couch.   In this NPV found
 in pink shrimp, were you able to establish Koch's postulates, were you
able to transmit and recover?

COUCH:  We're working on this presently.  The virus  actually was only
found about  a year and a half ago.   It has been consistently refound in
wild shrimp  as well as in those exposed to the chemicals.

R. B. JAOJJES:  I'd like to ask, Dr.  Couch, do you know whether this
virus is closely related to the insect virus?  Do you know whether
there is any relationship between  this and the insect virus?

COUCH:  I  have no evidence that it's related  to any  of the known NPV's
of insects.   There appear to be some differences in  polyhedral body sub-
structure, etc., in this virus as  compared with some of the insect vi-
ruses.  However, I don't think this  is too surprising when you look in
a crustacean or some of the other  arthropods  (other  than insects)  to
find many of these virus groups.  As people become more interested, fur-
ther developments are anticipated.   Dr. Hazard and Dr.  Anthony have in-
dicated to me that they have found  viruses of other  types  in Crustacea,
some fresh-water.

E. I. HAZARD:  The viruses that we found in fresh-water crustaceans in
Florida are  of the icosohedral (ICDV)  type, in daphnids and copepods;
we've not  seen anything at all similar to CPV's or NPV's.

D. W. ROBERTS:  There are three types of rod-shaped  viruses that have
been reported.  The Baculoviruses,  the Pox viruses,  and the Rhabdo-
viruses.  The latter are rod-shaped  in the general sense that they are
bullet-shaped.  There are many reports in virology and  plant pathology
of filamentous long rod-shaped particles.  So the occurrence of a  rod
is not unqiue.  It's been reported  in the literature for a long time.
I think initial  reports were in plant virology.


A. M. HEIMPEL:  I'd like to say that the occurrence of a DMA-bearing rod
is unique, and it's unique in the viral kingdom.   It may not be specific
in lepidoptera to use that term "unique" in terms of differences between
viruses, and the NPV's are unique within the virus group.

ROBERTS:  The point I  wanted to speak to is specificity.  I've taken
some flak in the years I  have worked with the pox virus in insects be-
cause people find this a very loaded term.   I  would think that if you
wanted to expose yourself to a virus, this  group  would probably be one
of the safer ones, myxomatosis being an example of one of  these viruses.
At our institute in recent months, we have  been frustrated by a pox
virus we produce in large amounts.   It comes from an insect, and has
several characteristics in common with the  vertebrate pox  virus.  It has
the same enzyme complement, the general morphology, general  morphogene-
sis, general size, all these parameters except host range, that seem to
place it with the known pox viruses.  We would like to do  some basic
biological comparisons for known characteristics  of pox viruses.  All
pox viruses except one are known to reactivate any other pox viruses;
that is, if you kill one with heat, and then put  it with another pox
virus, the heat-killed one will be reactivated.  This seems  a logical
way to look for an insect pox virus, but so far results with this have
been negative.  We can't get the insect pox virus to reactivate any of
the vertebrate pox viruses, nor can we go the other way.  We find that
you don't get interference with these viruses either.  Using electron
microscopy and bioassays, tissue cultures and supernatants and this type
of thing, it seems apparent that the insect virus is recognized by the
two vertebrate lines that we've tried.  They are  simply not  taken up,
the cells reject them, almost completely.  Yet the virus is  covered with
a "halo," which goes into the inclusion.  It is apparently made as the
virion goes into the inclusion.  This is a  rather nondescript protein
which the cell doesn't recognize and virions,  although uncoated, get
within the cell.  Animal  pox viruses occur  in the aquatic  environment,
but they have not been tested very well at  this point.  The existence of
this type of virus in insects should have no adverse bearing on the con-
cept of using viruses in insect control.

WILLIAM UPHOLT:  I hear much talk about "morphologically similar" viruses
occurring in widely different organisms.  I  am disappointed  there hasn't
been more comment on the rate of mutation.   What  I'm not clear on is
whether organisms with so similar morphological characteristics might be
subject to mutation that might make them susceptible to or make them in-
fective to quite different organisms.  This seems to me to be a very
important question.

IGNOFFO:  It's very difficult to answer that question in terms of rate
of mutations.  We know these mutations exist in nature and of course we
are exposed to many viruses, not only plant viruses, even  in our every-
day intake, and even tobacco viruses, of course,  not necessarily through
inhaled smoke but certainly through contact.  We're continually exposed

to insect viruses and these things have occurred in nature for a long
time, so whether one evolved into another form which eventually evolved
into one which man or higher primates may be susceptible to is a diffi-
cult question to answer.  We do have techniques, though, that will in-
duce mutations.  And there is a possibility of setting up an experimen-
tal program to induce mutations and then follow this up with testing to
see  if the host spectrum has changed.  Accompanying this, you not only
can  induce for mutation and test for a host spectrum change, but also
go through a process of continuing selection.  In a group of 20 or 30
different forms, or what we might call a collection or an isolate, there
might be forms that we can select for by mutation.   Then, by actually
exposing the mutagens or other types of mutating substances, we can se-
lect for those in each time test and see if the host spectrum has
changed.  We did do one type of test with NPV's.  Actually,  Dr. George
Allen and I, in two separate laboratories,  tried to do the reverse.
Could we select for resistance in an NPV?  Now it's the other side of
the spectrum.  We took it through 25 generations, under pretty heavy
pressure, and at the 25th generation and all the way along,  we had no
difference from our non-selective strain of insects nor did  we have any
difference from wild populations tested.  We have to ask ourselves if
the question is meaningful, if it can be put to the experimental  ap-
proach.  And if it is, then we should do it.

J. E. ZAJIC:  In one of Dr. Anthony's slides, I  believe there was a
polyhedral  inclusion which was the example  he was describing and then
there looked to be some spherical inclusions above it.  So my question
is:  Is it common to have two types of inclusions in the same system,
do you encounter this very frequently?

ANTHONY:  That was probably a dual infection of NPV and CPV.  We also
have seen dual infections of an NPV and a CPV in Uranotaen-ia sappharina.
I  don't think that this really occurs too often; however, in the par-
ticular epizootic Dr. Clark investigated, dual  infections were quite
common.  In one pond near our laboratory, where Ed  Hazard found the NPV
and CPV in Uranotaenia, a number of the specimens examined also had dual
infections.  But I  cannot give you any specific answer as to how often
this occurs in nature.

IGNOFFO:  A little enlargement on that question, with insects other than
aquatic insects:  it does occur, it's not uncommon  to find them,  but
it's not the general picture.  Those weren't both DNA viruses, were

ANTHONY:  No.  This is perhaps one thing that I  should have  brought out.
All of the nuclear polyhedrosis viruses investigated are DNA viruses,
while those cytoplasmic polyhedrosis viruses investigated are RNA vi-
ruses.   Also, the so-called ICDV's, of which the iridescent  viruses are
a  group, are all DNA viruses, whether they  are from vertebrates or from
insects or plants.

R. J. MIGET:  There has been much discussion about various biological
controls and it had to deal mostly with mosquitoes, it seems, as pests.
Now, aside from the health hazard problem with mosquitoes, encephalitis
or whatever, which we don't have now in this country,  my question is:
Has anyone looked at the ecological role that mosquitoes play?  Is the
thought to go out and spray the boondocks in south Louisiana, or to in-
fect with a biological agent, or to infect,  say, urban areas that have
drainage problems?  And this gets into how far mosquitoes can migrate.
Do you want to wipe out the ones in the towns or in the country?  If you
wipe out the ones in the country by mass application,  then what do you
do to the ecology?

IGNOFFO:  That's a good question, one to which people  are addressing
themselves.  What will come in to fill  the void of the ecological  bal-
ance once it has been eliminated?  I'll ask Dr. Chapman to answer it
since he has the msot experience in this area.

CHAPMAN:  That is a good question, we hear this quite  a bit at Rotary
Clubs and various things along this line.  They ask, What good is a  mos-
quito?—that's the next question.  Then I tell them it's given me a
pretty-good livelihood for 25 years,  my wife appreciates it.  We are not
attempting to eliminate mosquitoes, really.   All we would like to do is
to reduce them to a tolerable level,  be it for man or  animals down in
our area.  We haven't been able to eradicate mosquitoes by chemical
means and we don't expect to eradicate them with biological means.  We
also would like to think that our attempts to reduce mosquito popula-
tions would be in areas where this would be necessary, in areas where
we have shown we cannot reduce them by source reduction, and approaches
like this.  Too often we rush in to control  mosquitoes and similar pests
using a chemical  or something along this line, where we could just as
well empty the tin cans, drain the ditches,  or use impounded areas.  We
have to be very careful that first we try to eliminate mosquitoes by all
naturalistic means and then approach  the problem with, hopefully,  some
sort of dual approach, maybe some sort of chemical  and biological  con-
trol agent.


                         Donald  W. Roberts**


    Entomogenous fungi cannot be offered  at this moment as  alternatives
to currently employed methods of mosquito control.   However,  there have
been some very promising discoveries concerning these fungi  throughout
the world in the last five years.  Few people are working  in  this  area,
thus development has been slow.  Enough information is available,  how-
ever, to reveal that some fungi have considerable potential,  both  in
terms of controlling mosquitoes and reducing synthetic pesticide pollu-
tion in the environment.


    The discussion will be restricted to  five fungal  genera,  ranging
from small water molds, Coelomomyces (Blastocladiales) and  Lagenidiwn
(Lagenidiales), through higher Phycomycetes, Entomophthora  (Entomoph-
thorales), to Fungi Imperfect!, Bea.uver'La and MeixtTrhisium  (both Moni-
1iales)-  Other fungal pathogens of mosquitoes have been reported,  but
these either show little promise for mosquito control  or are  insuffi-
ciently studied at present to be evaluated.  The older literature  has
been covered (27).  More recent publications treat  Pythium  sp.  (7),
CoelomyGi,diim sp. (60), Fusariwn oxysponm (2k), and  an unidentified
Deuteromycete (61).


    There are approximately kO species of Coelomomyees, some  of which
are, as yet, undescribed.

    Host Range.—A1 1 Coelomomyaes spp.  are aquatic  insect  pathogens, the
majority of which infect mosquitoes.  Other than one  species  reported
from Notonectidae (3), their hosts are dipterous insects:   Culicidae,
including all major genera (e.g., Anopheles, Aedes, and Culex)  (13),
Psychodidae  (33), Chironomidae (38, A8, Ag, 69, 70, 71), Simulidae  (19) and
Tabanidae (2, 28).  There is one report of the fungus in a  non-insect
    *Excerpted from a chapter in J.-P.  Bourassa (ed.),  Mosquito Control,
Univ. of Quebec Press (to be published  Summer 197*0.

   **Boyce Thompson Institute for Plant Research,  1086  North Broadway,
Yonkers, New York 10701.

host.  Four specimens of a crustacean, Daphn-ia, collected along with C.
indiana-\nfected Anopheles subpiatus larvae, were found to contain a
few sporangia of the same Coelomomyaes (23) but it was not specified
whether they were in the gut or hemocoel.   Some species are reported
from a rather wide range of mosquitoes, but the majority appear to be
restricted to one or a few mosquito species.

    Pistri\Mt\or\.--Coelomomyces-infected  insects have been collected
from al1 continents except Antarctica.  Distribution has been discussed
(13, 27).

    Life Cycle.— Irregular shaped hyphae without cell walls develop in
the hemocoel of  infected larvae.  Sporangia are produced within the
hyphae at their  tips.  Motile cells are produced and released from the
sporangia.  The  process of motile cell differentiation and release is
called dehiscence.  The motile cells have been called "zoospores" in the
literature, but  since their function is unknown (they could be gametes,
zygotes, or zoospores) it is more appropriate to refer to them as
"planonts"  (37).  The role of these planonts in disease induction is un-
known.  Exposing larvae to suspensions of planonts alone has not re-
sulted  in rapid  development of disease, even though this would be ex-
pected to occur  if planonts were the infective unit of Coelomomyaes spp.
The release of planonts, however, is essential  to disease development
since  in laboratory studies, sporangial preparations with low percentage
dehiscence  induced fewer infections than  ones with high dehiscence
levels  (B. A. Federici and D. W, Roberts,  unpublished).  Pulse-label
type experiments were conducted in which  larvae were held for 6-day in-
tervals in containers to which dehiscing  sporangia had been added.
When removed to  clean water, larvae which had been exposed during the
first 6 days did not develop infections,  whereas those exposed during
the second 6 days were infected (17).  Since it is improbable that
planonts could survive 6 days, these observations are interpreted as
indicating Coelomomyoes may have an as yet undescribed stage in its life
cycle, and this  stage itself is, or is the producer of, the infective
units of the fungus.

    Sporangial dehiscence of C. psorophorae from Aedes taeniorhynohus
and Psovophora howardii was enhanced by exposing sporangia to homoge-
nates of mosquitoes and certain amines and amino acids, including Tris
[tris(hydroxymethyl)aminomethane], methionine,  citrulline, and glycine
(53).   Indolebutyric acid somewhat stimulated dehiscence of C, psoro-
phorae from a non-f loodwater species, Culi,seta i-novnata (A2) ,

    Infection.--Attempts to obtain infections in the laboratory seldom
succeed.  Exceptions include C. punotatus and Anopheles quadrimaeulatus
(10, 11, 17), C. indicus in Anopheles gamb-Lae (36), C. psorophorae in
Culiseta inornata (Chapman, personal communication; 17; H. Whisler, per-
sonal  communication), C.  psorophorae from Aedes taeniorhynahus (17), C.
tasmaniensis (=opifexi) in Aedes austvalis  (k$), and Coelomomyoes sp.  in
Aedes atropalpus (5*0-  A common feature of all successful trials seems
to be the presence of soil  or algae.


    Coelomomyoes-infected larvae have been collected from all  typical
types of mosquito habitats, including lakes, ground pools, treeholes,
tidal pools, husk pits, cesspools, discarded tires, and other  discarded
receptacles.  Fresh, brackish, clear, muddy, and polluted water are in-
volved (^7).  The latter is particularly interesting in view of the ap-
parent fragility of the fungus.

    Artificial Culture.—None of the Coelomomyoes spp.  have been cul-
tured free of their hosts.  Limited growth, which terminated after 36
hr, has been obtained with mycelium dissected aseptically from infected
larvae and  incubated in mosquito tissue-culture medium (M. S.  Shapiro
and D. W. Roberts, unpublished).  Also, spherical pigmented bodies were
produced on mycelium held for several weeks in association with mosquito
tissue-culture cells, but separated from them by a membrane (Roberts,
unpublished).  If Coelomomyoes spp. have a saprophytic phase which pro-
duces infective units  (17), then artificial culture of this phase is
theoretically more simply accomplished than culturing of the parasitic
phase.   It  is possible, however, that the phase outside mosquitoes may
develop on  some other organism, such as algae.

    Microbial Control.—Coelomomyoes has several attributes which make
it promising as a control agent for mosquitoes, either with or without
the help of man.  First, although the level of disease in most popula-
tions is low  (less than 10% infected), very high levels have been noted
in some  instances  (5, 63), e.g., 35%  in Culiseta inornata, 35% in
Psorophora  howardii, 37%  in Aedes triseriatus, and 85% in Culex peooa-
tor  (H. C.  Chapman, personal communication).  Second, current informa-
tion  indicates considerably more host specificity than that present in
available chemical pesticides.  Third, there are four examples of suc-
cessful  introductions  into new sites.  In one of these, the Tokelau
Islands, a  fungus from one mosquito species, Aedes aldopiotus, was suc-
cessfully colonized  in another species, Aedes polynesiensis, and re-
mained active in the new  locality for at least seven years (31).  The
other three examples involved no change  in  host.  Anopheles gambiae
larvae were infected in a  previously disease-free pool three weeks after
introduction of sporangia  (Al).  Mortality  approached  100% in later gen-
erations of mosquitoes.   Introductions of sporangia and larvae into ar-
tificial pools in North Carolina produced an average of 60% infected
larvae  (11).  Prior  to drying at  the end of the growing season, a rice
field in Egypt was treated with sporangia from Anopheles phzroensis
(18).  Larvae were collected from July to September the following sea-
son and  3k% of those collected  in August were found to be  infected.
Three other fields were treated early  in the season.   In  two of these,
infection  levels reached  90%  in early August and then  receded to 0%.
The  third field had  no  infected larvae.

    The  third field  illustrates one of the  current disadvantages of
Coelomomyoes as agents for mosquito control, viz.  their  performance is
unpredictable.  This situation  presumably  stems  from the  superficiality
of our knowledge concerning the group.   Perhaps  the most  serious  lack
of  information is our  failure, more  than 50 years  after  the fungi were


first found, to elucidate their life cycle(s).  Uncertainties concern-
ing the infective unit, site of infection, and appropriate chemical en-
vironment for disease induction, make predictable results in laboratory
and field infection studies virtually unobtainable.  Many lines of in-
vestigation are impeded at present by our inability to culture the fun-
gus in vitro.  Although field observations indicate Coelomomyoes are
safe for vertebrates and most invertebrates,  documentation by laboratory
experiments is needed.  This can be accomplished best after the infec-
tive unit has been identified and can be produced aseptically in large
amounts in artificial culture.


    Lagenid-iwn giganteum was described in 1935 (8), but for many years
it was thought to hold little promise for mosquito control (9).   One
isolate which was obtained from culicine larvae in 1969 in North Caro-
lina, however, has demonstrated considerable  potential for use in mos-
quito control (35).  Although references have been made to it as L,
eulicidum (3^, 6*0, it is properly called L,  giganteum (12).

    Host Range.—The original description of  L.  g-iganteum was based on
material from copepods, Daphnia, and mosquito larvae.  Umphlett's iso-
late, however, in a series of laboratory tests has infected only mos-
quitoes (35).  Late fourth instar larvae, pupae, and  adults were not
susceptible, whereas early fourth instar and  younger  larvae succumbed.
The non-mosquito invertebrate organisms exposed  to zoospores included
Cyclops, Daphnia, Scapholebevis, several  additional unidentified species
of copepods and cladocerans, crayfish, polychaetes, dytiscids,  chirono-
mids, and snails.  The vertebrates tested were fish (Lebistes and Gam-
bus ia), birds (chickens and quail),  and rodents  (rats).  The mosquito
species tested were Aedes aegypti, Aedes triseriatus, Aedes mediovitta-
tus, Aedes taen-ioThynchus, Aedes soll'i-C'itans, C.  pip-Lens qui-nquefaso-ia-
tus, C. fatigans, C.  tarsalis, C.  nigripaIpus, Anopheles albimanus,
Anopheles qitadrimaculatus, Anopheles stephensi,  and Anopheles sundaious.
All except the anophelines were susceptible (3*0-   Higher levels of in-
oculum resulted in some infection of Anopheles quadrimaculatus,  but the
100% mortality routinely obtained with culicine  species was not  at-
tained  (65; P. Giebel and A. A. Domnas, personal  communication).   Field
studies with C.  restuans, C. tarsalis, Aedes  ni-gromaauli-s, and  Psoro-
phora sp. indicate these species are highly susceptible (35,  65).   Nine
isolates other than Umphlett's have been obtained  in  Couch's laboratory

    Pi str i bution.--Mosqui to-parasit izing isolates  of  L, giganteum have
been collected in North Carolina and  India (8, 12, 6k, 66).   The fungus
was collected in England using termite wings  as  bait, but this  isolate
was not tested for pathogenic!ty to mosquitoes (72).

    Microbial Control.—The attractive features of L.  giganteum include
its wide spectrum of host mosquitoes, rapid rate and high level of
lethality, ease of production on artificial media, and its ability to
persist in mosquito habitats.  The latter is implied by Umphlett's iso-
lation of L. gigantettm from the same site after a 6-year interval.  Dis-
advantages  include its virtual restriction, at least at reasonable dos-
ages, to culicine mosquitoes  (it is possible, however, that isolates
other than Umphlett's will be better adapted to attacking anophelines);
lack of tolerance for NaCl and organic pollutants; short-lived sporan-
gia and zoospores which make  storage for more than a few days impracti-
cal; and the incomplete evaluation of the safety of the organism for
nontarget organisms.  The latter is particularly true for the isolates
originally obtained from non-mosquito hosts.

    Two field tests have been conducted, both of which gave promising
results.  Culex restuans disappeared from an artificial  pool  in North
Carolina 6 days after introducing 120 Lagenidium-infected larvae (65).
An untreated control pool was not included in this test but the fact
that A3% of  the C. restuans collected 3 days after initiation of the
experiments were  infected indicates the drop in population was due to
L. giganteum.

    The second trials were conducted in California in the late summer
of 1972 (35) with two insecticide-resistant mosquitoes, Aedes nigro-
maculis and C.  tapsalis,  The population of the former was declining as
the test was initiated, but introduction of the sporangia produced in
one infected larva (a potential of approximately 250,000 zoospores)  per
square foot of water surface greatly increased the rate of decline over
untreated sites.  After 3 days, 75 larvae were collected in the single
control site and 0-3 larvae in the three test sites.  With C.  tarsalis,
the population was more stable and sampling was continued longer.
Three sites were chosen, one  high in dissolved solids at pH 10, one
with conditions resembling that of rice fields at pH 8, and one with
high chloride ion levels.  Infections occurred in the first and last
sites, but after an initial wave of up to 25% infected 3 days after in-
troduction of the fungus, the levels dropped to 1-9%.   In sites of the
second type, numbers of C. tarsalis collected gradually dropped to zero
over a period of 5 days.  The population was still zero, as compared
with 111 larvae in control sites, 17 days after fungus introduction.

    The fungus presumably survives periods of adverse conditions as zy-
gotes produced by fusion of antheridial and oogonial nuclei.   Germina-
tion of this relatively heavily walled body has not been observed  in
the laboratory.  Elucidation of the factors which induce their produc-
tion and germination  would make available a form of L.  giganteum
which could be shipped and stored for use in mosquito control  tests.
Cadavers, if stored in fresh  tap water at 15-6 C (60 F) before sporan-
gia began forming zoospores,  remained useful for induction of disease
for up to 1^ days (3^0 .  If cadavers are to be used in extensive field
tests, considerable improvement will  be needed in methods of storage.


    Entomophthora infections take place primarily in adult rather than
larval mosquitoes.  The only exception is E.  aquatica ,  which has been
collected only in Connecticut (1).  The levels of infection, particu-
larly in overwintering populations of adults, frequently approach 100%
and epizootics have been noted in some sites  over periods of several

    Host Range. --Entomophthora aquatica was found in infected larvae and
pupae of Aedes aanadensis and Cuiiseta morsitans (1).  Attempts to  in-
fect  larvae in the laboratory failed.  Attempts to infect C. pipiens
with an Entomophthora sp. from C. pipiens using field-collected infected
adults as inoculum gave the following results:  third instar and younger
larvae = 0% infected, fourth instar larvae =  25%, pupae = 63-88%, male
adults = 33-67%, and female adults = 65-100%  (22).   Adults of Anopheles
maaulipennis messeae, Anopheles maoul-ipennis  atroparvus, Aedes dorsalis
and Aedes aegypti were resistant to this fungus.  The several reports of
either E. oonglomerata , E.  destruens , or Entomophthora  sp. infecting C.
pipiens  in nature indicate the fungal isolates involved may be somewhat
species specific.  Cuiiseta annulata adults were found  less susceptible
than C.  pipiens collected in the same overwintering  sites (k,
    D istr ibut ion. — Entomophthora destruens occurs in Czechoslovakia,
France, and England (68) in cool (1-20 C), moist (Mt-100% R.H.)  C.
pipiens resting sites all  seasons of the year,  but was most prevalent
in winter months (kk) .   The Entomophthora sp,  in the Netherlands also
occurred in overwintering  adult populations (62).  On the other  hand,
Entomophthora sp. and E, oonglomerata infecting C.  pipiens adults asso-
ciated with sewage disposal and water filtration systems have been
found  in the USSR in the summer months (20, 26, 30).  E. aquatiaa-
infected larvae were collected from pools near  a single stream in Con-
necticut during the summer months (1).  The levels of infection  varied
extensively (0.8-80%) from site to site.

    Microbial  Control . — The high levels of infection noted in nature in-
dicate Entomophthora spp.  hold considerable promise for control  of adult
mosquitoes, particularly C. pipiens.  Nevertheless, artificial  introduc-
tions of these fungi into  new mosquito breeding or overwintering sites
apparently have not been attempted.  In addition to causing high mor-
tality, these fungi are attractive because they can remain active in a
site year after year (44).  It is possible that a single introduction
would be adequate to establish a fungus for many years.

    Production methods, particularly production and germination  of rest-
ing spores, need improvement.   Conidia are short-lived.  Infected adults
were no longer useful as inoculum 8 days after  the first conidia were
produced (22).  E.  destruens has been cultured  on coagulated egg yolk
and other media, but there is  no mention of the infectivity of this ma-
terial  (68).   E. aquatica  could be cultured only on coagulated egg yolk,
but grew abnormally (l).  Neither cultures nor  infected field-collected


specimens induced infection in the laboratory.

    The safety of Entomophthora spp. for vertebrates and other non-
target organisms needs verification.  Aside from C. ooronatus, no infec-
tions of vertebrates have been reported for insect-parasitizing Ento-
mophthorales; but experimental exposure in the laboratory has been car-
ried out only with resting spores of E. thaxteriana from aphids (R.
Soper and F. Holbrook, personal communication).  The observation that
E. destruens and Entomophthora sp. from C. pipiens did not attack other
mosquito species (21, 43) indicates these fungi are more host specific
than C. ooronatus.

     Infected adults have been collected in overwintering sites, in sew-
age systems, in water treatment systems, and on vegetation surrounding
reservoirs.  These sites would appear to be the most promising types of
mosquito habitats for artificial  introduction of Entomophthora spp.
Such introductions, however, should be preceded by basic research on
the requirements of the fungus to be introduced.  For example, even
though E. destruens induced 100% mortality of C. pip-lens in two over-
wintering sites, it was not effective in similar sites which had been
painted recently.  Also, it was effective on brick but not on plaster
(43, 44).


    Various  isolates of Beauveria bassiana and B. tenella (B. brongni-
artii  [14])  infect a wide spectrum of insects, but there is little in-
formation concerning their effects on mosquitoes (27),  Only one epizo-
otic in natural populations of mosquitoes had been reported, viz.  B.
tenella in larvae of the treehole mosquito, Aedes sierrensis (46,  57),
near San Francisco, California.

    Host Range.--An isolate of B.  bassiana obtained from a non-mosquito
host was tested for virulence against larvae of Anopheles albimanus,  C.
pipiens, C.  tarsalis, Aedes aegypti, and Aedes sierrensis (6).  The
first three  species were infected, but both Aedes species were not.
Adults of the same five species, plus Aedes nigromaeulis, were all
highly susceptible to conidia applied directly to the insects or to  en-
vironments into which adults emerged from pupae.  B.  tenella isolated
from larval Aedes sierrensis was tested against other species using
blastospores produced in submerged cultures (46).  The rate of kill  was
slowest with the native host, Aedes sierrensis,

    Microbial Control.--Three small-scale outdoor tests with conidia of
B. bassiana caused 82, 95, and 63% reductions of C. pipiens larvae and
pupae present after 2 weeks (6).  The dosage used was equivalent to
3 Ibs/acre.  Treeholes treated with 5 x 10* or 5 x 10s blastospores  of
B. tenella per mi 11iliter of water had reductions of 53 and T\%, re-
spectively,  in production of Aedes sierrensis adults  (46).  Fifteen
treeholes were treated with each dosage level and 15 served as un-
treated controls.  Up to 32% of larvae collected from some treeholes


in which natural infection occurred were found to be diseased.  Readings
of treated treeholes were terminated after 72 days, but it is possible
that the fungus will remain active in the treated sites in future sea-
sons.  Field tests of this fungus against more susceptible mosquito spe-
cies may show greater population reductions.

    As mentioned previously, adults of all species tested were highly
susceptible to B. bassiana conidia.  Since mosquito adults tend to rest
in habitats favorable to fungal infection, treatment of such sites with
conidia has been suggested as a promising area for research (6).  Aedes
nigromaculis adults, which rest on grass after emerging and between
blood meals, have been mentioned as a species particularly amenable to
treatment as adults.
    Mass production of conidia is a relatively simple matter since the
fungus can be grown on a wide variety of artificial media, including
sterilized vegetable matter such as bran (39, 40, 58).  Conidia cannot
be produced in liquid culture with present technology, but production
is possible in aerated semisolid media (E. Westall, personal  communica-
tion).  Blastospores, on the other hand, are produced only in liquid
media (55, 56) and presumably could be produced using existing large-
scale fermentation technology.  Boverin, a mixture of B,  bassiana conidia
and mycelium which is produced in Russia for control  of leaf-feeding in-
sects, had little effect on mosquito larvae in a laboratory test (15).

    Larvae were susceptible to B,  tenella blastospores at any time, but
were susceptible to floating B. bassiana conidia only during the first
24 hours following molts.  In addition,  Aedes, a large and important
genus, was not susceptible to conidia as larvae; and conidia had no re~-
sidual effect because they germinated in mosquito habitats even when not
in contact with larvae.   These limitations, along with the high dosages
needed, led to the conclusion that the use of B. bassiana conidia for
larval control is not promising (6).   Nevertheless, from the results of
preliminary experiments  formulation with oil may overcome some of the

    The most serious problem concerning  Beauvevia at present is a ques-
tion concerning its safety to vertebrates.  Sensitivity has been re-
ported by persons exposed repeatedly to  massive amounts of B.  bassiana
conidia preparations (actually finely milled whole cultures grown on
vegetable matter) (25).   The active agent, whether a toxin or antigen,
was not isolated.  The sensitivity, however, could be overcome by the
simple expedient of using long-sleeved shirts, gloves, and respirators
while handling the preparations.  Long-term rodent tests with B.  bas-
siana conidia proved negative  (C.  Rehnborg and E. Westall, personal
communication).  These tests included inhalation, subcutaneous injec-
tion, and oral toxicity.  Sensitivity to blastospores has not been re-
ported .

    Host range as far as nontarget aquatic invertebrates and verte-
brates also needs close scrutiny.   Beauveria is one of the most fre-
quently isolated entomogenous genera, has cosmopolitan distribution,
and an extremely wide insect host range.  Isolates with minimal effects


on nontarget insects should be sought for field studies.


    Most isolates of Metarrhizium are identified as M. anisopliae or M.
brunneian.  M. brunnewn, however, is probably a brown-spored form of the
green-spored species M. anisopliae  (32, 67; M. G. Tulloch, personal com-
munication).  Although M. anisopliae has a very wide  insect host range
(200 species, primarily subterranean coleopterous larvae), it has not
been isolated in nature from aquatic insects  (67).  Nevertheless, mos-
quito larvae proved very susceptible to conidia of one isolate (F84-1-1)
in laboratory tests and preliminary outdoor trials (50, 51; F. Murphey
and D. W. Roberts, unpublished).  The conidia of this species are ex-
ceptionally hydrophobic and, unless treated with a wetting agent, float
on the surface of water for days or weeks.  Except where specified
otherwise, the spores used  in tests discussed below were not treated,
and therefore were floating.

    Host Range.—The species tested included Anopheles stephensi,
Anopheles quadimaculatus, Anopheles albimanus, Aedes aegypti, Aedes
atropalpus, Aedes taeniorhynchus, Aedes sollioitans,  Culiseta inornata,
Culex pip-lens pipiens  (k strains), and Culex restuans (51).  All  were
susceptible, but Aedes aegypti was susceptible to submerged spores
    Some field-collected aquatic organisms were susceptible to conidia
in the laboratory  (Roberts, unpublished).  Interpretation of these ex-
periments  is uncertain because of high mortalities of some untreated
controls.  Adult gyrinid beetles and broad-shouldered water striders
which live at the surface of the water were very susceptible and
slightly susceptible, respectively.  Damselfly and mayfly naiads were
somewhat susceptible and dragonfly naiads, amphipods and snails were
not susceptible.  Guppies exposed to massive amounts of conidia for two
months were not affected.

    Microbial Control. — In  laboratory tests the F84-1-1 wild type of M.
anisopliae routinely induced 98 to 100% mortality in larvae of several
mosquito species.  Populations of late ins tar C. pipiens pipiens larvae
were severely reduced  in outdoor tests conducted in 1972 using small
containers  (0.25 meter2 surface, ~20 liters of water).  More recent
tests with 300 and 600 mg of conidia/m2 in small artificial ponds re-
duced C.  pipiens pipiens by 91% and Sk% within 3 days and with Aedes
sollioitans by 85% and 98%  (F. Murphey and D. W. Roberts, unpublished).

    All  tests were conducted with pure conidia.  It is presumed that
formulations could be devised which would permit reductions in dosage.
Since inactivation of conidia by direct sunlight has been noted in
laboratory studies, if possible, formulation should include compounds
to protect against ultraviolet irradiation.

    Mass  production of M.  anisopliae conidia on artificial media was


 first  reported  in  the  late  l880's  (29), and has been successfully accom-
 plished  by a  number of  researchers  (39).  The fungus will grow on very
 simple artificial  media,  including  ones such as Czapek-Dox medium which
 contains no organic nitrogen.  For  mass production, however, as a matter
 of  simplicity,  sterile  vegetable materials  (usually bran or rice kernels)
 are commonly  used.  The medium on which the conidia are produced affects
 their  virulence.   In comparisons between conidia produced on a starch
 medium with yeast  extract  (YpSs  [16]), a peptone medium with yeast ex-
 tract  (SDAY), and  an  inorganic nitrogen medium  (CDB), the YpSs-produced
 spores consistently killed  larvae approximately kQ% more quickly than
 spores from the other  two media  (Roberts, unpublished).  Spores from
 YpSs also had the  highest  initial viability and retained viability long-
 est on storage.  This,  however, did not account for the increased rate
 of  kill  since viabilities of spores from the different media were equal-
 ized prior to testing  by addition of appropriate amounts of autoclaved
 spores to the two  lots  with higher  viabilities.
     As with the other  fungi, safety data are meager,  M. anisopliae  is a
 cosmopolitan  species,  but no infections of warm-blooded animals have
 been reported.  Substituting filtrates for water for two weeks had no
 discernible effect on  young mice (Roberts, unpublished), and long-term
 tests  with rodents indicated no adverse effects (C. Rehnborg and E.
 Westall, personal  communication).   M. anisopliae is neither toxic nor
 pathogenic to rats (59)-  Rats fed  a mixture of fungus-contaminated and
 uncontaminated  food lost weight, and a few of the  specimens fed only
 contaminated food died.   It is possible that these effects resulted
 from severe depletion  in nutritional value of the  food by extensive
 fungal growth.  There are no reports of human sensitivity to M, anisop-

     Since conidia are not produced  on fungus-killed mosquito larvae, M.
 anisopliae will be used in mosquito control programs as synthetic insec-
 ticides  are now employed, viz.  introduced repeatedly, rather than being
 colonized in new sites  through single introductions.  Since production
 on  simple artificial  medium is possible, this organism could be useful
 as  a local product for  nations desiring to conserve foreign capital.

     It  is apparent that there is insufficient information to assure pre-
dictable results with entomogenous fungi for mosquito control.  Types of
information needed are the same as those specified for fungi used
against all types of insects (52).

    The fungi known to infect mosquitoes offer a broad range of possi-
bilities for use in mosquito control (Table 1).   Organisms amenable to
colonization into new sites include Coelomomyees spp. , Lagenidium gigan-
teum, Entomophthora spp., and Beauveria tenella.  Metarphiziwn anisop-
liae is best suited for use as synthetic pesticides are now applied,
viz. introduced in large amounts each time the mosquito population


becomes excessively large.   In addition to fungi known to be pathogenic
to mosquitoes, it is possible that fungi with good potential for mos-
quito control will be discovered (or rediscovered) in the future.  Two
recent examples of rediscovery are the observations that certain iso-
lates of L. giganteum (65) and M. anisopliae (50, 50 are extremely
virulent for mosquito larvae.  The most recent report of a new discov-
ery  is an unidentified  imperfect fungus in Australia (61).
        TABLE  1.  Possible Uses for Selected Entomogenous Fungi
                          in Mosquito Control
Fungus Mos<1uito
3 stage
Coelomomyses La rva e
Lagenidiwn Larvae
Entomophthora Larvae,
aquatiea pupae
Entomophthora Ad u 1 1 s
spp. and E.
E. destruens Adults
Beauveria Larvae
B. bassiana Adults

Metarrh-i.zi.wn Larvae
Mosqui to
Al 1 major
Cul icine
Al 1 major
Al 1 major
Al 1 major
Aquatic, including brackish water
with some Coelomomyoes species
Aquatic, not brackish nor high
organic pol lution
Water surface and vegetation sur-
rounding water works and sewage
Overwintering and dark, moist
resting sites
Aquatic, including treeholes
Resting sites of adults

Aquatic, including brackish and
possibly organically polluted water
    Entomogenous fungi, without the assistance of man, have been found
affecting substantial proportions of some mosquito populations.   This
suggests that manipulation, such as modification of sites or increasing
inoculum potential, would make fungi more efficient in mosquito  control.
While success in such ventures can be expected, they must await  the
availability of basic information to guide us in making intelligent use
of the strengths and recognizing the limitations of each fungus.


     Appreciation  is expressed  to Dr.  C.  J. Umphlett  of  Clemson Univer-
 sity,  Clemson,  South Carolina,  and  Dr.  D.  E.  Pinnockof the University
 of California at  Berkeley for  providing  copies  of  manuscripts  prior  to
 publication.  This  work was  supported  in part by U.S. Public Health  Ser-
 vice research grants R01  Al  07383 and  R01 Al  10010 from the National
 Institute of Allergy and  Infectious Diseases.
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        infection of Aedes austvalis (Erichson) larvae.  Hydrobiologia

46. Pinnock, D.  E., R. Garcia, and C. M. Cubbin.   1973.  Beauver-ia te-
        nella as a control  agent for mosquito larvae.  J, Invertebr.
        Pathol.  22:143-147.

47- Rajapaksa,  N.  1964.  Survey for Coelomomyces infections in mosquito
        larvae  in the south-west coastal belt of  Ceylon,   Bull. World
        Health  Organization 30:149-151.

48. Rasm, K.   1928.  Coelomomyaes chironomi n. sp.  Vest. VI  Sjezdu
        Cesk, pi-f rodozpytcu v Praze. 3:146.

49. Rasfn, K.   1929-  Coelomomyaes ahironomi n. sp., houba cizopasTcf v
        dutine  telnf larev chironoma.  Biol. Spisy Vys. skoly zverolek.,
        Brno. B, 8:1-13-
50. Roberts, D.  W.  1967-  Some effects of Metaprhizium anisopliae and
        its toxins on mosquito larvae,  p.  243-246.  In P. A. van der
        Laan (ed.), Insect Pathology and Microbial Control.   North-
        Holland Publ. Co.,  Amsterdam.

51. Roberts, D.  W.  1970.  Coelomomyaes, Entomophthora, Beauveria, and
        Metarrhisiwn as parasites of mosquitoes.  Entomol. Soc.  Amer.
        Misc. Pub. 7(0:140-154.

52. Roberts, D.  W.  1973-  Means for insect regulation:  Fungi.  Ann.
        N.Y. Acad. Sci. 217:76-84.

53. Roberts, D.  W., M. Shapiro, and R.  L.  Rodin.   1973.  Dehiscence of
        Coelomomyaes psorophorae sporangia from Aedes taeniorhynahus:
        Induction by amines and amino acids.  J.  Invertebr.  Pathol.

5k.  Romney, S.  V., M.  M.  Boreham,  and L.  T.  Nielsen.   1971.   Intergeneric
        transmission of Coelomomyces infections in the laboratory.  Proc.
        Utah Mosquito Abatement Assoc.  24:18-19-
55-  Samsinakova, A.  1964.   Sporengewinnung  von Beauvevia bassiana (Bals.)
        Vuill.  aus Submerskulturen.   Naturwissenschaften 55:121-122.

56.  Samsina'kova, A.  1966.   Growth and  sporulation of submersed cultures
        of the fungus Beauvefia bassiana  in  various media.   J.  Invertebr.
        Pathol. 8:395-400.
57.  Sanders, R. D.  1972.   Microbial mortality factors in Aedes sierren-
        sis populations.   Proc. California Mosquito Control  Assoc.
58.  Schaerffenberg, B.   1964.   Biological and  environmental  conditions
        for the development of mycoses  caused  by  Beauveria and  Metarrhi-
        zium.  J. Insect  Pathol.  6:8-20.
59.  Schaerffenberg, B.   1968.   Untersuchungen  u'ber die Wirkung  der In-
        sektentotenden Pilze Beauveria  bassiana (Bals.)  Vuill.  und
        Metarrhizium anisopliae (Metsch.) Sorok.  auf  Warmblutter.   Ento-
        mophaga 13:175-182.

60.  Shcherban,  Z. P.,  and  A. M. Go!'berg.   1971.   Pathogenic fungi Coelo-
        myoidiwn (Phycomycetes, Chytridiales)  and Coelomomyces  (Phycomy-
        cetes,  Blastocladiales) in Culex  and Aedes (Diptera, Culicidae)
        in Uzbekistan.  Med. Parazit. i Parazit.  Bolezni  40:110-111.

61.  Sweeney, A. W., D.  J.  Lee, C.  Panter, and  L.  W. Burgess.  1973.   A
        fungal  pathogen for mosquito larvae  with  potential as a micro-
        bial insecticide.   Search  4(8):344-345.
62.  Teernstra-Eeken, M. H., and A.  Engel.   1967.   Notes  on entomophthor-
        ous fungi on Heleomyzidae  and Culicidae (Diptera).   J,  Invertebr.
        Pathol, 9:431-432.

63.  Umphlett, C. J.  1969-   Infection levels of Coelomomyces punctatus,
        an aquatic fungus  parasite,  in  a  natural  population  of  the com-
        mon malaria mosquito,  Anopheles quadrimaculatus.   J. Invertebr.
        Pathol. 15:299-305.

64.  Umphlett, C. J., and  C. S. Huang.  1970.  Lagenidium culicium  as  an
        agent of biological control  of  mosquitoes. Bull. Assoc. of
        Southeastern Biol.  17:68.

65.  Umphlett, C. J., and  C. S. Huang.  1972.  Experimental  infection  of
        mosquito larvae by a species of the  aquatic fungus Lagenidium.
        J.  Invertebr.  Pathol.  20:326-331.

66.  Umphlett, C. J., and  E. M. McCray,  Jr.   1974.  A  brief review  of
        the involvements  of Lagenidium, an aquatic fungus, with arthro-
        pods.  U.S. Nat.  Marine Fish. Serv., Special  Sci.  Rept. Fish.
        (In press)

67-  Veen,  K. H.  1968.   Recherches sur  la maladie, due Metarrhizium ani-
        sopliae chez le criquet pelerin.  Meded.  Landbouwhogeschool
        Wageningen 68:1-77-


 68.  Weiser,  J.,  and  A.  Batko.   1966.   A  new parasite  of  Culex pipiens
         L.,  Entomophthora destruens  sp.  nov.  (Phycomycetes,  Entomoph-
         thoraceae).   Folia Parasitol,  13:144-149.

 69.  Weiser,  J.,  and  V.  J. E. McCauley.   1971.   Two  Coelomomyoes  infec-
         tions of Chironomidae  (Diptera)  larvae  in Marion Lake, British
         Columbia.   Can.  J. Zool.  49:65-68.

 70.  Weiser,  J.,  and  V.  J, E. McCauley.   1972.   Validation of a new
         species  and  a new variety of Coelomomyees  (Blastocladiales,
         Coelomomycetaceae) described from Marion Lake, British Columbia.
         Can. J.  Zool. 50:365.
 71.  Weiser,  J.,  and  J.  Vavra,   1964.   Zur verbreitung der Coelomomyaes-
         Pilze in Europaischen  insekten.  Z.  Tropenmed. Parasitol.

 72.  Willoughby,  L.  G.  1969-   Pure culture  studies  on the aquatic phy-
         comycete,  Lagen-idLwn gigant&m.  Trans. Brit, Mycol . Soc.

E. M. McCRAY:  Prior to 1969 our efforts at the Center for Disease Con-
trol in control of vectoi—borne diseases had been directed primarily
toward use of conventional pesticides to control  the primary vectors of
malaria, yellow fever, filariasis, encephalitis and Chagas1  disease.
Some work was being done on genetic manipulation, nuclear radiation,
and chemo-steri1ization.  To a lesser extent, the potential  of  patho-
gens and predators was being evaluated.   We were not seeking new orga-
nisms, per se, but were looking at what  was being done by other labora-
tories.  At that time, most of the microorganisms lacked  certain quali-
ties that we were  interested in.  The aquatic fungi appeared to be the
group which would most nearly fit our needs.  We talked with two men—
Dr. Couch, at Chapel Hill, who was working on Coelomomyoes,  and Dr.
Roberts, at Boyce Thompson Institute, who was working at  that time with
Metavrhizium.  We fully expected one or  both organisms to be ready for
field trials within a few years.  The isolation of Lagenidiwn in the
fall of 1969 by Dr. Clyde Umphlett was totally unexpected.  Dr. Roberts,
I  think, has covered the life cycle very well, so what is the current
status of our investigations?

    In January of this year, 3 culex larvae were sent to  us  from our
station in El Salvador for examination.   These larvae were infected
with Lagenidiwn, probably L.  giganteum.   This is the first report of
this particular organism in that area.  Unfortunately, the larvae were
preserved in Bouin's fixative and were of no value in establishing a
fungal  culture.  The investigators returned to the original  site to
seek additional infected larvae, but it  no longer existed.  Adjacent
streams will  be examined in the future.


    Work at our laboratory is primarily on the dormant or resting spore
of this organism to develop methods for accumulating and storing large
quantities of the infective material.  Dr. Umphlett, now at Clemson Uni-
versity, is working on a practical method of in vitro culture of the or-
ganism.  Dr. Domnas, at Chapel Hill, is working on biochemical relation-
ships between host and parasite.  One small field test was started sum-
mer before last in an area outside Savannah, Georgia.  The local mos-
quito control commission had a Culex breeding area that had been a con-
tinuous problem for them for about 12 years.  We introduced spores of
Lagen-Lditon to see if we could reduce the natural population.   For the
past 2 years we've been unable to find a single mosquito larva in this
site, which we would like to think was the result of our fungus.

    What about the effect of this fungus on non-target organisms in the
environment?  The literature reports that the Lagenidiales and many of
the other aquatic fungi are parasites of a very large number  of aquatic
organisms, many considered vital in the food chain.   Obviously, a given
organism cannot possibly be screened against every single species in the
environment.  Two avenues of approach are readily apparent.

    One is to bring selected non-target organisms into the laboratory
for screening; the second is to take the organism into the field in a
carefully controlled environment and see what happens.  The latter ap-
proach will be necessary sooner or later.  Hopefully, some host parame-
ters will  be defined in the laboratory or by natural occurrences prior
to field introduction.

    There are two problems which we in the public health service feel
more research is urgently needed for:  (1) The development of a reason-
able set of guidelines for conducting laboratory tests with pathogens
on non-target organisms.  We have procedures for chemical  pesticides,
but not for pathogens.   Unfortunately, pathogens are not chemical com-
pounds which behave in certain clearly defined ways.  Each organism
will, of necessity, require unique approaches, but at least we should
be able to come up with some basic procedure or scheme which  will en-
able us to relate comparable tests.  We in the public health  service
want from those outside the public health service to offer >us such
guides.  (2) What is the natural occurrence and distribution  of the
more promising organisms and what are the environmental factors which
determine their presence or absence in any given site or area?  Within
the past few weeks, an agreement has been reached between CDC and Dr.
Washino at the University of California at Davis, to conduct  intensive
laboratory studies on the possible effect of Lagen-id-iim g-iganteum on
selected non-target organisms characteristic of rice fields.   With this
information, we should be able to reduce the unfavorable impact on the
envi ronment.

D. T. GIBSON:  Most of the safety tests that have been suggested today
have been directed toward infection of non-target organisms.   Are toxins
produced in the target organism?  As well as spores, perhaps  infected
mosquitoes also should have been given to the guppies to see  if there
was any toxic effect.


ROBERTS:  Some fungi are well known for their toxins, e.g. Aspergillus
flavus and aflatoxins.  This is an area we must examine in evaluating
fungi for insect control.  There are several problems.

     If a fungus makes toxins in vitro does this mean it is to be re-
jected out of hand for insect control?  Or is the way the fungus will
be used to be taken into consideration?  For example, the characteris-
tics of some entomogenous fungi are such that in insect habitats, un-
like artificial media where there are no other microbial competitors
for nutrients, there is virtually no saprophytic growth and, conse-
quently, no toxin production.  As mentioned by Dr.  Gibson, safety tests
should  include determination of whether amounts of  toxins present in
infected hosts are sufficient to be deleterious to predators or scaven-
gers.  The artificially disseminated units usually are spores.   Do these
spores contain significant amounts of toxins?  If not, and if the medium
on which the spores were produced is to be discarded, then toxins pro-
duced in vitro are pf little importance since they  will  be discarded
with the culture medium.  I  think all of us who work with fungi realize
we must solve these types of problems.  Lageni-dium and Coelomomyces
being lower fungi, a group not known to produce toxins,  probably will
require less stringent examination for problems from toxins than will
the higher fungi .

MARSHALL LAIRD:  Dr. Roberts' point is well taken about the obvious
need to understand the mechanism of infection in Coelomomyces as a
basis for studying effect on non-target organisms,  but perhaps  there's
another aspect of safety that one shouldn't lose sight of.  For example,
workers using DDT in the old days were supposed to  wear  face masks, and
I've seen teams working under extremely hot conditions in Pakistan
where the man applying the chemical or mixing it up had  a very  large
beard and a turban.  Wearing a face mask was grossly uncomfortable and
if the boss wasn't watching, he wouldn't wear it.  One day, when the
same gentleman or a successor is handling 100-lb sacks of Coelomomyces
sporangia, one wonders whether he'll be at risk in  terms of inhalation
and possible lung infection on the same basis.

S. R. DUTKY:   I have tested Metarrhisium anisopliae and  Beauveria bas-
siana against housefly and Aedes aegypti adults, and both are very
highly susceptible to these fungi.  A second point  of information is in
reference to Entomophthora aoi>onata, now known as Conidiobolus  oorona-
tus.  We are studying the sterols of two fungi, C,  coronatus and E.
apiaulata.  These two have identically the same sterols.  Cholesterol
is the major sterol, and I  don't.know of any other  case where this is
true with fungi, except maybe one in South Africa.    It would be unusual
for two unrelated fungi to have the same sterol composition.   I won-
dered if you were going to throw E. apiaulata out,  too.

IGNOFFO:  May !  address myself to that?  I  think each organism that
shows potential, and this is going to vary  from time to time as we de-
cide which ones  are the best, will have to  be examined as to whether
they are safe using the experimental  approach.  We are not necessarily
restricted just  to use of spores.  In some  instances, we're going to
have to test possible metabolites produced  in the host once infection
takes place.  I  think what should be done is  put the idea to the scien-
tific method to  determine its potential hazards and monitor it if neces-
sary to determine if it will retain its original specificity.   Because
some materials have wide ranges of specificity does not necessarily ex-
clude them from  consideration as potential  microbial insecticides.  It
makes us aware of a possible problem area,  but that should be corrobo-
rated or, in fact, refuted by direct experimentation.

JOHN BRIGGS:  This is a question to Dr. Singer.  We have a euphemism in
the western world for toxins from bacteria  and fungi, and the word is
"antibiotics."  There is a great need to be concerned about the sensi-
tivity not only  to particu late materials like  sporangia of Coelomomyees
but also to the  products of these materials.   We are all aware that the
risk-benefit ratio we exercise when we submit ourselves to antibiotic
treatment is a risk-benefit ratio because some individuals have a sensi-
tivity to metabolic products of fungi and bacteria.  Since we are talk-
ing about impact on aquatic environments, aquatic environments sometimes
carry metabolites to places other than where  they were applied.  When
such substrates  are removed, they are transferred to sites where non-
target organisms may exist that were not considered initially.  I  was
wondering, Dr. Singer, as a microbiologist, is there substantial  evi-
dence for degradation of metabolic products of bacteria and fungi?  For
example,  is there degradation of antibiotics, as we know them, through
sewer systems and others of this sort?

SINGER:  You have to be very careful  when you speak of antibiosis, the
effect of one living entity on another, and antibiotics as delivered by
various  industrial outfits.  The earth's soil is the great degrader,
and most  things  will disappear like we will,  fortunately (or unfortu-
nately).  When we speak about toxins we have  to be specific.  When I
write on  the sphaerious "toxin,"  I put it in  quotes--!  don't want to
call it an endotoxin, because that confuses it with Salmonella and so
forth.  We have to be careful how we use words like ecology, toxin and

ALLEN LASKIN:   I take issue with your euphemism, that toxins from bac-
teria and fungi  are antibiotics.  Antibiotic  has a very specific defini-
tion.   If I remember correctly, Dr. Waksman's original  definition was
that "an antibiotic is a substance produced by one microorganism which
inhibits the growth of other microorganisms in very low doses."  I
think we can clearly differentiate toxins from antibiotics in  that con-


                      OF AQUATIC  INSECT PESTS

                           Edwin  I.  Hazard*

    The most important and promising protozoan  diseases of aquatic  in-
sect pests are members of the Microsporida  (Sporozoa:  Protozoa).  These
diseases result in high mortalities in host larvae  and cause noticeable
reductions in the fecundity and longevity of  individuals which survive
to sexual maturity.  Several species,  evaluated in  laboratory studies,
show promise for control of mosquitoes, and others  have been described
which may be useful in control of black flies,  chironomids, and semi-
aquatic tabanids.   Hundreds of additional species undoubtedly will be
found in these insect pests as research programs are expanded to meet
the increasing demands of environmentalists to  replace chemical insecti-
cides.  A Nosema disease of anopheline mosquitoes,  the subject of con-
centrated research by the U.S. Department of  Agriculture for several
years, will be tested against field populations of  Anopheles albimanus
Wiedemann this year in the Canal  Zone.  The research on this Nosema not
only has demonstrated its potential use for the control of anopheline
mosquitoes, but has also shown that it is harmless  to all nontarget or-
ganisms tested including a local  species of crayfish, fishes of the
genus Garribusia, fresh-water shrimp, and several aquatic entomophagous
insects.  Microsporida (Sporozoa: Protozoa) have been reported in pest
species of the families Ceratopogonidae, Culicidae, Simuliidae, and
Tabanidae  (a family that includes both aquatic  and  semi-aquatic species).
Indeed more than 100 species of mosquitoes  alone are known hosts of
Microsporida.  Several thorough reviews of  these diseases in medically
important  insects  are soon to be published, some of which reviews will
establish new genera for morphologically distinct types found only  in
certain aquatic animal groups.  For the present, the reader may consult
Chapman (3) and Weiser (15, 16).
                           PROMISING SPECIES

    In 1966, the U.S. Department of Agriculture, ARS,  Insects Affecting
Man Research Laboratory in Lake Charles,  Louisiana,  began  intensive
surveys of aquatic insect pests for microsporidan diseases.  Many
    ^Insects Affecting Man Research Laboratory, Agr.  Res,  Serv., USDA,
Gainesville, Florida 32604

aquatic insect species have been found to be hosts of these enzootic
diseases, and nearly all species of mosquitoes examined are hosts of
one or more microsporidans.  Many of these, particularly species of the
genus Thelohania, were found to cause congenital  diseases, but none ap-
peared to be contagious or transmitted per os.  Few species, therefore,
were considered as promising biological  control  agents for aquatic in-
sect pests.  Recent studies have brought attention to development of
these microsporidans in surviving females that pass the disease to their
progeny via the egg.  In these instances, Thelohania produce spores in
adult females unlike those found in male larvae killed by the disease.
These Thelohania would be useful control agents  because they are all
carried via the egg and produce up to 50% mortality in the progeny of
infected females (10).  In some Thelohania species, spores from adult
females appear to be binucleate and may  be infectious to larvae that in-
gest them; spores in larvae are uninucleate and  probably never produce
new infections in healthy larvae.  All previous  attempts to infect
healthy insects by using spores from larvae have failed to produce dis-
ease symptoms.  However, no serious attempts have been made to infect
healthy mosquitoes by feeding them spores from adult females.

    In contrast, the survey of aquatic insect pests in the tropical re-
gions of Africa and Central and South America have recently uncovered
many microsporidans that are transmitted per os,   Perhaps some micro-
sporidans have not developed transovarian sequences in tropical hosts,
as is commonly seen in hosts in temperate regions, since many of these
tropical hosts breed continuously throughout the year.  To date, most
of our research on tropical forms has been concerned with the taxonomy
and distribution of these parasites, descriptions of which are soon to
be published.

    In addition to these new tropical microsporidans, we have six other
species in aquatic insect pests that soon can be evaluated in field
tests, two species in tabanids and four  species  in culicids.  The two
microsporidans in tabanids are (a) Thelohania tdbani (6) from larvae of
Tabanus atratus Fabricius and (b) a new  undescribed species discovered
by Dr. Donald Harlan and his colleagues  at Stoneville, Mississippi  (per-
sonal communication).  Tabanids readily  became infected, resulting in
high mortalities when fed other diseased larvae  in laboratory studies;
however, a means of distributing these microsporidans in tabanid larval
ecosystems must be investigated before they can  be evaluated in field

    Four microsporidans that offer promise for control of mosquitoes
are:  Nosema algerae (12) in anophel ine  mosquitoes; Pleistophora ouliois
(1*0 in both Anopheles and Culex mosquitoes; a Stempellia species (soon
to be described) in Culex pip-Lens quinquefasaiatus Say; and another un-
described species of Stempellia, found in larvae of Uvanotaenia sapphi-
rina (Osteen-Sacken) in Florida and Louisiana (k), that also causes
pathologies in the larvae of Aedes aegypti (L.).

    The pathologies produced by N. algerae in anophel ines have been de-
scribed (2, 5, 7, 8, 12).  High mortality has occurred in most Anopheles


species exposed  to  spores  in  laboratory colonies and  in small experimen-
tal test  plots.  Perhaps more  important is the ability of N. algerae  to
reduce  longevity of adult  mosquitoes that survive from less severe  in-
fections  (5).  Also, malarial organisms do not develop well in females
infected  with  this Noserna  (9,  13).  Our laboratory  is, therefore, con-
ducting field  evaluation studies with N. algeicae against Anopheles albl-
manus Wiedemann, a  primary vector of malaria  in the Canal Zone.  Large
test plots  (approximately  1.2  ha [0.5 acre]) will be  sprayed with IxlO6
spores/900  cm2 [1 ft2]).   This concentration of spores usually produces
10-20%  infection in larvae in  the laboratory.  Smaller plots will be
sprayed with 2 x 107 and 2 x  108 spores/900 cm2 to determine whether
higher  rates of  infection  and mortality can be produced.  Data from
these tests should  be available  in the summer of 197^.
    Canning (1)  studied the effects of P.  eullcls on Anopheles gambiae
Giles in  laboratory colonies and Reynolds (11) introduced the micro-
sporidan  into  field populations of Culex plplens fatigans Wiedemann on
an  island in the Pacific.  Reynolds found infections  in C. p,  fatigans
populations two  years after the breeding areas had been inoculated with
spores, but unfortunately  the  rate of infection was very low.
    The Stempellla  from C. p. qulnquefasalatus causes low levels of in-
fection in  larvae.  However, this microsporidan has a complex life
cycle that  results  in production of two types of spores.  One is pro-
duced in  low numbers and is believed to initiate infections in larvae,
while another  is produced  in large numbers which may not be infectious.
The Stempellla found in larvae of U, sapphlrlna caused serious patholo-
gies in A.  aegyptl  in experimental laboratory tests and is also highly
pathogenic  to  its natural  host.  However,  additional experimental data
are needed  before the potential of this microsporidan can be properly
    Additionally, two Plelstophora were found in two species of Cullao-
Ides (A), but  nothing is presently known about their mode of transmis-
sion.  Also, many microsporidans are known from simulid larvae; however
most are  apparently transmitted via the ovaries.   None of the described
species have been shown to be  infectious to healthy black flies in ex-
perimental  studies.
                           HOST SPECIFICITY

    During the  last two years, we have concentrated our studies on dis-
eases of a 11  invertebrates and some fishes that live in the breeding
areas of aquatic  insect pests.  These studies were deemed necessary to
determine  the host specificity of microsporidans under natural condi-
tions.  We have examined  116 species of aquatic animals, 60 insect pest
species and 56 nonpest species, and have found a total  of 122 micro-
sporidan species.  The latter represent species of the genera Glugea,
Mvazekla, Nosema, Parathelohanla, Plelstophopa, Stempellla, Telomyxa,
Thelohanla and several undescribed groups  (Table 1).  No examples were


found where pest species shared microsporidan parasites with nonpest
species and in only one case did species from different animal classes
share the same parasite.  A Nosema, a common parasite of amphipods, was
found once in a planarian commonly associated with these crustaceans.
All infected animals collected for these studies were prepared for elec-
tron microscopy, and the microsporidans examined in electron micrographs,
using diagnostic ultrastructural characters to identify the species.
      TABLE 1.  Taxa of Aquatic Animals in Florida and Louisiana
             with Species that Are Hosts of Microsporidans
Tax on
Amphi poda
01 igochaeta
Turbel lar ia
1 nsecta
Chi ronomidae
Cul icidae
Simul i idae
Tr ichoptera
Number of



Number of
Microspor idans



    Laboratory infection studies have also been conducted  to determine
host specificity (Table 2).   In these tests,  a  few Nosema  species showed
little host specificity, but most species of  Nosema,  and  particularly
Stempellia and Thelohania, were found to be host specific.   Many Nosema
species of lepidopterans readily invaded a variety of host  species of
this order, however, only four species (N. algerae, a Nosema species
from a mite and two Nosema species from two species of Ishnura [damsel


flies])  caused pathologies  in animals in  orders  other than  those of
their  natural  host.   The  Nosema  from  mites  readily  produced  light  infec-
tions  in A.  quadrimaculatus  larvae;  however,  the other  three Nosema  spe-
cies attacked  only the larvae of H.  sea  (corn earworms)  and  then only
after  the  worms  had  been  starved for  2k  hr  or longer.
        TABLE  2.   Experimental  Transmission Tests  with Microsporida
                                in  Ten  Insect  Species
> *
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Microsporida ^ $> -., <£"
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V? N*
V? -f
Af- •> ./
^ * *
- .& /
xf •/
Nosema algerae (Va"vra and Undeen)                    +
N.  apis (Zanders)
N.  hetepospomm  (Kellen and Lindegren)
N.  inoadens (Kellen and Lindegren)
N.  kingi (Kramer)
N.  necatrix (Kramer)
N.  plodiae (Kellen and Lindegren)
Nosema sp. from Arzama densa (Walker)
Nosema sp. from Carpocapsa pomonella  (L.)
flosema sp. from Cranganyx sp.
Nosema sp. from Ishnura sp.
Nosema sp. from Ishnura sp.
Nosema sp. from Neophelodes emmedonia (Cramer)
Nosema sp. from a mite (Kudo)
Parathelohania anophelis from A. quadrimaaulatus (Say)
P.  obesa from  Anopheles crucians  (Wiedemann)
Stempellia sp. from C. p. quinquefasaiatus  (Say)
Stempellia sp. from Chironomus attenuatus (Walker)
Stempellia sp. from G. holoprasinus (Goeldl)
Stempellia sp. from Uranotaenia sapphirina (Osteen-Sacken) +
Thelohania opaaita from Culex salinarius (Coquillett)
T.  tabani Gingrich from T. atratus Fabricius

    Subsequently, three of the Nosema species that showed exceptional
virulence in their natural host were chosen for infection studies with
crayfishes, fresh-water shrimps, and mosquito fishes.   The microspori-
dans used were N. neaatrix, N. algerae,  and an undescribed Nosema from
A. densa.  Corn earworms heavily infected with each of these microspori-
dans were fed to the test animals periodically.  None  of the test ani-
mals showed symptoms of infection during a 60-day test period,  thus mor-
talities in these animals were no greater than in animals used  in con-
trol tests.  Spores of N.  algerae were also fed to chickens, damsel fly
naiads, dragon fly naiads, dytiscid larvae, helgramites, and mice.   None
of the animals developed gross disease symptoms,  and no evidence of no-
sematosis was found in histological preparations  of dissected tissues
from these animals.

    Our field observations and laboratory infection studies, therefore,
have demonstrated some measure of natural  host specificity of microspo-
ridans in both aquatic and terrestrial animals; those  in aquatic animals
appear to be more host specific, especially species of the genera Stem-
pellia and 'Fhelohania.  Many microsporidan species infected only species
of one family or order; few species crossed host  classes.  Nevertheless,
starved corn earworm larvae are susceptible to N.  algerae while non-
starved larvae are more difficult to infect.   Thus, when starved for 2k
hr or more, they provide a good system for mass culture of many Nosema
species from insects.

    Since fishes of the genus Gambusia,  crayfish,  and  fresh-water shrimp
were not susceptible to N. algerae, N. necatrix,  or the Nosema  species
from A. densa, even though all three are virulent pathogens in  their
natural hosts, these three species of Nos-ema  may  not be hazardous to
other nontarget animals.  This apparent  host  specificity of certain spe-
cies of Microsporida from aquatic invertebrates will be documented  fur-
ther in manuscripts that describe new genera  restricted to the  species
of certain families of aquatic animals.

    Some of the Microsporida are promising  biological  control  agents  for
aquatic pest species, and most species  do not  appear  to  cause  patholo-
gies in nontarget animals other than insects.   Each  new  microsporidan
found in aquatic pests, however, should be  tested  on  nontarget animals
since degree of host specificity is variable among species.  Since new
information indicates that some microsporidans cause congenitally  trans-
ferred diseases in mosquitoes, they may be  useful  for control  of mos-
quitoes.  Basic studies pertaining to development  of  these microspori-
dans in female hosts carrying the disease via  the  ovaries  and  eggs are
needed.  The search for new and more efficient microsporidans  in pest
and disease vectors of tropical regions will be continued  since many  of
these appear to invade their host per os.

                            REFERENCES CITED

 1.  Canning,  E.  U,   1957.   Ple-istophora auliois Weiser:   Its development
        in Anopheles gamb-iae.   Trans.  Roy.  Soc. Trop.  Med.  Hyg.  51:8.

 2.  Canning,  E.  U.,  and R.  M.  Hulls.   1970.   A microsporidian infection
        of Anopheles gambiae Giles,  from Tanzania,  interpretation  of its
        mode of  transmission and notes of Nosema infections in  mosqui-
        toes.  J.  Protozool. 17:531-539.

 3.  Chapman,  H.  C.   1974.   Biological  control  of mosquito  larvae.   Ann.
        Rev.  Entomol. 19:33-59.
 4.  Chapman,  H.  G.,  T.  B.  Clark, J.  J. Petersen, and  D.  B.  Woodard.
        1969.  A two-year  survey of  pathogens  and parasites of  Culicidae,
        Chaoboridae, and Ceratopogonidae in  Louisiana.   Proc.  N.J.  Mosq.
        Exterm.  Assoc.  56:203-212.

 5-  Fox, R. M.,  and  J.  Weiser.   1959-   A microsporidian  parasite of
        Anopheles  gambiae  in Liberia.   J. Parasitol.  45:21-30.

 6.  Gingrich, R.  1965-  Thelohania  tabani  sp.  n.,  a  microsporidian  from
        the larvae of the  black horse  fly Tabanus atratus  F.   J.  Inver-
        tebr. Pathol. 7:236-240.

 7-  Hazard, E.  I.   1970.  Microsporidian diseases in  mosquito colonies:
        Nosema  in  two Anopheles colonies. In  Proc.  Fourth  International
        Colloquium on Insect Pathology.  College Park, Md.   August 1970.
        p. 267-271.
 8.  Hazard, E.  I.,  and C.  S. Lofgren.   1.971.  Tissue  specificity and
        systematics  of a Nosema in  some species of  Aedes , Anopheles, and
        Culex.   J.  Invertebr.  Pathol.  18:16-24.

 9-  Hulls, R. H.  1971.  The adverse effects of a microsporidian on
        sporogony  and infectivity of Plasmodium berghei.   Trans. Roy.
        Soc.  Trop.  Med. Hyg. 65:421-422.

10.  Kellen, W.  R.,  H. C. Chapman, T, B. Clark,  and  J.  E.  Lindegren.
        1965.  Host-parasite relationships of  some Thelohania from mos-
        quitoes.  J. Invertebr. Pathol. 7:161-166.

11.  Reynolds, D. G.   1972.   Experimental introduction of a  microsporidian
        into a  wild  population of Culex p-ip-iens fatigans Wied.   Bull.
        World Health Organization 46:807-812.

12.  Vavra, J.,  and  A. H. Undeen.  1970.  Nosema algerae  n.  sp.  a patho-
        gen in  a laboratory colony of  Anopheles Stephens!.  Listen.
        J. Protozool. 17:240-241.

13.  Ward,  R.  A., and K. E.  Savage.   1972.  Effects  of microsporidian
        parasites  upon anopheline mosquitoes and malaria  infection.
        Proc. Helm.  Soc. Wash.  39:434-438.

14.  Weiser, J.   1946.  Studie o mekrosporidlich z larev  hymzu nasich vod.
        Vestn.  Cesk. Zool.  Spol. 10:245-272.


15-  Weiser,  J.   1961.   Die Mikrosporidien  als  parasiten  des  insekten.
        Monogr.  Z.  Angew,  Entomol.  17.   1^9 p.
16.  Weiser,  J.   1963.   Diseases  of  insects of  medical  importance in
        Europe.   Bull.  World  Health Organization  28:121-127.

J. E. HENRY:  We have been working  for  a  number  of  years  with a  micro-
sporidan, Nosema loousti,  in grasshoppers  and  we have about  8 years  of
practical field experimentation behind  us.   In many areas, we've gone
a little further than Dr.  Hazard has with  his, and  perhaps this  is  the
reason I'm here.  I  was surprised that  our  dosage rates for  field appli-
cation are quite similar to those of Dr.  Hazard.  Our standard dosage
is a billion spores  in a pound  and  a half  of wheat  bran per  acre, and
this is quite close  to what the people  at  the  Insects Affecting  Man  and
Animals Laboratory were using in the Canal  Zone  against Anopheles mos-
quitoes.   We find that these dosages invariably  will  cause 50% mortality
in the grasshoppers  within k weeks  after  application, prior  to oviposi-
tion, and then 30-50% of the residual grasshoppers  will be  infected,  in-
hibiting  or reducing reproduction.   Nosema  loousti-  infects well  over  60
species of grasshopper, and it  will  also  infect  the Mormon and black
field crickets.  Attempts  to infect the eastern  house cricket, however,
have failed.  Also,  it will not infect  honeybees.   We've  done extensive
safety tests with Nosema locusti and this  may  apply perhaps  indirectly
to what Dr. Hazard is doing. We've done  acute dermo-toxicity, acute
ora1-toxicity, inhalation  studies,  long-duration feeding  studies, and
skin irritation studies in rats, but in all cases  none became infected.
These studies were in cooperation with  Dr.  Arthur  Heimpel at Beltsville,
Maryland, under contract to a private laboratory.   In no  instance did
we find any pathologies in rats. We also bioassayed  all  rat tissues  or
samples of rat tissue and  their gut contents,  in grasshoppers after  the
test.  We found spores in  rat gastric contents,  in  the rats  that were
killed immediately after treatment  with the acute oral toxicity  treat-
ment.  These treatments were 10 times our  normal dosage per  acre. When
first tested, we also found one rat with  a  small concentration of spores
that was sacrificed  three days  after treatment--these spores were pretty
well disrupted and the animals  were not infected.   But for the ones  we
got at 0-day time that were infected and  sacrificed immediately  after
treatment, we checked the  spores, and on  the first  run-through they
were not  infective.   We went back to find  out  why,  and found some in-
fectivity in those spores  after cleaning  them  up from the gastric con-
tents of  the rats.


    We've also tested  in fish, and found no pathology there.  So that's
about all I  want to say; if you want to know more about the grasshopper
work, I'll talk to you  later.  As it relates to safety, I  feel that No-
sema locusti at least  is safe, but as mentioned before, each organism
will be tested on its own merits.

IGNOFFO:  That was the major point in bringing Dr. Henry here, since to
date that is the only available systematic test of a known organism
against mammalian systems.

JOE MADDOX:  Most of the work that we do with Microsporida has also been
in terrestrial arthropods.  But I would like to make a few comparisons.
We are working with a few aquatic insects infected with Microsporida and
I'd like  to make some comparisons between what we've found in aquatic
and terrestrial microsporidans.  Dr. Hazard mentioned that he had about
120 microsporidan species and many of these, I  suppose, are undescribed.
But we've found many microsporida stored away because the order is in
such a state of confusion, as far as taxonomy and classification are
concerned.  I would imagine if this is clarified in the near future
there will be a vast increase in the number of described species.
    We found, for example, that some of the key taxonomic  characters
(and I've talked to a number of people about this over the years) vary
as a result of environmental conditions.  For instance, in one species
I  can vary the temperature at which the host is maintained; at low tem-
peratures it falls into one genus, at high temperatures into another.
We have another species and this same thing happens, except that it's a
function of tissue and  time.  If the insect goes into diapause, certain
tissues develop forms which are in completely different genera and yet
this, I feel certain,  is one species.  I won't elaborate on how we de-
termine that.  We also  find that some microsporida, when put into un-
usual hosts, will infect tissues that they don't infect in their origi-
nal host—they go into  these tissues and act differently.   So we have
some very confusing aspects of classification within the microsporida
that just have to be clarified before many of these species can be de-
scribed.  Fortunately, most of the microsporida we have examined have
stable base characters.  One noticeable difference between microspori-
dan-infecting terrestrial insects and aquatic insects is the ability to
be stored in liquid nitrogen.  We store all  of our terrestrial  micro-
sporida in liquid nitrogen and have done so indefinitely with no sig-
nificant  loss of viability.  Some have been stored now for over six
years and in the ones that we can bioassay we can't detect any differ-
ences in those that have been stored versus the fresh microsporida.
However, Al  Undeen has done this with Nosema algerae and we've done this
with some other microsporida from chironomids.   They won't withstand
storage in liquid nitrogen.  I don't know whether this just happens with
the ones we're looking at, or whether this is a unique difference be-
tween microsporida-infecting aquatic animals versus microsporida-infect-
ing terrestrial ones.   We found the same to be true with drying.  Most
microsporida from terrestrial  insects can withstand drying, some for


 considerable periods of time.  Those from aquatic insects that we^worked
 with could  not withstand drying, even for very short periods.  This is
 not surprising,  since freezing is a sort of drying process.
     I  think we need to define very carefully what we really mean when
 we say host range.  For example, we can frequently infect insects with
 a microsporidan  if we feed  it to them in very large doses as newly
 hatched  instar larvae.  If you feed it to them as second or third instar
 or older  larvae,  infection can't be achieved unless you define very
 carefully what stage you're trying to infect and how to go about infect-
 ing  it.  These host range statements don't mean a lot as far as host
 ranges go,  but in relation to what's happening in the field, they're
 significant.   In other words, if you can't infect a later instar larva
 with microsporIda, the chances are that particular species are not
 going  to be infected under field situations or conditions.  Frequently
 we can infect  insects by injecting into the hemocoel infectious stages,
 infectious  spores of which the polar filaments have been extruded or
 vegetative  forms from another insect.  But for some of these microspo-
 rida you can expand the host range enormously—it'll go into almost any
 arthropod you  inject it into if you inject the correct stage.  Others
 have a very specific host range and even by injecting you can't get it
 into other  arthropods.  Thus, I  think we need to look at some of the
 aspects of  host  range and what really determines host specificity.

 AL UNDEEN:  In the Biology Department of the University of  Illinois,
 we've  been  studying Nosema algerae, which is probably the same micro-
 sporida that Dr. Hazard has discussed.  We don't know for sure, we've
 never  really compared them  that closely.  We work with Anopheles ste-
 phensi since this microsporida infects better in A. stephensi than  it
 does in A.  albimanus.  The  required  infective spore dose per unit area
 is about the same as Dr. Hazard described in his Nosema algerae field
 tests.  We  have  been able to infect everything we've  injected with  this
 microsporida, we've injected the spores  in the hemocoel, anything that
 would  become infected, and  this has produced a good method  for mass
 producing spores.  The feeding tests we've done  in  the past have been
 with larger larvae, those that are easily handled, nymphs of aquatic
 insects and that sort of thing; we've not been able to get  infections
 by feed ing spores to these insects.  However, more recently we have  fed
 Heliothazia larvae as first  instars, without starvation or anything
 else,  and they were all infected.  So I think, as Dr. Maddox has stated,
 that we really need to look at the host range of this a little more
    I  want  to talk about the safety of these microsporida and verte-
 brate  systems.   We've used pig,  bovine,  and horse kidney cells  in tis-
 sue culture and they served  as adequate hosts for Nosema algerae.   This
 is simply inoculating the spores directly into the tissue culture medium
with the cells  as a monolayer on the bottom  of the flask.   This  Nosema
developed at 26 and 35 C  with production  of  viable spores,  and at  37 C
Nosema algerae  appeared  to multiply for  a short  time and  then die.   No
 infected cells  were seen  at  38  C.   We are presently  examining  the


possibility of a vertebrate nosematosis resulting from spores injected
by mosquito while obtaining a blood meal.  I  haven't examined the proba-
bility of this event happening, however, we have seen infected salivary
glands in female Anopheles stephensi, and Nosema algerae germinate very
well in mouse and human blood plasma.

    Since 37 C appeared to be the upper limit of this Nosema's tempera-
ture tolerance, mice were injected in areas suspected to be cooler than
the interior body temperature.  About one million spores were injected
at each site under the skin of the tail, the ears and the hind feet of
white laboratory mice.  At 2, 3, ^, 6, 8 and  12 day intervals a  mouse
was killed, the skin over the injection site excised, and the subdermal
layer scraped to obtain a microscope slide which was stained with giemsa
blood stain.  Nosema developmental stages were found at each injected
site, through the 8th day after injection.  No parasites were found on
the 12th day.  And no Nosema was found at any uninjected site, so this
means that the Nosema did not spread throughout the mouse and it also
means that there wasn't any other Nosema present in the mouse before it
was injected.  So, the host range in Nosema algerae seems entirely de-
pendent upon its ability to invade the host.   Since insects do not have
the same degree of resistance to invasion throughout development, sus-
ceptibility tests must include the whole period of exposure to Nosema
spores.  The immunity system of the vertebrate is an additional  factor
in attempting to destroy the Nosema once invasion has occurred,  that is,
in the vertebrates.  Experiments using cortisone to suppress the immu-
nity system are planned in order to see how severe a Nosema infection
can become in an immunologically incompetent  individual.  I  don't know
whether these results can be generalized to other microsporida but I
think it is here that these guidelines for safety standards are  really

IGNOFFO:  You might want to use very young mice, too, in addition to
the immuno-depressant drugs.

MARSHALL LAIRD:  I'd like to try a red herring at this point, arising
from the remark about giemsa bloodstain and the staining of microspori-
dans.  Those of us who work with mosquitoes will  be well  aware that we
have customarily fed mosquito larvae on dog biscuit.  Now, if you think
about that, it does seem a little strange and it did lead to tiny
amounts of DDT sprayed onto alfalfa, getting  into dog biscuit, and then
into mosquitoes, which allegedly have never been exposed to DDT, some
years ago.  I  mention this because eosin stain is the easiest of all
stains to use.   At the same time,  it gives infinitely variable results
and these are attested to by the fact that there is a literature of
several  hundred papers on yet another method  of attaining success in
staining with giemsa.  It seems to me that if we really are going to
get into the nitty-gritty of, for  example, the Microsporida, it  might
perhaps  be worthwhile casting around and endeavoring to develop  some
staining methods that are actually tailored to the microsporidans in-
stead of picking a stain from another group.


UNDEEN:  The giemsa stain as I  use it  did  show developmental  stage  of
the microsporida in which the cytoplasm and  nuclei  could  be  clearly
seen.  This was different from any other tissue I  saw  in  the mouse
slides or in the mosquito slides;  it was also identical  to  the stages
seen when taken from mosquito tissue or any  other  insect  into which it
was injected.
 IGNOFFO:  What was the source of nutrients  for  the development  of     ^
 spores  I assume you used?  And how far was  the  stage developed? Did  it
 just germinate?

 UNDEEN:  In the mouse it was difficult to tell  just how far  it  went be-
 cause  I gave them an inoculant, the temperature was high,  the develop-
 ment was rapid, and I  suspect the spores I  saw  on the slides could have
 either  been produced in the cells of the mouse  or could have been  from
 the original inoculant.  I  had no way of determining this.   However,  I
 did see the developmental stages which could only have come  through
 growth  from the spores.

 IGNOFFO:  The basic question  I was getting  at is, was the habitat  just
 a nice  localized  place for development of the spores or, in  fact,  did
 spores  develop on mammalian host tissue?

 UNDEEN:  I've never seen the microsporida in the tissue culture or in
 the insects to develop outside of  the cell, so  I imagine the development
 was i ntracel lular as  it was anywhere  else and  it must have been within
 a mouse cell.  The  spores  I  injected  were treated  to remove any extra-
 neous  amount of  insect  tissue.   The spores were  cleaned with a density
 gradient and several changes  of  distilled water, so  I'm sure they must
 have been within  the mouse  cells.   You  had  to  scrape the tissue and
 treat  it pretty  roughly  in  order to see anything so  I assume you had to
 break  open  the cells  to  find  anything.   Again,  this  is  preliminary work
 and I'm not sure  if all  aspects  of the  development occurred.

 IGNOFFO:   I think in most systems  if  you work  hard enough and put  no
 restriction on dose or what  site of  infection  to use, you probably can
 obtain  considerable material.   Now how,  then, does that relate to  speci-
 ficity?  This is  the question we'll have to address ourselves to.   You
 can go  to the literature on viruses and  bacteria and you find many in-
 stances of cross-transmission, using  roots of administration that  are
 far from the natural.    I think  in most of these  situations  what you're
 trying  to do is establish if,  in fact, the organisms can survive in
 those systems.   Another question is, can you retrieve the organism in
an infective stage and place  it back  into the normal  host?

HAZARD:  We have looked for  a natural  transmission  field because of
some of the difficulties we  have, mainly insufficient  personnel  for all


these laboratory studies.  Also, while we're collecting these non-target
organisms we have a chance to pick up other microsporidans which we can
add to our list and help us in our taxonomy, and this is one of our more
important studies right now.  We've got to find out what we're working
with.  We don't see this in the field, and we couldn't find any infec-
tions.   If there are any infections it's going to be less than one per-
cent.  We brought back numerous specimens to section and examine more
closely, but I  wonder in all these animals that you injected, did you
run this material through ludox?  This is going to pretreat these spores
if they're going to extrude, no matter what you put them into.

UNDEEN:  The ludox pretreatment, for those of you who don't know what it
is,  is a silica colloid, used in floor waxes and stuff.  I  use it as a
density gradient for separating microsporida spores from trash and mos-
quitoes, or whatever insect I  separate it from, but I  use it mainly in
this  instance for getting the microsporidan free of the bacteria, be-
cause if you run it through this gradient, all the bacteria are at the
top and the microsporida is in the band down by itself.  Under some con-
ditions  in vitro it does accelerate germination of the spores, however,
the untreated spores that were not run through the ludox gradient do ex-
trude, or germinate, in blood and in the tissue culture medium.  The
spores germinate, whether they're run through the ludox treatment, in
blood, serum, from these animals.

IGNOFFO:  I think what they're trying to stress is that there are tech-
niques in which you can handle the pathogen, handle the host and manipu-
late  it  in such a way that you can get data.  I think that's a good ap-
proach and should be considered.  It's been noted that there seems to
be a  limit in terms of upper temperatures at which these organisms will
complete their development to the spore stage.  But we also have to con-
sider the opposite area, that is, there are many vertebrates existing in
nature in which the body temperature is well below 37 C.  So the poten-
tial for infection will  have to be looked at on the basis of the host
and the pathogen.  The requirements are going to be slightly different.
We can establish guidelines which will assist us, but  the criteria for
measuring the effects will  be different depending upon the host and

                     PERSISTENCE  OF PATHOGENS
                    IN  THE  AQUATIC ENVIRONMENT

                             Y. Tanada*

    The persistence of pathogens in the aquatic environment  is an area
of  insect pathology that has received very little exploration.  This  is
especially true when pathogens, which attack terrestrial  insect pests,
occur in an aquatic environment.  [Terrestrial  insects are defined as
insects which spend or complete all of their life cycles  independent of
a body of water.  Aquatic insects are those which spend the  entire or a
portion of their life cycles in or on a body of water.]   A list of fac-
tors that may affect the persistence of pathogens in  the  aquatic en-
vironment is given in Table 1.
           TABLE 1.  Factors Affecting Pathogen  Persistence
                        in Aquatic Environment

           Physical                      Chemical                Biotic

Current  (waves, tides, etc.)      Solutes:                       Fauna
Density                             Acids,  alkalis  (pH)          Flora
Depth                               Gases (02, C02,  etc.)
Desiccation                         Inorganic  salts
Sunlight (UV)                       Organic compounds
Pressure                          _     , , .    , .  ,
_,..,_/    ^.           \        Suspended Insolubles:
Salinity (osmotic pressure)          inorganic compounds
Substrate (bottom, shoreline)               compounds
    A major difference between a terrestrial  and  an  aquatic environment
is the medium, air or water.   In the former,  the  pathogens are  less
likely to remain suspended in air unless  moved  by air  currents  and
other forces.  However, in an aquatic environment, the size,  buoyancy
and swimming appendages of the various pathogens  may enable them  to  re-
main suspended and move about in the medium for long periods.   The role
    "Division of Entomology & Parasitology,  University  of  California,
Berkeley, California 9^720.


 played  by  noxious compounds and contaminants  in the terrestrial environ-
 ment  varies  from rapid dispersion  in the air  to prolonged persistence
 in  the  soil.   In an aquatic habitat, the contaminants  in water may  re-
 main  for  long  periods depending on the rate of breakdown, dilution, and
 removal by water movements.  Thus, pathogens  in the aquatic environment
 may be  in  contact with these contaminants for  long periods.

    Pathogens  of terrestrial insects experience two physical factors
 that  play  major roles in their persistence.   These are sunlight, espe-
 cially  the UV  spectrum, and temperature.  These factors play less sig-
 nificant  roles in the aquatic environment, where UV would have a minimal
 effect, and  temperature, except in hot springs and volcanic pools,  would
 be  usually below air temperatures and at the  minimal, not many degrees
 below freezing.  On the other hand, the aquatic environment has factors,
 such  as pH,  osmotic pressure, dissolved inorganic and organic solutes,
 amd water  movement that may seriously affect  the pathogen persistence.

     In  the terrestrial habitat, the microenvironment that closely ap-
 proximates the aquatic environment is that of the soil, especially  after
 prolonged  wetting due to rainfall, irrigation, etc,  The microenviron-
 ment  of soil at  its maximum moisture-holding  capacity would be similar
 to  that of the aquatic environment, especially the sediment along the
 banks of  streams, rivers and lakes.


     Inasmuch as there is a serious lack of information on the persistence
 of  pathogens of terrestrial insects in the aquatic environment, it  is
 necessary  to extrapolate from laboratory studies and speculate on the
 survival of  such pathogens in an aquatic environment.  In some cases, a
 pathogen of  a  terrestrial  insect has been transmitted to an aquatic in-
 sect, and  such a pathogen, therefore, may become involved in an aquatic
 env i ronment.


    Viruses  of terrestrial insects maintain their infectivity for one or
 more  years when stored in water under refrigeration (k C).  Such viruses
 are mainly the occluded viruses, and they are stored at the stage of oc-
 clusion within the inclusion or occlusion bodies,  The nuclear polyhedro-
 sis virus  (NPV) of Bombyx mori has been maintained for 20 years in  hemo-
 lymph in flame-sealed glass tubes held at refrigeration temperatures
 (k$).   Cunningham, however, has observed, using the electron microscope,
 that prolonged storage for 6 years at 4 C causes holes to appear on the
 polyhedra of the NPV of Lambdina fisaellaria fiscellaria and the patho-
genicity of  the virus is greatly reduced.  A  similar observation has
been made with the scanning electron microscope on the polyhedra  of  the
NPVs of Galleria mellonella, Hyphantria aunea, and Bombyx mori  (60).
After  several months to years of storage,  the surface  of  the polyhedra

deteriorates, shows cavities and exposes the virions.

    The insect pox viruses within their spherules, as in the case of
the NPVs, may persist under refrigeration.  The survival of the granu-
losis viruses within the capsules is, in general, for not more than 1-2
years when suspended in water and held in the refrigerator.  We have
maintained the activity of the Tipula iridescent virus for about a year
under refrigeration.  There is very little information on the persis-
tence of other nonoccluded viruses in aqueous suspension.

    Earlier  it is noted that the soil of the terrestrial environment
may closely approximate that of the aquatic environment.  This aspect
is discussed, especially with insect viruses, because extensive studies
have been conducted along these lines by various workers (50, 51).  The
insect granulosis virus (GV) of Pieris brassiaae cannot be readily
washed out of soil and sand (9).  The prolonged persistence of viruses
in the soil  throughout the year even after rainfall  has been reported
(21, 23, 25, 52).  The NPV of Trichoplusia ni apparently persists for
more than k years in the soil  (2k), but the GV of T,  ni, after 2 years,
occurs only  in small amounts in the soil.  The NPVs of Hyphantria ounea
(20) and of T. ni (5*0 accumulate less than 5 cm from the soil surface
and polyhedra have been demonstrated in spaces between the soil  parti-
cles (20).

    The soil pH may play a significant role in persistence of the NPV
of T. ni (55).   In a loamy sand of various pH (A,83-7.17), the lower
the pH the more rapidly the virus is inactivated.  This would be ex-
pected, since the virus polyhedra are soluble in weak alkali and acids,


    Both nonspore-forming and spore-forming bacteria  would be expected
to live for short periods in an aqueous environment,  but this would
vary with the availability of nutrients, pH,  toxic substances, tempera-
ture, etc.  With spore-formers, the presence of water would increase
the likelihood of germination and thereby may reduce  the persistence of
the bacteria.  The spores of Bacillus popilliae, when suspended in
water and kept for 20, *tO, and 80 days, progressively lose their po-
tency with time  (k).
    Some pathogenic crystal 1iferous bacteria, e.g.,  Bacillus thuvin-
giensis and  its varieties, have a crystalline parasporal body, the
delta endotoxin, that is highly toxic to certain insects.  The endo-
toxin is proteinaceous and is soluble in weak alkali.  In an aqueous
environment with high pH, the endotoxin may be destroyed.

    The most favorable physical environment for bacteria of terrestrial
insects is the soil.  Both nonspore-formers and spore-formers have been
reported to persist in the soil.  Some facultative bacterial pathogens
may propagate in the soil.  The common bacterium, Bacillus aereus, has
strains which are pathogenic to insects.  With milky  disease bacteria,
these obligate bacteria infect soil insects in nature, and have been


 applied  to  the  soil and have persisted and controlled scarabaeid  beetles
 for many years.  Most  studies have been conducted with Bacillus popil-
 liae,  the Type  A organism of the Japanese beetle, Popillia japonioa  (4,
 69).   The spores of B. popilliae tend to remain  in the upper  layers of
 the soil, the first one inch (4), but there  is some vertical  spread to
 a  depth  of  4  inches.   After 18 weeks, there  is some loss  in the capacity
 of the soil to  produce disease, which suggests a decrease  in  the  persis-
 tence  of the  spores.   However, the loss of effectiveness  is due more  to
 a  diluting  or  leaching factor than to an actual mortality of  bacteria.
 In field-collected soil, Beard (4) reports that 30% of the grubs  become
 infected in soil  in the top inch, 5k% in the second inch and  46%  in the
 third  inch.   Low soil  pH (>6.0) may adversely affect the spores of B.
 popill-iae,  and  high pH (<6.0) may increase the potency of the bacterium.

    Several Rickettsiae have been reported from  larvae of scarabaeid
 beetles  which  live in  the soil.  These Rickettsiae produce infections
 in larvae and may be presumed to persist in the soil.


    Most fungi  of terrestrial insects, as with bacteria, may  not  persist
 for long periods in an aqueous environment,  The presence of moisture
 increases the likelihood of germination of resistant and non-resistant
 spores,  and only those capable of saprophytic growth would be expected
 to survive  in the absence of hosts.

    Some entomogenous  fungi are known to persist for long periods  in  the
 soil,  not only  in resistant stages, but the facultative form also may
 grow saprophytically in the soil.  This may occur with the green  muscar-
 dine fungus (Metarrhiziim anisopliae) and the white muscardine fungus
 (Beauveria  bassiana),  both of which are known to infect soil  insects
 under  natural conditions.  Species of Cordyaeps are known to  infect soil
 insects,  but there is  very little information of the mode of  infection
 and persistence in the soil.


    The  Protozoa are generally maintained in the laboratory in water and
 under  refrigeration.   This is especially true with spores of  Sporozoa,
which  may remain viable in water at 2-4 C for more than a year (31, 63,
 64).    The clean spores of the microsporidan, Nosema bombycis,  remain
viable for more than 8 years when stored in a refrigerator (64).  At 15
and 25 C, microsporidan spores are viable for a month or two  in water or
saline.  The spores of Oetosporea musaaedomestioae in water at 5  C re-
main  viable for at least 2 years (31).  The spores of Nosema neoatvix
persist  for only 6 weeks at -34 C, and at 4 C are viable for 2-1/2 years
 in water suspension, but there is a marked reduction  in  infectivity
 (Table 2) (33).

             TABLE 2.  Viability of Nosema neoatvix Spores
                        Stored in Water at 5 C*

          Length of Storage           Reduction of Infectivity

               1 day                               0
               6 months                          100 X
               1 year                          1,000 X
               2 years                        10,000 X
              *Maddox, 1973-
    The polar filament of a microsporidan spore is used to inoculate
the planont or sporozoite into the host cell or hemocoel  and  its extru-
sion is affected by certain chemicals,  pressure, and pH.   High K con-
tent and a pH 10.8 favor release of the polar filament (22,  36).  The
effective range of pH also varies with  different cations  (22), which
may cause premature release and death of the planont when no  host is
available.  The pressure developed at great depths of lakes and oceans
may also force the extrusion of the polar filament.
    When kept moist throughout storage, the microsporidan spores can
persist in water, but if they are first dried and then wetted, this may
cause the ejection of the polar filament.  This occurs with  the dried
spores of Nosema whitei, which when placed in water readily  extrude
their polar filaments (30, 30.
    The flagellate, Leptomonas etenoaephali, of the dog flea, Cteno-
cephalus aanis, becomes nonmotile in salt concentrations  of  k% and over,
but will revive if salinity is reduced  to below k% (13).

    Several protozoans, especially the sporozoans, infect soil insects,
and they would be expected to persist in the soil.  The larvae of sev-
eral species of lepidopterous insects of alfalfa develop  a microspori-
dan infection when fed the soil from the alfalfa field (52).


    Temperature and moisture appear to  be the major factors affecting
the persistence of nematodes (AO, 62, 67).  Nematodes are capable of
surviving in water but their length of  survival may depend on tempera-
ture, salinity, oxygen, pH, and other factors.  The nematode, DD-136
(Neoaplea tana carpoaapsae) can be maintained for up to 5  years in water
receiving periodic oxygenation and held at 7-1 C  (11).  It is destroyed
at temperatures above 38-^5 C (10, 66)  and at freezing temperatures in
water (66).  It can survive the pressures of a conventional  power
sprayer (200-300 psi) and can be safely combined with common  insecti-
cides and fungicides  (10).
    A survey of the natural distribution of Neoaplectana indicates that
insect parasitism is greatest at high soil temperatures and  with


abundant soil moisture (kO),   There may be a preference for calcareous
soil by mermithid nematode pathogens of insects (53).   The mermithid
nematode of the ant, Fheidole pallidula, appears to be associated with
a particular soil structure  (61).  The author has been able to predict
where the nematode may be found by the geological  structure of the soil.
    Certain fungi (Saprolegniales and Chytridial es) and protozoans  (Ac-
tinomyxida) are known to parasitize free-living stages of mermithid ne-
matodes, and they may also attack the entomophilic nematodes

    The persistence of pathogens of aquatic  insects will be treated
under two major categories:   (1) persistence  in the fresh water habitat
and  (2) persistence in a marine, including estuarine, habitat.  Inasmuch
as there are only a relatively few  insects in the marine habitat, the
pathogens of some invertebrates other than insects will be discussed in
this section.  Under both habitats, the discussion will be centered on
the  physical, chemical and biotic factors that may affect the persis-
tence of pathogens.  These factors are listed in Table  1, but some of
them will not be considered because of a  lack of information.

    Most pathogens of aquatic  insects are microsporidans, fungi and
nematodes, with a lesser number of bacteria and viruses.  Thus the dis-
cussion below will be directed largely toward the first three pathogens.


     Since  influence of the current or water movement  is closely associ-
ated with  the density  (buoyancy) of the pathogens, these two factors
will be considered together.  A buoyant or suspended  pathogen would be
expected to be carried or moved about by water movement.  Pathogens
 less than one micron may remain suspended in water.   Larger pathogens
may  have structures or appendages (cilia and flagella) that enable them
to move about  in water.  Still others, e.g., microsporidans, have spores
with caudal appendages, needle-like structures, and gelatinous capsules
that enable them  to float  (64, 65).

     The effect of depth on persistence of pathogens has received very
 little study.  The rate of infection by the mermithid nematodes, Hydro-
mermis oontorta and Limnomermis batkybia, on the midge, Ckironomus mo-
de stus , is  inversely correlated with depth (3*0, being highest at 0.5 m
and  lowest at 3~5 m.

     In general, desiccation would have drastic effects on the persis-
tence of most pathogens in the aquatic environment.   However, in some
 instances  the pathogens are able to resist desiccation and infect the
insect host when water becomes available again.  Chapman and Glenn (5)
in their study on the  incidence of the fungus, Coelomomyoes punatatus,
have reported its reappearance in larval  populations of Anopheles


crucians after an absence of 29 weeks, caused principally by a lack of
water in the ponds.  In other situations, alternate wetting and drying
may favor the pathogen.  The spores of the microsporidan, Thelohania
cali-fornioa, a pathogen of Culex tarsalis, when freshly removed from
infected mosquitoes are not infectious, but become so when alternately
dried and hydrated  (28).

    The sporangia of Coelomomyces spp. can be stored at 5~10 C on moist
filter paper for 5~8 months (7, 68).  The germination of these sporan-
gia is markedly affected by temperature (7), with  no germination below
10 and above 35 C, and optimum germination at 23 C.  Germination occurs
in tap and distilled waters, rain and lake waters,  in pure clean water
and in water contaminated with bacteria and protozoa (7).  Earlier
workers experienced difficulty in studying this group of fungi because
of failure of sporangial germination.  Roberts et al ,  (*tl) state:  "The
inability to induce dehiscence upon demand has been a  major deterrent
to the study of most Coelomomyaes species."  They report that the de-
hiscence of the sporangia of C, psovophova from Aedes  ixLenioThyruihus
decreases from 23% dehiscence to 3% in less than 1  and 1.5 months when
stored in water at  10 C.  They also note reduced dehiscence with use of
moist filter paper at 10 C as reported earlier (7,  68).  Roberts et al.
(4l) tested the dehiscence of C.  psorophova. with various salts, reduc-
ing agents, chelating agents, buffers, alcohols, carbohydrates, fatty
acids and derivatives, amino acids and derivatives, peptides, amines,
purines, pyrimidines, antibiotics, and plant hormones.  The most active
substance is Tris  [tris (hydroxymethyl) ami no-methane] at pH 8.9 and at
1-20 mM.  The active compounds have a basic requirement for -NH2 and
either -COOH or -CHaOH attached to the alpha carbon,  but only certain
amines and amino acids are highly active.  Sporangial  preparations free
of host debris are not responsive to Tris, but addition of bacteria-
free homogenates of A.  taenior'hync'hus larvae reactivates the material.

    Salinity would be expected to be one of the major  factors affecting
persistence of pathogens of fresh-water insects.  Data in this area ap-
pear to be seriously lacking.  The habitat of a mermithid nematode,
RomanomemrLs sp., on the southern house mosquito, Culex pipiens quin-
quefasoiatus, may be limited by salinity (37).   The mermithid does not
parasitize the mosquito when held in water with a NaCl  concentration
above 0.04 M (Table 3).  A sharp drop in parasitization is evident at
concentrations between 0.015 and 0.030 M NaCl.   This suggests that the
mermithid will not survive under a high salinity.


    Many pathogens of terrestrial insects commonly persist in the bi-
otic habitat (48, kS, 50).  Such persistence occurs in the primary and
secondary hosts, parasites, predators, and other animal carriers.
There is increasing evidence that this also is true with pathogens of
aquatic insects.  The host-parasite relationships of several microspo-
ridans (Thelohania spp.) have been studied in mosquitoes and four basic
types of relationships based on tissue specificity, sporogonic cycle to


the sex, and on the expression of patent infections of the host have
been classified (29).  The latter workers point out that in the Type  I
relationship, the transovarial transmission of the microsporidan through
the female parent, which  is not killed, enables the microsporidan to
persist throughout the year in the hibernating eggs of the univoltine
mosquito.  Since the host larva, pupa and adult are absent during the^
11-month period, it  is suggested (29) that the microsporidan spores will
not persist  in the environment in the absence of hibernating eggs.   In
hibernating  hosts, development of a microsporidan ceases or is greatly
delayed  (63).

        TABLE 3.  Effect  of NaCl on Parasitism of Culex pi-piens
             qui,nquefasei,atus Exposed to Constant Numbers
                          of Romanomermis sp.*
(M cone.)
Trial 1
Trial 2
1 1
1 nfected
Trial 3
— •»
            *Peterson and Willis,  1970.
    Transovarial transmission and subsequent persistence  in the host
also occur  in the mosquito  iridescent virus infecting Aedes
ohus (16).

    There are increasing numbers of  instances of pathogens infecting
several species of aquatic  insects.  The iridescent virus discovered in
the rice stem borer, Chilo suppressatis, can be transmitted per os in
the laboratory to the following aquatic genera:  Aedes, Anopheles,
Culex,  Culiseta, and Psorophora (12).  On the other hand, the mosquito
iridescent virus is  infectious only for Aedes and Psorophora.   The fun-
gus, Coelomomyaes maoleaijae, infects tree hole mosquito larvae of 3
Aedes subgenera in Australia, Fiji, and the United States (35), and
also attacks the predator mosquito, Toxovhynehites rutilus septentrio-
nalis.   A virulent aquatic fungus, Lagenidium sp., was isolated from
Culex restuans;  it also infects several  other species of mosquitoes
Anopheles quadrimaculatus, Anopheles sp., and Psorophora sp.  (58).


    Several fungi which predominantly infect terrestrial insects also
infect aquatic  insects.  Beauvevia tenella (B.  bassiana) infects Aedes
sierrensis, A. aegypti, A. dorsalis, A,  hexodontus, A, pipiens, Culex
tarsalis, and Culiseta inaidens (39).  A possible strain of the ubiqui-
tous green muscardine fungus, Metawhizium anisopliae,  is highly viru-
lent for the mosquito larvae of 3 genera, Anopheles, Culex, and Aedes
(47).  The fungus, Fusarium oxysporum, infects Aedes detritus and Culex
pipiens pipiens  (17).

    The microsporidan, Nosema algerae, isolated from Anopheles Stephens!,,
infects other species of Anopheles (59).   It is also infectious for
crayfish, whose gills are most heavily infected.  The mermithid nema-
tode, Hydromermis aontorta, has several  physiologically suitable hosts,
Proaladius dentiaulatus, and four species of Tanitarsus (3k).
    Some pathogens serve as food for other animals in the aquatic en-
vironment.  The  infective larvae of the nematode, Neoapleetana aarpo-
aapsae, are fed upon by larvae of Simulium vittatum and Culiseta inor-
nata (62).  The former feeds upon the nematode faster at 10 C than at
20 C, and  the latter eats more at the higher temperature.   The sporan-
gium of the fungus, Coelomomyaes punatatus, is eaten by ostracods, roti-
fers and other small invertebrates, and  is destroyed by parasitic fungi
(6).  The  invasion of the ciliate, Tetrahymena pyrifovmis,  into the
larva of Culex tarsalis is affected by the presence of the larvae of
Aedes aegypti, which may be consuming the ciliate (14).


    During the past several decades, there has been a great increase in
the number of studies on diseases of marine animals, but these studies
are widely scattered in the literature (42).  Most of the reports are
concerned with the taxonomy and biology of the pathogens,  with only
limited studies on the epizootiology of  the diseases.   I have not made
an exhaustive search of the literature on the persistence of pathogens
of marine  invertebrates and my review is far from complete.
    In the terrestrial environment, sunlight, especially the UV radia-
tion, plays a major role in reducing the persistence of pathogens in
the host habitat.  However, in the aquatic habitat, penetration of the
UV radiations into the water is limited,  and therefore  is  relatively
ineffective.  This is indicated in the rearing of bivalve mollusks
where the UV treatment of water and phytoplankton greatly reduces the
incidence of infection of mollusks by fungi (32).  Certain  soluble com-
pounds, such as antibiotics, phenols, insecticides, weedicides, and
such inert substances as silt and kaolin may improve the growth rate of
bivalve larvae.    Loosanoff and Davis speculate that these compounds re-
move the toxins from water or assist in  killing pathogenic  and other
competing microorganisms (32).

    Salinity may  be a significant factor in the persistence of patho-
gens in the marine and estuarine habitat.  Johnson and Sparrow (27)
state:   ". . . The status of knowledge on fungi in salt water is such


that while considerable is known about their structure and occurrence,
the mechanisms permitting them to exist in a 'salty environment1  are
not understood or at most only dimly apprehended."  This statement would
also apply to other pathogens of marine invertebrates.  The fungus,
Lagenidium aall-ineetes, which infects the blue crab, Calli-nectes  sapidus,
has a wide tolerance range of salinity and can sporulate in water of
5-30 %o salinity  (26).  The mosquito, Aedes australis, is capable of
breeding in pools ranging from fresh water to hypersaline, but the infec-
tion by the fungus, Coelomomyaes opifexi, is associated with less than
approximately 20 %„ salinity and more particularly below 10 %o  salinity
(38).  The high concentrations of sea salts inhibit the dehiscence of the
fungus sporangia, whose walls become thicker and darker as a result of
exposure to higher salinities.  The germination and zoospore activity of
the  sporangia have been studied at various temperatures and different
salt concentrations  (Table 4) (38).  Germination occurs at 5, 10, 23 and
28  C in distilled water, brackish water (4.2 700 ), and  in salinities of
8.5  %0 and lower.  Zoospore activity decreases at 5 and 10 C.  Regard-
less of temperature, germination is variable at the higher salinities,
but  none occurs  in pure sea water  (35 %0)•

           TABLE 4.  Mean Averages of Sporangial Germination
                        of Coelomomyaes opifexi*

               ..  ,.                       Temperature
               Med i urn	
Distilled H20
Tap H20
Sea H20:

             *Pillai and O'Loughlin, 1972.

            **Numerator = zoospore activity (Scale:  0-4)
              Denominator = sporangial  germination (Scale:  0~5)

    Although the sporangial wall of C.  opifexi increases  in thickness
under high salinities, the thick wall does not protect the sporangium
from desiccation, unlike the thick-walled sporangia of Coelomomyaes of
fresh water mosquitoes (38).  Thus, complete drying of pools can lead
to elimination of the fungus, but  if the soil  remains moist, the fungus


may survive for several months in intact larval cadavers.
    The general observation is that the fungus, Dermooystidium marinttm,
which produces epizootics in oysters, is absent in all very low salinity
areas (less than 10-15 %0) as well  as in a few high salinity areas
(greater than 30 %o), but is prevalent in virutally all moderate to
high salinities.  Hoese  (18) has  investigated the possible reason for
the absence of this fungus in oysters in Port Aransas, Texas, and be-
lieves that an inhibitor which occurs in the water stops development of
the fungus hypnospores (resistant spore).  The origin of the inhibitor
is not known.  D. marinim is considered to be a pathogen which kills
oysters during the warm  periods of July through October  (2, 3).  The
author claims that D. marinwn proliferates readily only at temperatures
above 25 C and overwinters in oysters as subclinical cases (2),
    Another serious pathogen of oysters is the sporozoan, Minchinia nel-
soni, which commonly was known as "MSX" prior to its identification.
This sporozoan favors high salinity  (greater than 15 %o) and its preva-
lence in the estuaries may vary with salinity changes (1, 2, 3).
    Infection of the protozoan, Paramoeba pernieiosa, on the blue crab,
Callineotes sapidus, appears to be limited to high salinity waters (44).
High temperatures (30 C and higher)  appear to favor the bacterial patho-
gens (Vibrio and Pseudomonas) of  bivalve larvae (15).
    In the case of Coelomomyoes opifexi, field observations in the win-
ter months have corroborated the  laboratory study that this fungus can
infect Aedes australis larvae when the mean temperature  is lower  than
7 C (35).
    The micrococcus, Gaffkya homari, which causes Gaffkemia in American
(Homarus americanus) and European lobsters (H. vulgaris) may live as a
free-living organism, since Gaffkya-} ike bacteria are readily isolated
from mud samples of tidal pools (46).  G, homari have been isolated
from the mud of tidal pools and also from sea water several miles from
infected ponds (42).


    Pathogens of marine  invertebrates are known to have alternate hosts.
Gaffkya homari causes fatal septicemia not only in the American and
European lobsters, but also in other crustaceans, such as shrimps,
crabs, etc.  (46).  "The fact that G. homari can infect a variety of
hosts, with some of these species able to carry the pathogen for  ex-
tended periods (80 days or more in C. irroratus), coupled with its
ability to survive in the absence of a host, gives G. homari an ex-
tremely broad-based potential for survival" (46).  This statement would
also apply to other pathogens with alternate hosts and carriers.
Strains of marine Vibrio sp. are  pathogenic to several species of bi-
valve mollusks, and can be isolated from overtly healthy, diseased or
moribund bivalve mollusks or their environments (56, 57)-

    The fungi, Atkinsiella dubia and Pythiwn thalassium, which infect


Pinnotheres p-iswn, have an extensive host range which includes Gonoplax,
Trypton, Crangon, Leander, and Portunus (26).  The flagellate, Hexamita
nelsoni, is a pathogen of several oyster species (43) .   The gregarine,
Nematopsis ostreaawn, infects oyster, pecten, and other marine gastro-
pods  (43).  The microsporidans, Nosema legefi and N. dollfusi, have sev-
eral hosts belonging to different species of marine bivalves.  Gregar-
ines, coccidians, microsporidans, and haplosporidans have several host
species of decapod crustaceans  (44).

    Although no secondary host of the damaging  sporozoan, U-inokinia nel-
soni, is known, the  failure of  the epizootic caused by this  sporozoan
to decline, even with the severe reduction of  the oyster populations,
suggests that another unknown host may be  involved  (3).  With the fungus,
Dermoeystidium marinum, "the decline of Dermocystid-ium with  the disap-
pearance of oyster populations  strongly  implies that in  its  life cycle
the parasite is not  dependent for winter survival or transmission upon
other hosts or resting stages living outside oysters"  (l).   Two species
of ostracods, which  feed on the exoskeleton of  moribund mosquito larvae
infected with Coelomomyoes opifexi, expose the  sporangia which germinate
readily  (38).  It  is believed that  in permanent pools  the ostracods and
other scavengers and predatory  organisms,  including predatory mosquitoes,
may play a crucial role in maintaining a cyclic infection of coelomomy-

    Although data are  limited, there  is evidence that some pathogens of
 terrestrial and aquatic  insects are capable of persisting in the host
 habitat, outside of  living organisms, for several years.  There is a
 serious  need,  however, for more studies and data, especially under field
 conditions.  This review reveals  that information is scarce or lacking
 in  nearly  all  of the factors  listed  in Table  1.  Studies should be con-
 ducted also on problems  associated with the aquatic environment, such
 as  industrial  and agricultural pollutions, the eutrophication of lakes,
 and the  tremendous outbreaks  of marine organisms, such as the red tide.

     Some pathogens of  terrestrial  insects, i.e., Bao-Lllus thuring-iensis,
 Beauveria  bassiana,  and  Metarrhizium anisopliae, are facultative patho-
 gens  and may develop outside  their hosts.  Since they have been applied
 to  control aquatic  insects, especially mosquitoes, their persistence  in
 the aquatic environment  should be more fully  investigated.

     Insect pathogens may persist  in  the aquatic environment for long
 periods,  but their persistence would  not be expected to cause problems
 similar  to those of  chemical  insecticides, such as DDT, where the chemi-
 cals  accumulate in various  levels of  the food chain and increase in
 quantities, attaining  a  very  high concentration at the topmost trophic
 level of the predatory animals.   In  the case of pathogens, their host
 specificity would restrict  their  abundance mainly to one trophic level.
 Moreover,  the  pathogens  would be  much more readily degradable than the


 chemical insecticides because of the more numerous and varied external
 forces, living and nonliving, involved in their degradation.
                            REFERENCES CITED

 1. Andrews, J. D.  1965.  Infection experiments in nature with Dermo-
        cyst-idium marinum in Chesapeake Bay.  Chesapeake Sci.  6:60-67- '
 2. Andrews, J. D.  1966.  Oyster mortality studies in Virginia.  V.  Epi-
        zootiology of MSX, a protistan pathogen of oysters.  Ecology
 3. Andrews, J. D., and J. L. Wood.  1967.   Oyster mortality studies  in
        Virginia.  VI. History and distribution of Minohinia nelsoni,  a
        pathogen of oysters, in Virginia.  Chesapeak Sci.  8:1-13.
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MARSHALL  LAIRD:   I didn't know what I  was supposed to discuss until my
arrival here.  And that arrival coincided with Hank Aaron's 715th homer
which quite unfairly robbed me of the attention that an 8-minute dash
between the two furthest points of Atlanta airport really warranted.
And  thanks to my athletic achievement between flights last night I
wasn't really  in a condition to concentrate on my topic until after
breakfast and we had to wait an hour for that, and then the various
speakers  during the day have kept the lights off so I  haven't been  able
to write  very much.  And because Joe Tanada has so competently covered
the  scattered  literature on this subject, I'm going to restrict myself
to some hopefully provocative generalizations.

     First of a 11, I want to emphasize how little we know of pathogen
persistence in the aquatic environment by reference to an old favorite
of mine,  Coelomomyaes, once thought confined to mosquitoes.  It's now
known from some other Nematocera, from blackf1 ies and  from chironomids,
and  it's  known from Tabanus, too.  But there's an often overlooked  old
Russian record that goes almost back to the first description of Coelo-
momyaes,  and this one concerns an utterly unrelated insect, a Notonec-
tid  bug.  And Notonectid bugs prey on mosquitoes so the question arises,
was  that  Notonectid infected following eating a mosquito larva?  Oddly
enough, nobody anywhere seems to have followed this up in any real  detail
by undertaking really comprehensive surveys of the total  animal fauna of
even one  selected vector habitat in order to see whether, in fact,  infec-
tions do  occur in non-transparent aquatic hosts that are rather less
easy to handle than transparent dipterous larvae.  I'm really going to
address myself to the text "seek and ye shall find."  Earlier preachers
today have emphasized our still enormous lack of knowledge as regards
pathogens of aquatic stages of insect disease vectors  and pests.  I was
particularly struck this morning by Dr. Anthony's tables of our newly
acquired  knowledge,  none of it dating back much more than a decade, on
the entomopathogenic viruses of mosquitoes.  Of his 15 references,  11
concerned work involving quite a few species of mosquitoes, and seemed
to provide a basis for beginning to consider occurrence and incidence
of these  viruses.   However, more thoughtful consideration of his data
showed that what they really indicated was the occurrence and incidence
of Dr. Harold Chapman and his colleagues.  In fact, much of what we
know of the distribution of pathogens of vectors relates  equally well
to the success in obtaining research support and travel  grants  on the
part of Dr. Couch and such other long-term enthusiasts as Dr. Weiser,
Mr.  Muskrat, Dr.  Vago,  and so on.  If  we have to say this for mosquitoes
and  other vector pathogens we're in much greater ignorance as regards
pathogens of non-target aquatic organisms associated with mosquito
larvae in the aquatic environment.   While we know that some Nosema  of
lepidopterans  have several  hosts, and  that between order  infections
aren't common,  nothing  has yet been reported  on,  for example, whether
the  microsporidans of blackfly larvae  are able to parasitize their  ar-
thropod  predators.  And what about  their vertebrate predators,  since


many fish are commonly parasitized by microsporidans of genera parasi-
tizing aquatic insects?  We still don't pay sufficient attention to the
stabilization and standardization of terms, the use of which extend be-
yond frontiers.   I have the greatest respect for the fine work of the
early 19th century American entomologist Say, but like all non-Americans
I  don't accept his name "quinquefasciatus" which many of those who have
spoken earlier still quite wrongly apply to Culex pipiens fatigans.
Thus, I tend to divide my American friends into good Americans and bad
Americans, and the good Americans who use "fatigans" are still greatly
outnumbered by the ones continuing to use "quinquefasciatus."  I'd sug-
gest that perhaps it is a little more important that we take more care
about using the same name.  Especially when the particular case of a
cosmopolitan insect which in many parts of the tropics is of supreme
importance as the major vector of bancrofti and filariasis.  Workers in
a number of countries in different parts of the world would have diffi-
culty in recognizing the relevance to their problem of a Stempellia
which seems to have been described from something called "quinquefas-
ciatus" in the United States of America.

     I've really used that argument over what's in a name as a red herr-
ing because if we don't care enough to be tidy about standardizing in
this respect, one wonders whether we're really not a very long way away
from solving the very much more complex and demanding problems of what
constitutes specific criteria in pathogens, whether those pathogens are
look-alike viruses that may well have distinctive morphological criteria
beyond our present capabilities to detect, or microsporidans like Dr.
Maddox's that seem to move from genus to genus with temperature,   That
one smacks of Lysenkoism, I  must say.  Without a very much better appre-
ciation of the pathogen texts that are found on the various organisms of
complete aquatic ecosystems selected for individual  study, we are not in
a position to do more than venture educated guesses on questions  of host
specificity or indeed of the persistence of pathogens of insects  in
aquatic environments.  Among the relevant things we perhaps should be
doing are making really purposeful collaborative surveys for, and ap-
praisals of, candidate pathogens of mosquitoes and other aquatic  vectors.
There is a very effective mechanism (which has been mentioned today) to
aid in this connection.  I refer to John Briggs'  WHO international ref-
erence center for diagnosis of diseases of vectors.   This seems to me to
be a very valuable mechanism which perhaps deserves more support  than
it's currently getting for rapidly obtaining the kind of information on
candidates which we so clearly need.  I  believe we should also be as-
sembling really exhaustive inventories of the total  fauna and flora, and
their parasite-pathogen faunules, of individual and representative
aquatic habitats selected for intensive study.  So far, there isn't a
suitable pattern of international collaboration existing in this  par-
ticular connection.

    Finally, it might be observed that none of us and none of our organi-
zations are going to achieve the desired results in this field alone.
We're entering an era of integrated control that is immensely more com-
plex than anything ever tackled in applied entomology in the past.  It's


my firm conviction we need to call for sustained liaison and collabora-
tion among all interested parties in the relevant governmental, aca-
demic and industrial sectors.

R. B. JAQUES:  I'd  like to discuss the possibility of viruses used
against terrestrial insects entering the aquatic environment.  In this
I'm going to talk about the nuclear polyhedrosis virus  (NPV) of the
cabbage looper and  the granulosis virus  (GV) of the cabbage worm.
These are nuclear  inclusion viruses, of  the types which we are consider-
ing for use  in insect control.   I'd like to emphasize a point  that Dr.
Tanada made, namely that the GV and NPV  remain active stored  in water
for  long periods.   Here we are talking about distilled water,  I believe.
Naked virions, on  the other hand—virions taken out of  the  inclusion
bodies--don't survive long.  Here, it  is interesting that proteolytic
bacteria, found  in  the bottom of  ponds,  etc., have very little effect
 in decomposing the  polyhedral protein.   Stronger acids and weak alkalis,
of course, do dissolve proteins.  However,  I think it might be quite
 safe  to assume that inclusion viruses could persist  in fresh water, and
 I don't  know anything about marine water.
     I concluded  that viruses and  other pathogens as well reach the
 aquatic  environment by three paths.  The natural one is applying viruses
 directly  to  the  water surface.  The second path would be application of
 viruses  to  trees or crops growing in or  near water that may reach the
 water.   The  third  path, of course, would be infected insects dying  in
 locations from which cadavers could reach the aquatic environment.   I
 wish to  discuss  briefly why  I doubt that pathogens applied to crops or
 trees would  reach  the aquatic environment by other means.  We  know  that
 viruses  are  inactivated quickly by exposure to sunlight and so forth,
 and  over  5Q% of  the activity is lost  in  2 days.  If this is true, the
 virus is  not going  to survive long after we apply it.  Another consider-
 ation, of course,  is that  insects feeding on the foliage would die
 there and the  leaves might fall and end  up  in streams, eventually in
 ponds and so on.  However, virus  within  the cadavers does not  inactivate
 as  quickly as  this.  Another point in  relation to inactivation or loss
 of  virus activity  on foliage is that viruses are not washed off appre-
 ciably.   For example, several years ago  I put some deposits of virus on
 cabbage  leaves and  put them  in a  photoprint washer for about k days,
 and  I only  lost  about 25% of the  original activity.

     In regard  to inactivation of  virus in the aquatic environment,  how
 far  does UV  light  penetrate  into  water,  particularly into murky water,
as you would find  in a pond?

     Work done  in 1964 showed that in  regard to persistence of virus  in
 streams,  it  is significant that nuclear  polyhedrosis and granulosis vi-
 ruses remain active in soil for  long  periods.  A nuclear polyhedrosis
virus of cabbage looper remained  active  for 318 weeks.  There was still
a fair amount of activity after that  length of time.  Virus accumulated
 in the environment  over a period  of time, and these viruses, once put
 in the environment, do persist.   Concerning the persistence of these


viruses in soil, are they going to get into the ground water, etc.?
There  is little leaching of virus from the top layer of soil down into
the lower levels.   It  is interesting, in experiments carried on by my-
self and Dr. David  in  England, that when we pass immense amounts of
water, through columns of soil on which we'd put virus on the surface,
there  is little virus  percolating through the column of soil.  There-
fore, we would assume  viruses do not leach from the soil.   In clear
water or in suspensions of soil, it's rather interesting that NPV and
GV remain in suspension for about 2 weeks or more.   Therefore, in a
stagnant pool or pond  the virus would remain in suspension.   Dr. Anthony
and Dr. Clark mentioned that NPV of Aedes sollicitans accumulated or at
least  persisted in  the aquatic environment between epizootics.  But if
the pond dried up,  the virus was lost.  I  was wondering whether the
virus was actually  inactivated or had settled to the bottom, became ad-
sorbed onto the soil particles and remained there,  and when  the water
came back to the pond  it would not be on the surface.

H. C.  CHAPMAN:  Our Gulf Coast mosquito research lab has some data on
the persistence of various species of fungi, the Coelomomyces , in spe-
cific  ponds over a number of years.  Coelomomyces punctatus  has per-
sisted in larval populations of Anopheles crucians  in one particular
site for 8 years.  Both Coelomomyces dodgei and Anopheles crucians in
two particular ponds and Coelomomyaes pentangulatus and Culex peccatov
have persisted in these sites for over six years.  We first  found the
mermithid nematode, Rhisomermis nielseni,  in larva  of Uranotaenia sap-
phirina in 1965 in one particular pond, around Lake Charles, Louisiana.
Seventy-one percent of the larvae were infected in  collections from
this site last week, nine years later.  We have data showing persistence
of this nematode in mosquitoes in one other pond, for over 8 years.
Myxomermis petersoni, another mermithid nematode that happens to be spe-
cific  to Anopheles, has been found in larvae in a cypress swamp pond,
since  1967, though  infection levels have generally  been quite low.  This
nematode also has occurred in another swampy site for a period of 6
years with rather high levels of infection.  One hundred percent of a
collection of anophele larvae made last week showed multiple infection.
This was 6 years after we had initially found these pathogens in this
area.  Such persistence of species of Coelomomyces  and nematodes is
rather remarkable, considering our rather  frequent  droughts  and the ab-
sence of hosts for  long periods.  A number of years ago we made up a
very simple plastic container with an 80-mesh plastic screen in the
sides and bottom.  This made a good surveillance tool for studying per-
sistence of pathogens  in nature.  With this container and host larvae
from the lab, we've been able to determine the presence of various spe-
cies of Coelomomyces, mermithid nematodes, occluded viruses, and also
some microsporidans.  This is a good way to check for pathogen persis-
tence  in treated areas where the hosts do  not occur.

WILLIAM UPHOLT:   In general, with predators and parasites used for bio-
logical control  it  is fairly common that you can find some place in the


world where that particular parasite or predator actually keeps the
host organism so well under control  that the host organism is, for prac-
tical purposes, not a pest.  The only suggestion of this that I've heard
today in the case of pathogens is in the last comments,  by Dr. Chapman.

IGNOFFO:  Bacillus popilli-ae is an example of an organism which gives
long-term control.  There was a pine sawfly introduced into Canada and
areas of the northern United States  in which the virus associated with
that was also introduced, and that has kept that species in check, in
balance.  There is another record of a cabbage looper virus,  an isolate
from the United States, that was introduced into Colombia about 3 years
ago which has in the third year been successfully controlled.  In one
localized field they took the virus  from this and distributed it over
large areas, over an entire valley.   For the first two years  it signifi-
cantly held the populations down.  I'm sure this approach can be uti-
lized and I  think Dr. Roberts mentioned that somewhat in some of his
presentation.  I  wouldn't be surprised if we went back and looked at
our  introduced pests (some of which  are pests now and some of which are
no longer pests)  and may find diseases associated with these  introduced
pests, which have reduced the population below that constituting a
serious economic  threat.

TANADA:   I  would  like to enlarge on  this aspect.  In almost every study
which has been made on an insect population, disease plays some role  in
regulating  the insect population. With some pathogens,  the insect popu-
lation is maintained at a very low level, with others at a fairly high
level.  This is also associated with the dispersion of the pathogens.
If a pathogen has a wide dispersal,  it may maintain the  insect popula-
tion at a low level.

                        OTHER THAN INSECTS

                          Thomas  C. Cheng*


    Use of microorganisms for the control  of aquatic  pests other  than
insects is in its infancy, with the only known  field  trial based  on a
highly dubious premise that the bacterium under consideration  was patho-
genic.  Specifically, it was claimed that Baa-illus p-Lnottii, a Gram-
variable species, could be used to control  BiomphataP'i-a .glabrata,  the
major transmitter of the human blood fluke Schistosoma  mansoni; however,
critically conducted laboratory tests have revealed that  this  bacterium
is nonpathogenic.

    Except for molluscs, practically nothing is known about possible
use of microorganisms in biological control. What little has  been done
is directed almost exclusively to those species responsible for trans-
mission of disease-causing helminth parasites.   Microorganisms suggested
include bacteria, fungi, and protozoa,  with at  least  one  nematode, Dau-
baylia potomaaa, reported as a possible candidate.  Unfortunately, none
of these potential biological control agents currently  are in  culture.

    We recently have isolated four species of bacteria  from the desert
snail Theba p-Lsana originating in Israel.   Epizootiologic evidence sug-
gests that at least one bacterium is a  latent pathogen,  i.e.,  it  will
kill the host but only under lowered ambient temperature.  This evi-
dence, coupled with earlier studies on  the flagellate Hescamita nelsoni
in the oyster Crassostrea virginiaa, emphasizes the need  for increased
information on the internal  defense mechanisms  of molluscs before a
rational  search for candidate microorganisms can be initiated.  Spe-
cifically, we must know if the phagocytes of molluscs and other inver-
tebrates are chemotactically attracted  to invading organisms,  what in-
duces phagocytosis, and what is the fate of the phagocytized organism?
Such information is essential since an  effective biological control
agent should not become phagocytized, but if it does, it must  not be
degraded intracellularly.  Information  pertaining to  this systematic
approach to developing potentially useful  biocontrol  agents for aquatic
invertebrates other than insects is reviewed.
    *lnstitute for Pathobiology,  Center for  Health  Sciences,  Lehigh
University, Bethlehem, Pennsylvania 18015.


    Use of microorganisms as biological control agents of aquatic pests
other than insects has gained popularity during the last decade; however,
realization of this approach is still futuristic.  The major reasons why
such research is still in its infancy are (a) there is a lack of economic
pressure to develop programs with this objective; (b)  the traditional
type of undergraduate and graduate training being offered in the biologi-
cal and agricultural sciences has not stimulated students toward this
endeavor; and (c) although a rational approach has been developed and
followed for development of biocontrol agents for destructive insects
 (37), a comparable program has not been developed for  noninsect inverte-


    Certain  insects are well known as agricultural pests, vectors of
pathogens to man, animals, and plants, and as undesirable molesters of
humans  in such recreational areas as beaches, camp sites, and parks.
Consequently, the economic implications of their presence are vivid and
have been translated  into monetary losses.  On the other hand, although
a  number of  invertebrates are known to be noxious and  undesirable, the
full impact of their presence usually is not felt in those countries
which can afford to support research leading to their  eradication.  Cer-
tain species of non-insect, aquatic invertebrates are  associated with
seasonal problems in certain restricted areas, for example,  the occur-
rence of "swimmer's itch" or cercarial dermatitis in both fresh and salt
water areas of North America where recreation is an attraction.  Cerca-
rial dermatitis, however, is a seasonal problem and the resulting eco-
nomic loss is difficult to document.  Furthermore, chemical  eradication,
rather than biological control, is practiced.  Why, then, should agen-
cies sponsor a relatively long-term research program to control the
aquatic snails responsible for the emission of the dermatitis-causing

    Let us examine another example.  The occurrence of both  gastropod
and pelecypod molluscs in reservoirs and drainage ditches can result in
blockage of water flow (6A, 65).   Such problems essentially  are "in-
visible" to the general public and therefore policy makers are not im-
mediately concerned; thus support for research leading to new methods
of eradication is usually not available.  Where the situation is suffi-
ciently severe to warrant attention, traditional methods involving
chemical control  are applied with satisfactory results,  hence there are
those who question the feasibility of biological control.

    It  is well known that numerous species of aquatic  molluscs serve as
intermediate hosts for helminth parasites of man and animals.   Further
it is  agreed generally that the most effective method  of disrupting the
life cycles of these parasites is through eradication  of the molluscan
vectors.  With between 200 and 300 million cases of  human  schistosomiasis


in the world (7), there should be sufficient economic pressure to cause
national and international health agencies to support research on bio-
logical control of the gastropod vectors.  Unfortunately, this is not
the situation.   Human schistosomiasis is caused primarily by three spe-
cies of trematodes of the genus Sahistosoma, namely, S.  mansoni, S.
haematob-iton, and S. japon-ioum.  Like almost all parasitic diseases,
schistosomiasis, although debilitating and at times lethal, is a chronic
rather than an acute disease.  Consequently, although its role as a  pub-
lic health problem is generally recognized, it is extremely difficult
to assess its economic impact.  Furthermore, even if an  assessment is
made (72), the parameters employed are faulty and the correlations and
implications open to question.  Thus, without the expression of schisto-
somiasis in monetary terms,  it is difficult to convince  agencies to  sup-
port research  in chemical molluscicides, let alone biological  control.

    A similar situation is true for a number of other human helminthic
diseases such as clonorchiasis, paragonimiasis, and heterophyidiasis,
all involving aquatic molluscan intermediate hosts.  These serious para-
sitic diseases are endemic to developing countries whose limited budgets
cannot fund the necessary research.  Those interested in control of  ani-
mal fascioliasis are more apt to gain support than those concerned with
human helminthic diseases.  This is due to the fact that the loss to
the cattle and sheep industries, attributed to fascioliasis, can be
readily measured in dollars.  Cattle and sheep owners have considerably
more political  clout than the poor of underdeveloped nations who are
the primary victims of other  helminthic diseases.


    In American universities  the "core" of each institution is usually
the College of Arts and Sciences and the discipline of biology is
usually housed within this administrative unit.  Such colleges gener-
ally teach biology in the Oxbridge or the Germanic or Continental Euro-
pean tradition, i.e., great prestige is heaped upon esoteric research.
In recent years, trends of molecular and cell biology have swept the
academic scene.  Although these aspects of biology are valuable, they
also tend to breed a narrow viewpoint.  As a result, invertebrate path-
ology, as far as I can determine, is only offered at one American uni-
versity.  Of course, because of the mission of Colleges  of Agriculture,
insect pathology is taught formally at some of the land  grant colleges.

    There is no need to attempt to justify or dignify such disciplines
as invertebrate pathology or  parasitology.  This has been done in a  re-
cent publication (6).  However, development of biological control agents
for aquatic pests other than  insects can be equally as sophisticated and
challenging as any of the more esoteric areas of biology.  But it will
take more than a "popularity contest" to put invertebrate pathology  into
the mainstream of biology.  To establish a lasting mark, those of us en-
gaged  in research in this area must practice carefully designed and  me-
ticulously conducted scholarship.  It is only when this  becomes the rule
that our discipline will  be regarded as respectable.



    The development of successful biological control  agents should be
based on understanding:  (a) mode of entry,  (b) chemotaxis, (c) recogni-
tion of self from nonself,  (d)  intracellular degradation, and (e) mecha-
nisms of pathogenic!ty.

Mode of Entry

     If the target organism  is an aquatic  invertebrate, entry of  the
pathogen can only be effected via  the oral, body surface, and anal
routes.  Of course, in the  case of  poriferans, cnidarians, ctenophorans,
and  platyhelminths  the last mentioned  route is not available.

     Oral Route.—The feeding  habits of non-insect aquatic  invertebrates
have been studied intensively,  and  comprehensive reviews are  available
(25, 36).
     The feeding  response of cnidarians, which  include a  number of  toxic
species, has been the subject of considerable  investigation  (*»7).   Dur-
ing  feeding, most cnidarians  first  capture  and pierce their  prey with
their  nematocysts.  Subsequently,  a substance  escaping from  the  nemato-
cyst wounds causes  the tentacles to contract  toward  the  mouth and  the
mouth  to open.   Finally, upon making contact with the mouth,  the food
is  ingested.   In view of this series of feeding reactions,  it would  ap-
pear that a microorganism  for  biocontrol to be introduced via the oral
route  must be  accompanied  by  the molecule(s) that will cause  the host's
mouth  to open.   Some  information is available  on activators of the feed-
 ing  response.   Loomis  (51)  has  demonstrated that the reduced  form  of the
tripeptide glutathione is  the activator for Hydra littoralis, Fulton (29)
has  shown  that the  amino acid proline  is  the activator for Cordylophora
lacustris, Pardy and  Lenhoff  (59)  have demonstrated  that  the  marine hy-
droid  Pennaria t-iarella  gives a feeding response to  proline at concen-
trations as  low  as  10~6  M  and that  the proline analog pipecolic  acid
also serves as an activator.  Lindstet et al.  (50) have  shown that the
sea  anemone Boloceroides sp.  is stimulated  to  feed by valine.  Since the
 response  to valine  is  inhibited by  isoleucine, and not leucine,  it would
appear that  Boloaeroides  responds  to  the amino-n-butyric acid moiety
with a branch  point at the 3  carbon.

     The presence of certain ions in the aqueous environment  is known to
affect activators.  For  example, it has been shown that  ionic calcium
must be present  for the  feeding response  to glutathione  to occur (kB),
whereas Lenhoff  (kj)  has reported  that magnesium and sodium competitively
 inhibit the  feeding response.   Magnesium  ions  also inhibit  the feeding
 responses of Anemonia suloata (57)  and the  proline-stimulated feeding
 response of Cordylophova (29).   Among  other substances,  chelating  agents,
such as EDTA,  will  also  inhibit feeding of  cnidarians; however,  this in-
hibition can be  completely reversed by addition of calcium  ions  and  to
some extent by strontrium  ions  (48).   Potassium ions will decrease the
maximum response of hydra  to  glutathione  (k7)•  The  role of activators


and inhibitors of the feeding of cnidarians is important if biological
control agents are to be  introduced into these aquatic invertebrates
via the oral route.  Attention must be given to providing an appropri-
ate activator to stimulate ingestion.  Furthermore, the presence or ab-
sence of certain ions and molecules may dramatically alter the feeding
response.  This is true also of temperature (45) and perhaps other pa-
rameters.  Fresh-water cnidarians are not usually considered pests;
however, a number of marine species, especially the Portuguese man-of-
war, Fhysalia physalia, is an extremely toxic pest.  For a review of
species of cnidarians known to be toxic to man and animals, see Cleland
and Southcott (11) and Hal stead (30).

    Considerably less is  known about the feeding behavior of aquatic
platyhelminths, although  certain species of turbellarians are said to
be venomous  (30-  The major problem concerning flatworms is cercarial
dermatitis and schistosomiasis, both of which are contracted when free-
swimming cercariae invade the host's skin.  The biological  control of
cercariae has not been attempted or even investigated, although it is
known that hyperparasitism of larval trematodes by microsporidans will
cause abnormally developed cercariae (12, 13)i  However, since these
cercariae were situated within molluscs, it remains undetermined how
the microsporidans enter  the cercariae, although the oral route is sus-

    Nothing  is known of the factors that control or influence feeding
of the aschelminths and nemertines that occur in the aquatic environ-
ment, although certain species of infective larval nematodes that tem-
porarily occur in water certainly are health hazards to man and domes-
tic animals.  Cheng and Alicata (8) have reported that the third-stage
larva of the meningoencephalitis-causing nematode of the Pacific basin,
Angiostrongylus cantonensis, could be transmitted via the water route.
Whether the control of such larvae is feasible is moot.

    It  is known that certain species of marine annelids are toxic (11,
30, 58), however, no attempts have been made to control them chemically
or biologically.  Consequently, nothing is known relative to the oral
route of infection among  these aquatic invertebrates.

    Molluscs have been the principal focus from the standpoint of con-
trol, an interest stemming almost exclusively from attention to control
of schistosomiasis.  Furthermore, although biological control has been
tried periodically on a model.scale, the primary approach is still
chemical (7)-  As reviewed in a later section, several species of bac-
teria, fungi, and protozoa have been reported as potentially useful
biocontrol agents, but the available information does not indicate the
route of entry.  However, in the case of the microsporidan Steirihausia
(=Coaoospora) brachynema, Richards and Sheffield (61) reported that the
parasite occurs primarily in the intestinal epithelial cells of Biom-
•p'halar'la gldbrata, suggesting that its transmission is probably by in-
gestion.  Consequently, bait formulations should be given special at-
tention as they have with chemical control agents.

    A number of species of marine echinoderms are known to be toxic  (11,


31), but the only major echinoderm eradication program is that against
the crown of thorns starfish, Acanthaster planoi, in the Pacific (see
Branham  [3], for review).  The eradication was effected through direct
human efforts, rather than by biological or chemical means.  It has been
reported, however, that a few species of animals, such as the fishes
Abudefduf curaeao, Pseudobaliste flavimarginatus and Chellinus undulatus;
the triton shells, Charonia tritonses and Cymatiwn lotorium; the helmet
shell, Cassis aornuta; and the painted  prawn, Hymenoaeva elegans, may
serve, to a very  limited extent, as biological control agents because
of  their predatory habits.  No microorganisms have  been  proposed as
suitable control agents.  There  is  some information on the  feeding
mechanisms of echinoderms  (23, 28), although  the  implications of this
basic  information from the standpoint of  biological control  remains un-
determined .

    Body Surface  Route.—The composition  of the  body  surfaces of practi-
cally  all groups of aquatic  invertebrates is  known  to  some  extent  (25,
36).   The  importance of  such surfaces from the standpoint  of entry by
microorganisms remains unexamined.  With  aquatic  gastropods, Michelson
 (55) was able to  infect  k out of 20 Helisoma  trivolvis,  3  out of 5 H.
trivolvis fallax, 2 out  of 5 Biomphalaria glabrata, and  3  out of 5 B.
pfeifferi with an acid-fast bacillus by stabbing  each  snail  with a con-
taminated  needle.  Furthermore,  12  out  of 20  B. glabrata and  A  out of  10
B.  pfeifferi were  infected with  the same  bacterium  by  inoculating  the
snails.  Although these  studies were experimental and may  not reflect
what occurs  in nature, it  is of  interest  to note  that  the  bacterium was
 introduced  through the body surface.   In  natural  infection  of gastropods
by trematode miracidia,  one of the  most common methods of  entry is
 through  the epidermis.   Such entry  occurs with schistosome  miracidia
and their  molluscan hosts.  The  body surfaces of  molluscs,  and  possibly
 other  groups of  non-insect aquatic  invertebrates, are  known  to  serve as
 routes for  infection.  Only add! tional, .specially  designed  experiments
will  reveal  the  feasibility of  introducing biocontrol agents  through
 the body surface.

     Anal  Route.--Nothing is known relative to the  importance of this
 route  for  the entry of pathogens; however, the possibility  should  not
 be overlooked.

     Theoretically,  if  a  species  of  bacterium  is  to  be  effective  as  a
 biological  control  agent,  upon entry,  it  should  not  be immobilized  by
 the internal  cellular  reactions  of  the  target organism.   In  other words,
 it should  not be pnagocytized by the  host's hemolymph  cells.   One of
 the questions that  needs  to  be raised  is  whether  the phagocytes  of  the
 target organism are attracted to the  invading organism.   From  the stand-
 point  of biological  control,  this  is  a  very important  point  since chemo-
 tactic attraction,  at  least  in some instances, may  be  considered as a
 prelude to phagocytosis.   Unfortunately,  experimental  evidence correlat-


ing chemotaxis and subsequent engulfment of the foreign material is
currently not available; however, Cheng et al. (10) have shown that
chemotaxis between molluscan hemolymph cells and certain foreign mate-
rials does occur.  Specifically, it has been demonstrated that cells of
the American oyster, Crassostrea virginica, are attracted to the meta-
cercarial cyst of Himasthla quissetensis.   Recently in our laboratory
we isolated four species of bacteria from the desert snail Theba pisana
and found them to be lethal to this gastropod when the ambient tempera-
ture  is  lowered  (10-18 C).  These species of bacteria, particularly a
coccus,  are recognized as "self" by T. pisana and are not phagocytized.
In fact, this bacterium  is widely dispersed throughout the tissues, es-
pecially the muscles, of the snail   (Fig. 1).  The working hypothesis at
this  time is that this is a case of a latent lethal infection, which
becomes  activated when the ambient temperature is lowered, at which
time  the microorganisms  kill the snail.  The fact that leucocytosis is
not apparent in  the vicinity of the bacteria suggests that the host's
cells are not chemotactically attracted to the bacteria.   Consequently,
these microorganisms theoretically could be considered as a potentially
useful biological control agent.
FIGURE 1.  Photomicrograph showing nonphagocytized bacterial  (B)  inter
  mingled among myofibers of Theba pisana.   (Brown and Brenn  stain)
Recognition of Self from Nonself

    It appears reasonable that if positive chemotaxis occurs, engulf-
ment of the foreign agent by phagocytosis should follow, but this need
not be the case.  Chemotaxis and phagocytosis are two different phe-
nomena controlled by different mechanisms.  Phagocytosis is essentially
a manifestation of the cell's ability to recognize self from nonself.


However, exceptions do exist.  Michelson (55)  has reported  that in pla-
norbid snails, acid-fast bacteria can multiply within phagocytes and
presumably can be carried in them to uninfected tissues.   Pan (56) has
reported occurrence of yeast-like organisms in nerve cells and amoebo-
cytes of Biomphalaria glabrata that apparently were not destroyed.
Nevertheless, to find suitable biocontrol agents, one should search for
microorganisms recognized as self by the target organism and hence are
not phagocytized.   In vitro  testing of phagocytosis may be used as a
second screening for potential control agents.

    Relative  to phagocytosis, encouragement must be given to basic he-
matologic studies on invertebrates.  Although  the types of hemolymph
cells present in insects have been fairly well defined (33, 40),  this
is not true of other groups  of invertebrates.  Only  recently have the
types of hemolymph  cells of  two  common species of molluscs, Crassostrea
virgin-ica, and Meraenaria mercenaria, been defined  (26, 27).   It has been
shown that there are three types of cells  in  the hemolymph of  the mol-
lusc Meraenaria mevoenaria:  granulocytes, fibrocytes, and hyalinocytes
(27).  Furthermore, based on phagocytic  index  data when the cells are
exposed to Baa-Lllus megatevi-im,  it is known that the granulocytes are
the most actively phagocytic.  The ambient temperature influences the
rate of phagocytosis, all categories of cells  being more actively phago-
cytic at 22 and 37  C and being essentially non-active at k C  (Fig. 2).
Such studies  should be carried out with aquatic molluscs that are the
targets of biological control.   Those microorganisms potentially  useful
as control agents should be  applied under ambient conditions least fav-
orable to phagocytosis.

Intracellular Degradation

    Microorganisms  that become phagocytized are usually degraded  intra-
cellularly, although exceptions  are known.  Thus, as a part of  a  rational
approach to  the discovery and development of  biocontrol agents  informa-
tion must be  available on the enzymes present  in the phagocytes of the
target organisms.   Such  information should include optimal pH,  salt de-
pendency, temperature dependency, and other characteristics of  the en-
zymes.  Such  information could serve as guidelines for utilization of
microorganisms which are not susceptible to the degradation enzymes.
    We have  studied a number of  enzymes  in several species of molluscs
including Biomphalar-ia glabrata, the major vector for Schistosoma man-
soni.  Table  1 presents  findings relative  to  the specific activities of
several lysosomal and other  enzymes.  These data show that lysozyme oc-
curs in the  snail's hemolymph.   Consequently,  if a bacterium is to be
employed for  the biological  control of this gastropod, it should  be a
species which will  not be affected by the  lysozyme.

FIGURE 2.   Idiograms  showing  percentage of granulocytes,  fibrocytes,
  and hyalinocytes of Meroenaria meroenavia  that  have  phagocytized
  Bacillus megater'iwii at  A, 22, and  37 C,
  G = granulocyte;   F = fibrocyte;   H = hyalinocyte.
      TABLE  1.  Specific Activities of  Enzymes  in Whole Memo lymph
                       of Biomphalaria  glabrata
    Specific Activity
Alkaline phosphatase
Acid phosphatase
SGPT (serum glutamic-oxalacetic
SGOT (serum glutamic-pyruvic transaminase)
(3-g lucuronidase
 0.S   my/mg
 2.S   mu/mg
 7.3   Sigma-Frankel  y/mg

16.7   Sigma-Frankel  y/mg
29.8   Sigma y/mg
 0.13  Somogyi y/mg
 0.16  Sigma-Tietz y/mg
 0.035 AOD/mg

Mechanisms of Pathogenicity

    Since an effective microbial biological control agent for noxious
aquatic  invertebrates other than insects has yet to be established,
nothing  can be said relative to mechanisms of pathogencity.  Neverthe-
less,  in view of what  is known about viruses, bacteria,  and fungi  that
hold promise as control agents for  insects, it  is extremely important
that the mechanisms responsible for pathogencity be ascertained  for mi-
croorganisms  to be employed for control of molluscs and other aquatic
                      KNOWN PATHOGENS OF MOLLUSCS

    As  noted,  nothing is known about potential and real microbial con-
 trol  agents  for  non-insect aquatic  invertebrates except for species of
 molluscs  that  serve as  intermediate hosts for the human-infecting spe-
 cies  of schistosomes.   This topic has been adequately reviewed by Mich-
 elson (5A) and Malek and Cheng (53).  However, brief accounts of micro-
 organisms suggested as  possible biocontrol agents are presented below.


    No  virus has yet been  identified or  isolated from any species of
 medically important mollusc although virus-like particles have been re-
 ported  from  the octopus, Octopus vulgaris (62), and the oyster, Crasso-
 strea virginioa  (22).   Successful establishment of cell lines from mol-
 luscs has not  been achieved, although Cheng and Arndt  (9), Hansen and
 Perez-Mendez (32), and  Basch and DiConza  (l) have contributed media for
 maintaining  cells of B-iomphalcccia glabvata.   The medium of Cheng and
 Arndt has been employed successfully for transfer of cells through 30
 passages.  Since viruses are intracellular, obligatory parasites of
 their hosts' cells, molluscan virology has yet to become a reality.


    A few species of bacteria have been  reported from fresh-water gastro-
 pods  of medical  importance.  Berry  (2) reported a Gram-negative bacterium
 that  caused  an epizootic with high mortality in laboratory colonies of
 Biomphalaria glabrata, B. pfeifferi, and Physopsis africana.  This bac-
 terium, unfortunately, has not been maintained in culture.  Dias (17, 18,
 19) reported a Gram-variable bacterium from the ovotestis of Biomphala-
T-ia gldbrata.  This bacterium was originally designated as BET (bacilo
de esporo  terminal) and later (15) named Bacillus pinottii.  The orga-
nism  is saprophytic and develops a high  degree of virulence after re-
peated  serial passage in B. glabrata.  Although Texera and Scorza (69)
 in Venezuela and Dias and Dawood (20) in Egypt have reported killing B.
gldbrata  in  the field and  in the laboratory with B,  pinotti-L, Tripp (70),
 in carefully controlled laboratory experiments involving B.  pinott-ii.


from Dias1 laboratory, reported that this bacterium apparently is non-
pathogenic to B. glabrata.


    Although no pathogenic fungus has been cultured from a medically im-
portant species of mollusc,  it is of interest to note that Malek (52)
has reported that a species of the Fungi Imperfect! will kill both Bi-
omphalaria boissyi and Bulinus trunaatus in aquaria.  In addition,  Cow-
per  (14) has observed a species of Catenaria invading and destroying
the egg masses of Biomphalaria glabrata (Planorbis guadeloupensis), and
Michel son  (54) has reported that in a personal  communication from De
Meillon an unidentified fungus, originating from the hay and grass  in-
fusions used to feed the snails, has been observed to invade the tis-
sues of unhatched, embryonic Physopsis  in the laboratory.


    A number of ciliates have been reported associated with fresh-water
gastropods (16, 38, 41, 44, 70 but none appear to be parasites, or at
least pathogens, and hence hold little promise as biological control

    Hoilande and Chabelard (33) have reported heavy mortalities among
laboratory colonies of Lymnaea, Bithynia, Biomphalaria, and Bulinus due
to a flagellate, Dimoeriopsis destructor, and have suggested that this
protozoan may hold promise as a biological  control agent.  Another  fla-
gellate, Cryptobia (=Trypanoplasma) isidorae, has been reported from
the fresh-water pulmonate Isidora tropioa (21), but its pathogenic!ty
is unknown.  Cryptobia heliois is known to occur in the reproductive
organ of various species of pulmonate snails including Triodopsis albo-
labris, T. tridentata, Anguispira alternata, Helix aspersa, and Mona-
denia fidelis.  This flagellate is apparently nonpathogenic.

    Richards  (60) reported the occurrence of two species, Hartmannella
biparia and H. quadriparia,  in Bulinus globosus and Biomphalaria pal-
lida, respectively.  Because of the general structure of these amoebae,
the occurrence of contractile vacuoles  (which as a rule are not present
in parasitic amoebae), and their sporadic occurrence in aquaria, Rich-
ards concluded that these are free-living amoebae that have invaded the
gastropods as facultative parasites.

    Both H. biparia and H. quadriparia cause pathologic reactions within
their hosts.   Specifically, H. biparia occur as intracellular parasites
within the host's amoebocytes, which, in turn,  are surrounded by fibro-
blasts to form nodules.  These nodules, each enclosing several infected
amoebocytes,  occur throughout the digestive tract, digestive gland,
heart, kidney, reproductive system, and mantle.  Parasitized snails may
become moribund, and the presence of H. biparia is believed to affect
the growth and reproduction of Bulinus globosus.

    H. quadriparia also occurs within amoebocytes within nodules, which
occur in the foot, tentacles, along the edge of the mantle collar, and
in tissues lining the mantle cavity.  Infected Biomphalaria palHda
have been reported to be commonly sluggish and pale and may become mori-
bund.  Again, the growth and reproduction of infected snails are believed
to be interfered with.

    Richards has attempted to infect a number of other species of gastro-
pods through exposure to amoebae.  Results are summarized in Table 2.
Both species of amoebae could be cultured for several months in medium
NCTC  109 diluted tenfold with autoclaved tap water.

    Whether these two species of Hartmannella are of any use as biologi-
cal control agents remains to be tested.
    TABLE 2.  Occurrence of Hartmannella bipapia and H. quadbiparia
                     in various species of gastropods*

         Molluscan  Species            H. biparia**  H.  quadriparia**
Bulinus globosus
Bulinus forskalii
Bulinus guernei
Bulinus jousseaumi
Bulinus trap-Lous
Bulinus tmmoatus
Biomphalavia glabrata
Biomphalapia tenagophila
Biomphalapia pfeifferi
Biomphalara helophila
Biomphalaria dbstruota
B-iomphalaT-ia pall-Ida
B-tomphalaT-ia r-i-ise-i
B-iomphalap-la strami-nea
Drepanotrema simmonsi
Helisoma sp.
Indoplanopbis exustus
Bithynia sp.
Physa s p .
         *After  Richards,  1968.

        --I,  found  infected  in  laboratory aquaria;  E, found  infected
     after  experimental  exposure;  -, experimental  infection  unsuc-
     cessful;  0, experimental  infection not attempted.


    Microsporidans have been advocated as a major group of microorga-
nisms with promise as biological control agents but the available  in-
formation suggests that this group of parasites may not be effective as
control agents for molluscs.  Specifically, as pointed out by Cheng et
al .  (10), although the microsporida are widely distributed as intra-
cellular parasites of invertebrates (42) where they are commonly patho-
genic, they are rarely found in molluscs.  Even those species that have
been reported from molluscs occur as hyperparasites in trematode sporo-
cysts and rediae, e.g., Nosema eahinostomi in echinostome rediae within
Lymnaea limosa (4), Nosema dollfusi in Bucephalus auaulus sporocysts
within Crassostrea virginiaa (67), Perezia helminthorum in trematode
larvae in Malayan snails  (5), the unidentified microsporidan in Malayan
echinostome rediae (49),  the unidentified species reported by Schaller
(63) in larval trematodes in Tropi.di.saus planorbis, and Nosema stri-
geo-ideae in the intramol luscan stages of Diplostomum flexicaudum in
Stagnioola emarginata angulata  (34, 35)-  As hyperparasites, these mi-
crosporidans are protected from the phagocytic action of the mol luscan
hosts' hemolymph cells and most probably the intracellular enzymes.
The same holds true for the microspor idans Chytridiopsis myt-itovum and
C. oviaola reported from within the ova of Mytilis edulis and Ostrea
edulis, respectively  (2k, 43, 66).  Both C, mytilovum and C. ovicola
have been transferred to  the genus Steinhausia by Sprague et al. (68).
The only known exception at this time is Coeaospora braahynema,  which
Sprague et al. (68) also  have transferred to the genus Steinhausia.
This microsporidan is found primarily in the intestinal epithelium of
BiomphoLlavia gldbvata.  Thus, with this, except ion, it would appear that
the microsporidans, although potentially useful  as biological control
agents against insects, have not been able to overcome the internal de-
fense mechanisms of molluscs and only have been able to survive in this
group of invertebrates as hyperparasites.

    What  I have attempted to do in this presentation is to stress the
need for goal-oriented fundamental research that will lead to the find-
ing and development of microbial biocontrol agents for non-insect
aquatic invertebrates.  Only certain essential elements of what has
been termed a rational approach to attaining this objective have been
cited.  A variety of other factors need to be examined and a number of
new "tools" need to be developed.  Those concerned with microbial
agents for the control of insects know that studies aimed at under-
standing nutritional and other growth requirements of candidate micro-
organisms are essential.  For viral  control agents,  invertebrate tissue
culture must be developed to make viral cultivation possible.


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D. G. AHEARN:   I'll not try to add comments directly to Dr.  Cheng's ex-
cellent review on parasites of a variety of phylla.  I  will  supplement
by commenting on selected predation as a possible control  of blue-green
algae in aquatic habitats.  At Georgia State University we have been
studying the fate of blue-green algal blooms occurring  in the eutrophic
region of a  large fresh-water lake.  The blooms occur periodically in
an embayment region which is receiving secondarily treated sewage ef-
fluents.  Dense concentrations of the algal filaments float  to the sur-
face and in small coves.  At times several square miles of the lake sur-
face have been covered with a thick algal scum.  Shortly after this scum
appears, it rapidly changes in texture and within 2 to  3 days the sur-
face of the water develops a short-lasting, white scum.  The white scum
is composed mainly of an algophagous amoeba.  The large trophozoites are
capable of ingesting k to 5 algal trichomes at once.  Digestion of the
trichome, based on the disappearance of all but the heterocyst from the
time of ingestion, is about 2-1/2 minutes.

    This large free-living fresh-water amoeba is capable of  consuming
extremely large numbers of anabaena and more or less dissolving a nuis-
ance bloom within 2 or 3 days.  We have been unable to  grow  the amoeba
on bacteria or stock cultures of anabaena which we have obtained from
type culture collections.  We have had laboratory blooms by  feeding the
amoebae with anabaena from the lake.  We hope to rear this amoeba in the
lab to the extent that we are able to seed algal blooms and  initiate di-
gestion.  Our preliminary report has been published (197^0 in Water, Air
and Soil Pollution 3=71-80.

R. CHARUDATTAN:  I was very much interested in what Dr. Cheng mentioned
in the case of snails and the Mycobacterium, the recognition of self vs
non-self.   I  assume that the recognition of self vs non-self in this
example and similar examples is based on the presence of common antigens
appearing in the bacterium and the host.  I was involved in  a study simi-
lar to this with plant pathogens and higher plants.  Perhaps you could
devise a simple and efficient serological test to denote the presence  or
absence of common antigens.  I suppose where you do have common antigens
you don't have phagocytosis.  Where common antigens are absent, the


bacterium is destroyed by the host.  Perhaps this would be an efficient
method to screen hundreds of organisms in the laboratory.   I  would like
to do this with plants, but the plant system is a little difficult to
handle.  Invertebrate  immune systems are more organized,

CHENG:  Searching for common antigens is a good preliminary screening
method, I agree, but  I'm sure you also recognize that phagocytosis
could be due to factors other than common antigens.  There is some in-
formation in the literature that mucopolysaccharides elicit endotosis
by the host cell.  The unanswered question at this time is what induces
phagocytosis, especially in invertebrates?

A. M. HEIMPEL:   I have two points.  One  is that  I don't think Dr. Cheng
completely covered the area of possible biological control, and we have
this  in insect pathology as well, of the diseases of beneficial animals.
In shrimp, oysters and crabs there are a few diseases, some of which
are  bacterial.  The possibility of using phages against these should be
     The second point  is your concern about the number of schools that
teach  invertebrate pathology.  We in insect pathology and invertebrate
pathology have a lot of basic research to do for there is a dearth of
good  information to work from.  When insect pathology started in this
country about 25 years ago under Ed Steinhaus, there was only about one
school offering a course in this topic.   It is my opinion that we
shouldn't train too many invertebrate pathologists.  As Steinhaus said,
both pathology fields cross over disciplines.  I  think it's better if
you  have  individuals  that have no knowledge of insect pathology from
separate disciplines who enter invertebrate pathology and contribute
their  specialties.  If you look around at the insect pathology labora-
tories, for example,  the one in Beltsville, Maryland, biochemists, ento-
mologists and microbiologists are all contributing to the field.

SAM  SINGER:   I don't want to belabor the point in terms of B. pinottii,
but  throughout the symposium we've heard of this or that lost culture.
In my own work,  it's  very difficult to maintain active cultures.  We
were interested  in this B.  pinottii culture and spoke to Dr. William
Haynes of the Northern Regional Laboratory of ARS, especially when his
and  Ruth Gordon's new monograph was published.   I asked him for the cul-
ture, which he sent along with a letter saying that they had corresponded
with the Oswaldo Cruz  Institute and had the feeling that the B. pinottii
sent  to the ARS was the wrong culture.  Diaz died in the late fifties
and  the work of Tripp was in 1961.   It could very well be that the cul-
ture with which he worked was B. sphaevicus.  The point is that there
are  bacteria, fungi, etc.,  which are obviously active, otherwise we'd
be flooded with all these diseases.  Thus, let us not overlook the pos-
sibility of these things.  When we hear of Bacillus matthesi and so
forth being "lost," we should be a little more optimistic and hopefully
we will find either B. pinottii or some organism like it.


S. R. DUTKY:  Dr. Cheng mentioned that the lack of phagocytosis is a
favorable characteristic of a pathogen.  That probably is true but the
best pathogens aren't necessarily those that exhibit all  the best points.
I  think  it would not be a good  idea to eliminate consideration of a
pathogen on the basis that  it was phagocytized, because many of the
"good" pathogens of man are "bad" ones from this standpoint.  The occur-
rence of phagocytosis only  increases the number that is required to pro-
duce a threshold infection.  This does not have anything  to do with its
efficiency.   If it can produce  these numbers by multiplying in the host
and destroying the phagocytes,  then the phagocytes only become carriers
of the pathogen to various areas.

CHENG:  What you say is quite true, but from our experience with mol-
lusks, if you introduce a bacterium and it becomes phagocytized, almost
all become  intracellularly degraded.

DUTKY:  That's only a question of numbers.  If the exposure level  is
raised by a factor of one decade, then perhaps not all  of the intro-
duced bacteria will become degraded.

CHENG:  That  is, if they multiply within phagocytes and/or are not sus-
ceptible to the cells1 enzymes.

DUTKY:  No, only if the initial exposure gives you a number which will
saturate the capacity of the phagocytes to take them up then the phago-
cytes are overwhelmed.  In other words, you may require ten times  the
dosage in order to get the  introduced bacteria to be effective.

CHENG:   I am not sure such a phenomenon has been demonstrated in mol -
lusks.  However, I  would think  that it could be demonstrated easily by
inter-hemocoelic injections.  One could introduce a number of bacteria
and the mollusks are able to recover.  However, beyond  that number they
do not recover.  Specifically, a possible relationship  between the mor-
tality and  ineffective phagocytosis should be checked,

MARSHALL LAIRD:  Somebody mentioned Bacillus matthesi and black flies.
As I  recollect, what was called Baateviim matthesi was  actually isolated
in 1935 from Guacina, in what's now Tanzania, and this  bacterium was
said to have been lost.  This phrase keeps cropping up  about all these
lost cultures and I wonder whether sometimes we sound unduly pessimistic
in that some of the earlier work in these areas was really so trivial
that there never was any attempt to establish a culture in the full
sense of the word.   Perhaps we should go and look again and do now what
wasn't done 20 or 30 or whatever years ago.

IGNOFFO:   From what I've heard reported from the review and what little
review I've done in this area,  I have yet to find a systematic search


that selects a particular pest and  then go to see  what's  there.   Ob-
viously, those that will  be observed will  be the virulent types  of
pathogens.  But, in fact, the ones  you might want  to  use  are the de-
bilitative type, which reduce the host population  down  to a  low  level
and yet survive in the aquatic environment.  I  think  one  of  the  first
things to do is select the model, and search for that model.  Concur-
rently, what you can do is utilize those known pathogens  that are
available.  And from what we've heard here, there  are some groups that
don't have the specificity of other groups.  Is it possible  that one of
these microsporidans might in fact  be an excellent pathogen  for  the
snail that transmits schistosomiasis?  I'm just suggesting this, ac-
tually, as a systematic approach to solving a particular  problem. Now
if you select the right experimental animal, the basic  information you
want to elucidate will be forthcoming.  A  lot of us do  not do this.
The  impact we can make from our findings is not as great  as  it can be
because of our poor selection of the model  to begin with.


                           R.  Charudattan**


    Among natural enemies of plants the most versatile and  ubiquitous
are pathogenic and antagonistic microorganisms.  In this category are
fungi, viruses, bacteria, nematodes, mycoplasms and possibly Rickett-
siae.  Each species of plant is subject to its characteristic  diseases
(38).  That diseases can be utilized to combat unwanted plants is shown
conclusively by successes in controlling terrestrial  weeds  like North-
ern jointvetch (10), skeleton weed (9, 15),  and others (37).   Indeed,
biological control of water hyacinth with a  Fusarium disease was  con-
templated as early as 1932 (1).  However, despite the long  existence of
aquatic weed problems in the United States and abroad, successful  con-
trol of water weeds using microorganisms remains to be accomplished.
The reasons for this are many, among which lack of  studies  on  aquatic
plant pathogens is perhaps the most important
    Several species of aquatic weeds pose serious problems  in  Florida.
The more significant ones in order of importance are:   water hyacinth
(Eichhomia erass-ipes [Mart.] Solms.), hydrilla (Hydrilla vertiaillata
Royle), al 1 iga tor weed (Alternanthera philoxeroides [Mart.]  Griseb.),
and Eurasian water milfoil (Myr-iophyllwn spicatim L.).   Studies  at  the
University of Florida to seek and evaluate plant pathogens  and phyto-
toxic organisms as biocontrols of water weeds were started  in  1970.
Since then, we have been concerned with all  four species mentioned
above, with major emphasis on water hyacinth and hydrilla.

    Our work has consisted of six major facets:  a)  establishing a
quarantine facility and developing procedures for bringing  foreign
pathogens of aquatic plants  into the United  States from various  geo-
graphic regions; b) developing techniques to study diseases of aquatic
plants; c) conducting local  and foreign explorations and seeking effec-
tive pathogens of the four target weeds; d)  screening available  patho-
gens of aquatic weeds to determine their efficiency, host range, and
suitability as biocontrols;  e) testing on a  limited  basis two  fungal
pathogens on water hyacinth  in the field; and f) testing and evaluating
microbial metabolites toxic to hydrilla.  The following general  proce-
dure is used for testing and evaluation of pathogens:   a) isolation  in
    ^Florida Agricultural Experiment Station Journal  Series  No.  5A83.

   **Assistant Professor, Plant Pathology Department,  University of
Florida, Gainesville, Fla. 32611.


pure culture, identification, and artificial inoculation on weed hosts;
b) establishment of pathogenic!ty and aggressiveness on target hosts;
and c) determination of host range from published information and from
our own studies.  Once a promising pathogen is found, limited field
tests are designed in order to determine its performance under natural
conditions, and to ascertain possible environmental  impacts of its arti-
ficial introduction.
                            REVIEW OF WORK

    A detailed review of research conducted at the University of Florida
 is available  (13).  The following is a summary of our findings concern-
 ing the four  target weeds.


    Several local and foreign explorations were undertaken to collect
 and test  pathogens of aquatic weeds.  Some pathogens of water hyacinth
 and alligator weed were found locally (17, 29), although several patho-
 gens reported abroad were not located in the United States.  Diseases
 of hydrilla and  Eurasian water milfoil were not encountered in Florida.
 As these  weeds entered this country, their pathogens were not necessarily
 among them.   Once here, and free of their pathogens, such plants may be
 free of major diseases for long periods of time until original pathogens
 are introduced (14).  Foreign explorations were considered important
 because these plants are likely to be in balance with their natural
 enemies in their native habitats.  Thus, the probability of finding
 suitable  pathogens is considerably greater in areas from which these
 plants originated.  The following countries were surveyed for plant
 pathogens:  Barbados, Jamaica and Trinidad of the West Indies, the Do-
 minican Republic, El Salvador, Guatemala, India, Mexico, Panama, Puerto
 Rico and  Venezuela.


    Introduction of foreign microorganisms into the United States must
 be approached with utmost caution.  The potential dangers to local agri-
 culture and to environment from such  introductions must be evaluated
 before any large-scale use of imported pathogens can be contemplated.
 For this  purpose, a quarantine greenhouse facility was built in Gaines-
 ville in which all foreign isolates suspected to be pathogens of the
 four target weeds were housed with maximum security.  The quarantine fa-
 cility was approved by the U.S. Department of Agriculture and the Florida
 Department of Agriculture and Consumer Services.  To date, about 600
fungal  and bacterial isolates and two suspected viruses of foreign ori-
gins have been tested on water hyacinth and hydrilla in this  greenhouse.

                        PATHOGENS OF TEST  PLANTS


    A list of  identified  pathogens of water  hyacinth and  their  presence
or absence  in  the  United  States on water  hyacinth and other hosts ap-
pears in Table  1.   Of  these,  isolates of  Rhizoatonia solani Kuehn from
India,  Panama,  and Puerto Rico have been  studied  intensively  (5, 11,
18, 29), and  in artificial  inoculations .was  found to be the most aggres-
sive of the pathogens  on  water hyacinth.  R. solani  induces severe,  ir-
regular lesions on leaves,  blighting, and often death of  plants  (Fig. 1).
Studies by Joyner  (18)  indicated that isolates of this fungus from
Panama, Florida, and  in culture collections  in this country varied with
regard  to virulence,  temperature optima for  infection, host specializa-
tion, and ability  to  infect emersed versus submersed portions of aquatic
plants.  All  isolates  tested were capable of attacking 1^ species of
aquatic plants  of  9 families.  According  to  an early report (k) Rhizoo-
ton'La solani. was responsible for destruction of valuable aquatic plants
in Virginia and North  Carolina that were  a source of food for ducks.
In areas where  this pathogen was found on water hyacinth, it did not
seem to affect  other vegetation (29).  The potential of this fungus as
a biocontrol agent of  water hyacinth cannot  be ignored (13).

    A zonal leaf spot  of  water hyacinth caused by Cephalosporiwn zona-
twn Sawada was  found  in Puerto Rico, Florida, and Louisiana (Fig. 2).
A disease with  identical  symptoms has been ascribed to C. e^Lohlnofniae
Padwick in  India (2k).  The two species,  as well as C. fiat Tims and
Olive,  the causal  agent of  leaf spot of fig, are probably synonymous
(30).  The pathogen is  quite virulent and destructive on  leaves of
water hyacinth, often  causing necrosis of most of the leaf area. Green-
house tests on  this fungus  indicated a broad host range.  Of 17 plants
(12 families)  tested,  16  were susceptible, including one submersed
aquatic plant  (30).  Despite this wide host  range under laboratory con-
ditions, C. zonatum attacks naturally only fig (3*0 in North America.
In view of  its  narrow  host  range in nature,  this pathogen has good po-
tential as a biocontrol agent of water hyacinth.

    A leaf blight  of water  hyacinth caused by AlternaTia eiohhorni-ae,
Nag Raj and Ponnappa,  from  India, may hold promise as a biocontrol
agent (21).  This  fungus  was among those  isolated from that country by
this investigator.  Tests  in India indicate  it is highly host-specific.
Of Al species  (19  families) tested, including 7 aquatic plants, only
Monoahoria vaginalis Pers.  of the same family as water hyacinth was
susceptible to  this pathogen.  In addition, A.  eiahhovn-iae produces a
metabolite of pigment origin toxic to water hyacinth (21).  Tests with
this pathogen  in our quarantine facility  have been encouraging.  It is
a  virulent pathogen of water hyacinth (5) and may prove useful  in Flo-
rida as a biocontrol of the plant.

       TABLE  1.   List  of  Water Hyacinth Diseases, Causal Agents
                     and  Their Presence or Absence
                          in the United States

                                                    Presence or absence
                                                      in United  States
Di sease
Leaf spot
Zonal leaf spot
Leaf spot
Leaf spot
Leaf spot
Leaf spot
Leaf spot
Thread bl ight
Leaf spot
Leaf spot
Leaf spot
Leaf spot
Leaf spot
Bl ight
Causal agent On water On other
hyacinth hosts
Altemaria eiahhorniae *
Apiooarpella sp. +7
Cephalosporium zanatwn (= C. eichhorniae) + +
(= C. fid)
Ceraospova piavopi +
Curvularia clavata
C. lunata + +
Doassansia eichhorniae
Dreahslera sp.** ~ 7
Fusarium rosewn (= F. equiseti) + +
Helminthosporium biaolor
Marasmiellus inoderma
Myaoleptodiseus terrestris** + +
Myrotheaium roridwn - +
Nigrospora sphaeriaa + +
Pestalotia sp. + +
Phoma sp.** + +
Rhizoctonia solani (= Cortioiwn solani) + +
(= Bypoahnus sasaki)
Sigmoidea sp,** + ?
Uredo eiohhorniae
          *Compiled from references 29, 35,
         **Charudattan, unpublished data.
     A variety of Myrotheciwn roridum Tode ex Fr. from India was re-
 ported to be pathogenic to water hyacinth (20).  Several isolates of
 this pathogen were  collected and tested in Florida (5).   It is highly
 pathogenic to water hyacinth, but is also known to attack a wide va-
 riety of other plants  (27).   In this country it is a significant patho-
 gen of ornamental plants  (35)-   Further tests on the host range of this
 organism are continuing.   Recently,  an  isolate of  Dreahsleva sp. has
 been obtained from water hyacinths  in the  Dominican Republic.   In pre-
 liminary tests,  this organism equaled R. solani in  its pathogenicity
on  water  hyacinth.   Further  tests are underway.  Cercospora piaropi.
Tharp. was described in 1917 on Piaropus crassipes  (= E. ovassipes)
from  Texas  (33)-   Its occurrence in this country has recently been  re-
confirmed  (12).   Since species of Cercospora are frequently host

FIGURE 1.  Blight of water hyacinth incited by Rhizoctonia solani:
  a)  Blighted leaves of anchoring water hyacinth (Eichhornia arassipes)
  from Panama.  b) The fungus from leaves A;  sclerotia,  marked by
  arrows, can survive in water for over 26 months,   c)  Dead water hya-
  cinth plants (E. crassipes), inoculated with B (right)  and noninocu-
  lated controls  (left).  From Rints (29).

                                   E  Wi
FIGURE 2.  Symptoms and morphological characteristics of Cephalosporium
  zonatum:  a) Zonate lesion due to C,  zonatum on  water  hyacinth  leaf
  b)  Lesion on narrow-leaved pickerel weed  (Pontederia lane eo la to.) .
  c)  Lesions on a leaf of variety Celeste of  fig.   d)  Single  branched
  conidiophore with spore heads (X360).   3) A  specific diagnostic fea-
  ture:   a strand of funiculose hyphae  bearing  con idiophores and spore
  heads  (X80).   f)  Con idiophores  and  conidia  (X800).   From Rintz (29)

 specific,  tests are being  conducted on  the  usefulness of  this  pathogen
 against water hyacinth.  Several other  pathogens listed  in  Table 1  have
 been tested  and maintained  in our collection.   Their use  in control of
 water hyacinth may be obscure due to their  lack of aggressiveness on
 the plant.

     A number of bacterial  isolates have been obtained as  potential
 pathogens  of water hyacinth.   However,  none of  the isolates tested  so
 far has proven pathogenic.   Numerous plant  pathogenic viruses  were
 tested on  water hyacinth,  hydrilla and  alligator weed (Table 2),  but
 none infected any of these weeds.
             TABLE 2.
Plant Viruses  Tested on Water  Hyacinth,
 Hydrilla and  Alligator Weed*
 Virus Group**
Indicator Plant Species***
Bromovirus     Brome  mosaic
Cucumovirus    Cucumber mosaic
Nepovirus      Tobacco  ringspot
Potexvirus     Clover yellow mosaic
               Cymbidium mosaic
               Papaya mosaic
               Potato virus X
               Papaya ringspot
               Sugarcane mosaic strain E

               Tobacco  etch
Tobamovirus    Odontoglossum ringspot
               Tobacco  mosaic  (aucuba strain)
               Tobacco  mosaic  (type strain)
Monotypic      Tobacco  necrosis
Other          Barley stripe mosaic
               Bacilliform virus isolated
                 from Southistle
               Hippeastrum mosaic
               Da sheen  mosaic
               Onion  yellow dwarf
               Southern bean mosaic

               Wheat  streak mosaic
                         Horedeum vulgare  'Moore1
                         Chenopodium amarantiaolor
                         Niaotiana tabaaum 'Turkish1
                         Pisum sativum 'Alaska1
                         Cassia tora
                         Gomphrena globosa
                         G. globosa
                         Comphrena globosa
                         Zea mays va r.  saaaharata
                           'Golden Cross Bantam1
                         N, tabaaum 'Tu r k i s h'
                         C, amarantiaolor
                         N, tabaaum 'NN'
                         N. tabaaum 'NN'

                         N. tabaaum 'Turkish'
                         H, vulgare 'Moore'
                         N, olevelandii x
                           N,  glutinosa
                         Hippeastrum sp.
                         Philodendron selloum
                         Allium aepa
                         Phaseolus vulgaris
                           'Kentucky Wonder Wax1
                         Tritiahum aestivum
                           'Georgia 1123'
    *F.  W.  Zettler, unpublished.
   **After  P.  Wildy's Classification and Nomenclature of Viruses,  1971
(Monographs in Virology.  S. Kargar, Basel).   "Other"—not in Wildy's  list.
  ***Inoculated before and after each trial and also used for back inocu-
lation  attempts.


    Diseases affecting hydrilla were unknown prior to our study.  In
1973, two diseases of hydrilla from India, caused by a Pythium species
and Sclerotiim rolfsii were found (5).  They induced chlorosis, yellow-
ing and lysis of test hydrilla plants (Fig. 3).  In addition, about 15
isolates of Aspergillus, Penicillium and Triahoderma that produce me-
tabolites toxic to hydrilla have been found from Florida and  India
(Table 3).  Twelve of these have been reported to produce substances  in
liquid cultures toxic to hydrilla  (6).  The toxic culture solutions in-
duced chlorosis of test plants followed by death and  lysis of  the dead
remains of  plants  (Fig. k).   The effects of toxins were visible within
six  days after mixing the  culture  solutions.   The  toxin of four of  the
isolates was  oxalic  acid  (6),  known to  be  involved  in  diseases caused
by Sclerotinia schlerotiorum  (19)  and Selerotium rolfsii  (3).   Since
oxalic acid is a  potent biotoxin,  its potential  in controlling hydrilla
is considered poor.   Studies  are underway to  identify and determine the
nature of other  toxins  from fungi,  hopefully  with  more specificity  than
oxalie acid.
                 TABLE 3.   Fungi  Pathogenic to Hydrilla*

               Fungi                              Source

             Pythium  sp.                        India
             Sclerotium rolfsii.                 India,  Florida
             Cha.etomi.wn sp.**                   India
             AspevgiHus spp.                   India,  Florida
             Peniaillium spp.                   India,  Florida
             Tviafaoderma vivide                 India,  Florida
                 '•Charudattan  (5) •
                **Charudattan,  unpublished data.
     A  virus-induced  stunt of alligator weed, characterized by an over-
all  stunting of  the  plant, was  found  in  the Ortega River near Jackson-
ville  in  1971  (17).  Affected  leaves were smaller than normal, reddish
 in  color, and often  distorted.   Electron microscopy of affected leaves
showed  presence  of a flexuous  rod  type virus (Fig. 5).  The nature and
length  (1587-1781 nm) of the virus particles suggested that the causal
agent  belonged to the beet yellows group of viruses (17).  Members in
this group are aphid-transmitted.  However, laboratory transmission was
not  accomplished using Myzus persicae and Aphis gossypii which normally
colonize  alligator weed.  The virus was graft transmissible (Zettler,
unpublished), but other methods  of mechanical transmission were unsuc-
cessful.  The virus  persists in  plants vegetatively propagated from

    FIGURE  3.   Damage  to  hydrilla  caused  by  Sclerotium rolfsii and Pythium sp,   1)  Hydrilla with discolor-
     ation  especially of  the  growing  tip induced  by Sclerotium,  2)  Damage due  to  Pythium.  Arrows  point
     to development of green  axillary shoots.   3)  Control.    From Charudattan  (5).

    FIGURE 4.  Test tube assembly used for testing pathogenicity of organisms to hydrilla  (a).   Control
      (b) and Peniailliwn-\ nf ected hydrilla  (c).  Note the  pale appearance of hydrilla  in  (c) due  to  the
      fungus.  Lysis of hydrilla caused by toxin from Penicilliwn  (d) and control  plant  (3).    From
      Charudattan (5,  6).

FIGURE 5.  Virus stunt of alligator weed.   A)  Healthy (right)  and  dis-
  eased  (left) alligator weed plants;  infected plants are markedly
  stunted and grow at a slower rate than healthy plants.   B)  Virus par-
  ticles (arrows) as seen with the electron microscope.   C)  Magnified
  view of shoot apex of infected alligator weed plant showing  pro-
  nounced foliar distortion and twisting as compared  with healthy  shoot
  apex of this plant (D) at the same magnification.   From Zettler, un-
  publi shed .

diseased material, and holds promise for controlling alligator weed.
Unfortunately, this virus is not readily transmitted in nature, as re-
peated searches subsequent to 1971 in the Ortega River failed to dis-
close its presence.  Table k includes known pathogens of alligator weed,
of which the significant one is the virus.

                 TABLE A.  Pathogens of Alligator Weed
Occurrence in
United States
      Alligator weed stunt virus
      Rhizootonia solani
      Heterodera marioni
      Anguillulina dihystera
      Uredo nitidula
      Meloidogyne sp.

     Surveys  in Florida for diseases of Eurasian water milfoil did not
 disclose any  pathogen of this plant.  Pathogenic!ties of several fungi
 from other plant hosts were tested on this plant.   Included were four
 species of Fusarium, 11 species of Pythium and seven species of Phytoph-
 thora.  The  latter two taxa are phycomycetbus genera and produce zoo-
 spores motile in water.  Another phycomycete, Aphanomyaes euteiches,
 also was tested  in Eurasian water milfoil because of its high degree, of
 virulence on  Echinodorus brevipedicellatus, a submergent aquarium
 plant (28).   None of these pathogens  infected Eurasian water milfoil,
 however  (16).  Several bacteria obtained from diseased Eurasian water
 milfoil plants were non-infective under  laboratory  inoculations  (16).
 Only a Panamanian  isolate of Rhizoctonia. solani from Eichhornia azurea
 was  pathogenic to this plant and Myriophyllum brasiliense.  The fungus
 caused local  necrosis on submersed portions of the  stem and consequent
 toppling of  portions above necrotic zones.  Usually healthy side shoots
 emerged from  uninfected portions of the  stem.  Due  to its inability  to
 kill  the plant, this isolate of Rhizoctonia was not considered a likely
 biocontrol for Eurasian water milfoil (16).

     Limited field  tests have been conducted with R. solani and C. zona-
 tum  on water  hyacinth by T. E. Freeman  (unpublished).  None of the
 other  pathogens or  toxic isolates are currently being field tested on
 any  of the other  three target hosts.  The test site for field trials
 with water hyacinth  is in Lake Alice,  situated within the boundaries


of the University of Florida campus in Gainesville.  The area involved
is approximately 30 ha in size and has been infested by water hyacinth
for several years.  For  inoculum, fungi were grown in flasks containing
Czapek-Dox broth for l*t days.  Mycelia and spores were harvested, ho-
mogenized  in a blender, and sprayed on water hyacinth plants with a
pneumatic  sprayer.  Pathogens were applied singly and in combination.
Plants became readily  infected within two weeks, and characteristic
symptoms were visible as early as a week with R. solani and in two
weeks with C. zonatwn.   In two months, secondary spread of C.  zonatum
was apparent.  The disease incited by R.  solani was, however,  less than
expected.  These trials showed that the two organisms can be utilized
for artificial induction of diseases on natural populations of water
hyacinth.  Additional field tests with C.  zonatwn are in progress to
determine  its pathogenicity to several terrestrial crop plants.
                          FUTURE PLAN OF WORK

    A top priority  in our work is to obtain rust and smut pathogens of
water weeds reported from South America.  Rusts and smuts, theoreti-
cally, are the most  ideal pathogens to control weeds.  Due to their
host specificity, virulence, and ability to invade reproductive organs
of plants rusts and  smuts are likely to be effective as biocontrol
agents.  Uredo eiahhorntae Frag, and Cif.--a rust, and Doassansia eieh-
horniae Ciferri--a smut, have been reported on water hyacinth from the
Dominican Republic  (8, 7).  A rust, U. nitidula Arth., was discovered
on alligator weed from Guatemala (2).  However, a recent search in the
former country (Charudattan, unpublished) and an earlier survey by Hill
and Rintz (unpublished)  in the latter did not reveal the presence of
any of these three pathogens.  Literature on South American rusts is
scanty, and personal correspondence with 25 plant pathologists in 13
Central and South American countries did not establish either the pres-
ence or absence of these three or other obligate pathogens of target
weeds.  Currently, attempts are being made to rediscover the above
three pathogens, but excessive reliance on them for biocontrol of
aquatic weeds may be unwise.
    Another current area of intensive investigation is possible use of
C. sonatwn in an integrated control of water hyacinth along with the
weevil, Neochetina eichhorn-iae, and the mite, Orthogalwnna tevebrantis.
This is a joint study with Dr. B. D. Perkins, USDA-ARS, Fort Lauderdale.
Evidence suggests that water hyacinths in Florida infested with the
above arthropods are invaded readily and colonized by a variety of
facultative pathogens and wound parasites resulting in severe leaf ne-
crosis, senescence and/or root damage.  Dead plants were often seen
among severely infected water hyacinths.  Among fungi isolated from
insect-infested plants, the most significant pathogens are C. zonatwn
and Nigrospora sphaerioa (Sacc.) Mason.  As mentioned earlier, the
former is more virulent on water hyacinth than the latter.  Spraying C.
zonatwn on insect-infested plants might increase the biological stress


and  reduce  the vigor of water hyacinths, leading to a gradual decline^
 in populations.  Laboratory tests are underway to prove this hypothesis.
The  effect  of C. zonatum on the two arthropods, and the role of other
facultative parasites  in causing disease on insect-infested plants^also
are  being evaluated.   Cephalosporium zonatum is perhaps the most likely
candidate for large-scale field tests.  Current studies on  its host
range, evaluation of its effectiveness on water hyacinth under natural
conditions,  and  the analysis of its role in the decline of  insect-
 infested plants  would  be coordinated  in justifying a large-scale field
use  of C. zonatum in Florida.
     Search  for newer pathogens and phytotoxic microbial products for
use  against submersed  aquatic plants are being continued.   Field test-
 ing, or applications of any of these newer organisms or their products
for  biocontrol of weeds, will be carefully reviewed by experimentation
to assure safety.

    Adequate  studies on the  host  range of any potential biocontrol
agent are  required  to  insure that  it will not seriously affect economi-
cally and  ecologically  important  plants and animals.   It  is preferable
to use a biocontrol agent with extreme host specificity.   Rust pathogens
are among  the most  highly host-specialized.  But even  rusts generally
require alternate hosts to complete their life cycles,  Though serious
attempts to find newer  rusts of aquatic weeds have  not been made, such
endeavors  when  undertaken might prove futile.   It was  pointed out ear-
lier that  rusts of  water hyacinth  and alligator weed have  not been
found since their early discoveries about 50 years  ago.  At present,
choice of  plant pathogens to control aquatic weeds  is  limited to facul-
tative pathogens, which generally  infect a number of hosts.  Under
these conditions, safety to  other  plant species can be assured by use
of sufficiently narrow  host-range  pathogens similar to C.  zonatum.

    Overall,  usefulness of plant  pathogens in biocontrol  is too signifi-
cant to arbitrarily dismiss  on the basis of unsubstantiated fears con-
cerning safety  to the environment.  Since none of our target weeds are
useful as  fresh food for man  or his animals,  dangers of ingestion of
plant pathogens via infected  plants are limited.   The potential  dangers
of plant pathogens  to fish or other aquatic  animals  or  to  the  food
chain in water need to be studied.
    In determinations of the ability of  R. solani to survive in  lake
water, Freeman  (11)  found  that sclerotia  were  viable for over  26  months.
This is a much  longer duration of  aquatic  survival of  this  organism
than reported by other workers (25, 26,  36).   Though the pathogen may
be capable of surviving and   infecting  water  hyacinth from  submersed  in-
oculum,  its persistence in  water  is likely to  be  limited to a  shorter
length of time than  in natural soils  (23,  32) under  comparable tempera-
tures.   Moisture certainly  reduces the viability of  sclerotia  of  this


pathogen  (22, 23, 32) and possibly of soil fungi in general  (31).  Ac-
cordingly, irrigation reduces the viability of soil organisms like Eel-
minthosporium and Alternaria, while aiding in the dispersal of aquatic
fungi  (31).

    Plant pathogens are present  in soil, air and water as resting struc-
tures.  Of these, soil and water are normal habitats for survival of
microorganisms.   In a study, "Pollution of irrigation water by plant
pathogenic organisms," J. R. Steadman of the University of Nebraska has
found  the natural occurrence of many potential plant pathogens in irri-
gation waters of  North Platte Valley of Nebraska (Steadman, personal
communication).   Special attention will be given to the presence of
Sclerotinia salerotiomun (white mold of field beans), Xanthomonas pha-
seol-L  (bacterial  blight of field beans) and plant pathogenic nematodes.
Of concern to us  is the effect artificial  inoculations will have on
natural populations of pathogens, in water and in soils surrounding
bodies of water.  What effects would the survival of a substantially
higher number of  pathogenic propagules have on the environment?  If the
organism produces a phytotoxin, would the  higher levels of toxin have
adverse effects on nonhost plants and animals?  These and other related
questions should  be examined before large-scale uses of any microorga-
nisms  are initiated.  More studies in this direction are needed.  Our
studies in Florida would include evaluation of survival, persistence,
and safety of any organism used  in biocontrol of aquatic weeds.  Con-
cerning use of purified phytotoxic microbial  metabolites, persistence
in the environment will perhaps be the least troublesome problem.
Since microbial toxins are products of metabolism, they should be sub-
ject to biodegradation, unlike some man-made chemicals.
                        SUMMARY AND CONCLUSIONS
    The term "microbial herbicide" has been coined by T. E, Freeman to
denote any microbial agent used in control of a plant, and includes
microorganisms as well as their products.  The potential values of
plant pathogenic microorganisms as bioherbicides against water weeds
are many.  First, most plant diseases have the ability to destroy any
plant species but rarely do so  (39).  This could be an advantage in
that, once a balance is established between populations of the host
and the pathogen, the system might be self maintaining.  Microbial
populations can be mass produced commercially, although no aquatic
plant pathogen is currently being manufactured on a commercial scale.
The presence of technology to produce pathogens commercially is also a
favorable point while considering biocontrol  of weeds with plant patho-
gens.  Generally, animals are non-susceptible to plant diseases and
thus might be safe from aquatic weed pathogens used in biocontrol.
Strict adherence to such safety procedures as use of fairly host-
specific organisms and monitoring of their survival and persistence in
the field would be necessary.  Although we are looking forward to ex-
pediency in field trials, and possibly in large-scale tests, safety to

 the environment and mankind is of prime concern.
     We have at our disposal facilities of the University of Florida Ag-
 ricultural Experiment Station and excellent cooperation of the^following
 agencies:  Florida Department of Agriculture and  Consumer Services, U.S.
 Department of Agriculture, Florida Department of  Natural Resources, U.S.
 Army Corps of Engineers, and Office of Water Research and Technology
 (formerly Office of Water Resources Research) of  the Department of the
 Interior.  The latter three organizations have also provided financial
 support for our program.  Hopefully these studies will contribute to-
 ward establishment of procedures and standards for future researchers on
 biological control of aquatic weeds with plant pathogens.

 1. Acharkar, S. P., and S. N. Banerjee.  1932.  Fusarium sp. causing
        disease of Eichhornia arassipes Solms.  Proc.  Indian Sci. Cong.

 2. Arthur, J. C.  1920.  New species of Uredineae XII.  Bull. Torrey
        Bot. Club 47:465-480.

 3. Bateman, D. F., and S. V. Beer.  1965.  Simultaneous production and
        synergistic action of oxalic acid and  polygalacturonase during
        pathogenesis by Selerotium rolfsii.   Phytopathol. 55:204-211.

 4. Bourn, W. S., and  B. Jenkins.   1928.  Rhizootonia  disease on certain
        aquatic plants.  Bot. Gaz. 85:413-426.

 5. Charudattan, R.  1973-  Pathogenic!ty of  fungi and bacteria from
        India to hydrilla and water hyacinth.  Hyacinth Cont. J. 11:44-48.

 6. Charudattan, R.  1974.  Isolates of Penioilli-wn, Aspergillus and
        Trichoderma toxic to aquatic plants.   Hycacinth Cont. J. (in

 7- Ciferri, R.  1928.  Quarta contribuzione allo studio degli Ustilagi-
        nales.  Ann. Mycol. 26:1-68.
 8. Ciferri, R., and R. G. Fragoso.  1927.  Hongos parasites y sprofitos
        de la Republica Dominicana.  Bol.  Soc. Espan. Host.  Natur.
        (Madrid) 27:68-81.

 9. Cullen, J.  M.,  P.  F.  Kable,  and M.  Catt.   1973.   Epidemic spread of a
        rust imported  for biological  control.   Nature (London)  244:462-464.

10. Daniel, J.  T.,  G.  E.  Templeton,  R.  J.  Smith,  Jr.,  and W.  T.  Fox.  1973.
        Biological  control  of  Northern  Jointvetch  in  rice with  an endemic
        fungal  disease.  Weed  Sci.  21:303-307-

11. Freeman,  T.  E.   1973-   Survival  of  sclerotia of Rhizootonia  solani  in
        lake water.  Plant  Dis. Reptr.  57:601-602.

12. Freeman, T. E., and R. Charudattan.  1974.  Occurrence of C&TOO-
        spora piavopi on water hyacinth  in Florida.  Plant Dis. Reptr.
        (in press).

13. Freeman, T. E., R. Charudattan, F. W. Zettler, R. E. Rintz, H. R.
        Hill, B. G. Joyner, and H. F. Hayslip.  1973.  Biological con-
        trol of water weeds with plant pathogens.  Publ. 23, Tech.
        Compl. Rep. OWRR Proj. B-011-FLA.  Water Resources Res. Center,
        Univ. of Florida.  55 p.

14. Gaumann, E.  1950.  Principles of Plant  Infection.  Crosby Lock-
        wood, London.  543 p.

15- Hasan, S.  1974.  First introduction of a rust fungus in Australia
        for the biological control of skeleton weed.  Phytopathol.
16. Hayslip, H. F., and F. W. Zettler.   1973.  Past and current re-
        search on diseases of Eurasian water milfoil (Myriophyllum
        spicatum L.).  Hyacinth Cont. J. 11:38-40.

17- Hill, H. R., and F- W. Zettler.   1973-  A virus-like stunting dis-
        ease of alligator weed from Florida.  Phytopathol. 63:443.

18. Joyner, B. G., and T. E.  Freeman.  1973.  Pathogenic!ty of RhizoG-
        tonia solani- to aquatic plants.  Phytopathol. 63:681-685.

19. Maxwell, D. P., and R. D. Lumsden.   1970.  Oxalic acid production
        by Solevotin-ia saleroti-orwri in infected bean and in culture.
        Phytopathol. 60:1395-1398.

20. Nag Raj, T. R., and K. M. Ponnappa.  1967-  Some interesting fungi
        of India.  Tech. Bull. Commonw.  Inst. Bio. Control 9=31-43.

21. Nag Raj, T. R., and K. M. Ponnappa.  1970.  Blight of water hya-
        cinth caused by Alternaria eichhorn-iae sp. nov.  Trans. Brit.
        Mycol. Soc. 55:123-130.
22. Newton, W.  1931.  The physiology of Rhizocton-ia.  Sci.  Agr.

23. Nisikado, Y., and K. Hirata.  1937-  Studies on the longevity of
        sclerotia of certain  fungi, under controlled environmental fac-
        tors.  Bericht. Ohara Inst. Landwirts Forsch. Kurashiki
2k. Padwick, G. W.  1946.  Notes on Indian fungi.  Mycol. Pap. 17:1-12.

25. Palo, M. A.  1926.  Rh-izoaton-La disease of rice.  I. A study of the
        disease and the influence of certain conditions upon the via-
        bility of the sclerotial bodies of the causal fungus.  Philip-
        pine Agr. 15:361-376.

26. Park, D., and L. S. Bertus.   1934.  Sclerotial diseases  of rice in
        Ceylon.  3- A new Rhizoctonia disease.  Ceylon J. Sci. (A)

27-  Ponnappa,  K.  M.   1970.   On the pathogenic!ty  of  Myvotheaiim roridum-
        Eichhornia erassipes isolate.   Hyacinth  Cont.  J.  8:18-20.
28.  Ridings,  W.  H.,  and F.  W.  Zettler.   1973.  Aphanomyces blight  of
        Amazon sword plants.  Phytopathol.  63:289-293-
29.  Rintz, R.  E.   1973-  Location, identification,  and  characterization
        of pathogens of the water hyacinth.  Ph.D.  Thesis, Univ. of
        Florida.   53 p.
30.  Rintz, R.  E.   1973-  A zonal leaf spot of water hyacinth caused by
        Cephalosporium zonatim.  Hyacinth Cont.  J.  11:41-44.

31.  Rotem, J., and J. Palti.  1969.  Irrigation  and plant diseases.
        Ann.  Rev. Phytopathol. 7:267-288.

32.  Sherwood, R.  T.  1970.   Physiology of Khizootonia solani, p. 69-92.
        In J.  R.  Parmeter,  Jr.  (ed.), Khizoatonia solani, Biology and
        Pathology.  Univ. Calif. Press, Berkeley.

33.  Tharp, B.  C.   1917.  Texas parasitic fungi.   Mycologia 9:105-124.

34.  Tims, E.  C.,  and L. S.  Olive.  1948.  Two interesting leaf spots of
        fig.   Phytopathol.  38:707-715.

35.  USDA.  Crops Res. Div.  ARS.  I960.   Index of Plant Diseases in the
        United States.  U.S. Govt. Printing Office, Washington, D.C.
        531 P-
36.  Wei, C. T.   1934.  Khizootonia sheath blight of rice.  Nanking Univ.
        Coll.  Agr. Forestry Bull.  (NS) 15.  21 p.

37-  Wilson, C. L.  1969.  Use of plant pathogens in weed control.   Ann.
        Rev.  Phytopathol. 7=411-434.

38.  Yarwood,  C.  E.  1962.  Some principles of plant pathology.  Phyto-
        pathol.  52:166-167-
39.  Yarwood,  C.  E.  1973-  Some principles of plant  pathology.  II.
        Phytopathol. 63:1324-1325-
40.  Zettler,  F. W., and T.  E.  Freeman.   1972.  Plant pathogens as  bio-
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GEORGE ALLEN:  The importance of exotic aquatic weeds was not fully rec-
ognized in the southeastern United States until a national environmental
pollution control program was initiated in 1970.  The severity of the
problem continues to increase yearly, requiring increased funding to
control various aquatic weed species.

     In 1971, the Florida legislature appropriated funds to develop
methods of aquatic weed control  under the direction of the State Depart-
ment of Natural Resources.  In order to cope with immediate aquatic weed
problems and to develop long-range plans for the state, a cooperative
program including the U.S.D.A. and state universities was organized.
Although the majority of aquatic weed pests are currently controlled by
the use of herbicides, several approaches, including importation and in-
troduction of various natural  enemies, are being investigated to reduce
future use of chemicals.

    The U.S.D.A. has been actively engaged in research on biological
control of aquatic weeds since 1959.  Program emphasis until  1967 was on
alligator weed, resulting in successful introduction of three insect
species from Argentina.  In 1967, the U.S.D.A. established a laboratory
in Argentina to isolate and evaluate insect enemies of water hyacinth
and  later establish PL-480 projects  in Europe and Asia to conduct simi-
lar research on Myriophylltm spioatum and Eydrilla vevtio-illata.  In the
inter-agency cooperative program, the University of Florida was assigned
the responsibility of conducting new foreign exploration programs.

    Some of our most successful  introduced and potential species are in-
sects, therefore, we are very much concerned with their interaction with
pathogens.  The introduction of insects free of their predators and para-
sites  is an important rule in biological control, however, their associ-
ation with pathogens is often overlooked or not considered important.
In the case of aquatic or semi-aquatic insect enemies of water weeds,
pathogens are often the most important biotic factors involved.

    Currently, water hyacinth, E'ia'tihor'n-La crassipes, is our number one
aquatic weed pest in Florida.   It has been proposed to be native to
South America where it has an extensive range centered on northern Bra-
zil and Venezuela.
    Several insect species have been isolated and screened for importa-
tion at the U.S.D.A. laboratory in Argentina.  The first introduction
was the^weevil, Neochetina eichhorniae.  Limited releases were made in
Florida in 1972, however, due to the possible simultaneous introduction
of a microsporidan disease associated with the insect in Argentina,  fur-
ther releases have been delayed.   Extensive surveys have been conducted
in Argentina to determine the interrelationship of the pathogen, a No-
sema, and Neoohetina eiehhorniae and N. bruchi, also scheduled for re-
lease  in Florida.  A selective breeding program was developed in Argen-
tina to produce disease-free stock material which was examined in the
United States before field release.  Recently, we have encountered a

 potentially more  important  parasitic nematode  in both Neoahetina spp.
 in  Argentina.   The  nematode  is a member of the order Tylenchida, many
 of  which  may  cause  a  significant reduction in  fecundity or adult mor-
 tality.   Natural  populations of both weevil  species are susceptible  to
 Beauveria bvongniartii  (= B. tenella)  in Argentina.  We have recently
 isolated  this  pathogen  from field collected  N. eiahhorniae  in Florida.

     Another area  of interest concerns  the microsporidan diseases of Aoi-
 gona infusella and  Epipagis albiguttalis; two  lepidopterous  insects
 being considered  by the U.S.D.A. for introduction on water  hyacinth.
 Preliminary  indications are  that the pathogen  associated with the  lar-
 vae of both these species  is Nosema neaatrix,  a major natural control
 agent of  our  native Arzama densa.  The disease is so severe  in  both
 Aeigona and Epipagis  in Trinidad and Argentina that it has  prevented
 laboratory rearing  for  basic studies.   If a  final diagnosis  substan-
 tiates our findings,  the decision to continue  quarantine studies of
 these two insects should be  reconsidered  in  view of the fact that  both
 species inhabit the same niche as Arzama densa.   It is also  associated
 with the  lily borer,  Beltura goTtynoides, a  close relative of A. densa
 which attacks  the yellow water lily, Nuphar  advena, in  Indiana.  This
 species is also susceptible  to a multiploid  virion  nuclear  polyhedrosis
 vi rus.

     The submerged plant Eydrilla vertioillata  was first observed  in
 Florida fn I960 and is  rapidly becoming  our  number  one aquatic  weed
 pest.  We are currently involved  in establishing  the distribution  and
 area of origin of hydrilla  to enable us  to develop  foreign  research
 projects  in the search  for  natural  enemies of  the  plant,

     A U.S.D.A.-sponsored PL-480  project  in  Pakistan has yielded several
 insects associated  with hydrilla.   Three species,  the dipterous leaf-
 miner Eydrillia sp.,  the weevil,  Bagous  lutulosus,  and  Nymphula diminu-
 talis, show potential as biological control  agents.  Diseases have not
 been reported  in any of these  insects, however,  based on our experience
 with hyacinth insects,  we  expect  to encounter  pathogen  involvement.

     Water lettuce,  Pistia  stratiotes,  is a  potential pest  in many  coun-
 tries including the United  States;  therefore we  are in  the  process of
 surveying for  potential biotic factors.   It  is attacked in  Florida by
 the Pyralid moth, Samea multipliaalis, by  the  weevil, Neohydronomus
 pulahellus, and a Noctuid moth  in East Malaysia.  Samea multipliaalis
 has  been  reported to  be attacked by a  nuclear  polyhedrosis virus as
 well  as by several  parasites.
     Although Salvinia spp., water fern, are  not considered  major world-
 wide or Florida aquatic weed pests, considerable research  has been  con-
 ducted  to  evaluate  insect enemies as biotic  agents as  the  result of the
 explosive  growth  of S.  molesta on Kariba Lake  in  Africa.  Three  insects,
 the  Acridid grasshopper Paulinia acuminata,   the weevil Cyrtobagous  sin-
gularis, and S. multiplicalis have been evaluated.   Metarrhizium ani-
sopliae is a significant pathogen of P. acuminata as well as  Cornops
sp., another grasshopper that attacks  water  hyacinth.


    Aquatic plants provide both habitat and food for animal  vectors of
human diseases such as malaria, filaria, fascioliasis, and schistoso-
miasis  (bilharziasis).  Anopheles, Culex and Mansonia mosquitoes breed
in the calm, still pockets of water created among stands of  floating
and emergent vegetation.  In a study made in the Tennessee Valley Au-
thority reservoirs,  it was determined that A. quadvimaculatus breeding
was related to the extent that plant organs intersect the water surface.
Pistia stratiotes and E. crassipes are  important species involved.

   ^The intermediate  hosts of schistosomiasis are species of the aquatic
snails Bulinus, Biomphar-ia, and Onoomelania, which live in the micro-
habitats provided by  aquatic vegetation in which they find both shelter
and food.

    In Egypt,  bilharzial snails are closely associated with  several
aquatic plant  species including water hyacinth.

    In summary, the control  of aquatic weed pests is directly related to
use of microorganisms to control aquatic insects.  Insects have been and
will  be introduced in the future to control water weeds.  Many of these
insects are attacked  by disease organisms, therefore, it would be wise
to consider their susceptibility to any microorganisms proposed for
aquatic insect control.

G. E.  TEMPLETON:   I want to begin with a compliment and a word of en-
couragement to Dr. Charudattan on his work with plant pathogens for con-
trol  of water weeds,  and Dr. Howard Ohr, who is also working in the area
of biological weed control  with plant pathogens.  I  commend  both of
these young men for their courage to enter this new and challenging re-
search area and to set out on the arduous task of developing the scien-
tific base of data with which to make a rational judgment on whether to
introduce foreign pathogens for biological weed control purposes.  As
many of you know, plant pathologists are from 30 to 50 years behind en-
tomologists in this area of introducing microorganisms for biological

    Perhaps a reason  is that in the history of plant pathology we have
so many examples  of introduced pathogens that have been devastating to
man's crops and trees.  I'm sure you're aware of the Dutch Elm disease,
of Chestnut Blight, and  Late Blight of Potato that precipitated the
great  Irish Famine.   Every beginning plant pathology student knows these
tales.  I  remember, too, how coffee rust completely devastated this crop
in Ceylon and now threatens coffee crops in the Western Hemisphere be-
cause of its recent introduction into South America.  There is also the
story about how Charles Valentine Riley, an entomologist from Missouri,
played a role  in  introducing downy mildew into France and nearly deci-
mated the wine industry there while importing Phylloxera resistant root
stocks from this  country, so you can readily see why pathologists have
been reluctant to undertake biological control  in the classical sense.
    I  would like  to offer another observation that  I feel is very impor-
tant in planning  for  future work in biological control.  Progress can


be most rapid when the team approach is used with one specialist on the
target organism and one on the biocontrol agent.  This is how Dr. R. J •
Smith, USDA-ARS weed scientist, and I  have worked as we developed a bio-
control system for a leguminous weed in Arkansas rice fields.  The team
approach largely accounts for the success of this project.
    Our objective  is to demonstrate that biological control of weeds  in
cultivated crops  is practical with fungal plant pathogens.  Specifically
we are seeking to  demonstrate that endemic,  rather than  introduced,
plant pathogens can be used for biocontrol of weeds  in cultivated  crops
by employing  them  as bioherbicides, e.g., a  mycoherbicide  in  this  par-
ticular case.  Our host-parasite combination is  northern  jointvetch
(Aeschynomene virgini-aa  (L.) B.S.P.) and  the  endemic  fungal  pathogen of
jointvetch anthracnose, Colletotr-iohwn gloeospori-o'ldes  (Penz.)  Sacc.  f.
sp. aesahynomene.
    The weed  problem that can develop  in rice fields  can reduce the
rice  yield but probably of equal economic  importance is  the dockage
farmers take  because of the trouble millers  must  go  to  so as  to remove
the black  shiny  seeds  that are  intolerable  in packaged  rice.   The  weed
can be controlled  with 2,4,5-T  but  there are several  disadvantages to
its use.   As  in  this case the farmer did not want  to spray his  rice
with  2,4,5-T  for  fear of  it drifting to  and  damaging  his neighbor's
soybeans.  Also,  if 2,4,5-T applications are not  properly timed the
rice, too, can be damaged.
    Careful  examination of plants  in a weed  colony will  reveal  wide-
spread and often  uniform distribution of the anthracnose disease.   It
occurs naturally  in practically every colony we have  looked at, yet  it
 is  not particularly damaging  to the weed.   Thus  it fits  well  the defi-
nition of  an  endemic disease.   Interestingly, we  have another related
weed  species, A.  -indiaa, quite widespread  in our  state but we have
 never been able  to find this  disease occurring  on  it.  We have  been
able  to  infect A.  indi,oa  in the field and greenhouse  but  the fungal
 isolates  are  not very  virulent  to  it.

     The  typical  anthracnose  lesion of  this  disease has pustules (ascer-
 vulae) of  spores  erupting from  the  tissue.   These  spores  (conidia)  are
 quite sticky, not readily disseminated by wind  but are distributed in
 a passive  way by insects of various types.

     We isolated  the fungus  in  pure culture  on PDA or  Lima  bean  agar and
masses of  spores are  readily  produced on the surface of  the agar.   It
 is  a  fungus  that is very  easy  to work with  in this respect.   Photomicro-
graphs show  that  the  spores are relatively  fragile.   They are suffi-
 ciently  stable for easy culture and harvest  and  spread  in the field,
 yet  surely not a  type  that would over-winter well  in  our  climate.

     In order  to  determine  the  host  specificity  of  this pathogen, we
 have  tested  about 150  species and  breeding  lines  of  various crop and
native plants in  greenhouse and field  tests. We've  tried  to  emphasize
 soybeans,  other  leguminous plants  and, of  course,  all the  commercially
 important  crops  in our area such as cotton,  rice,  corn,  etc.


    We store  isolates of the fungus in sterile soil, a standard proce-
dure for storing fungi of various types.  Transfers are made to modi-
fied Richard's solution, which  is a sucrose-potassium nitrate medium
supplemented with V-8 juice.   It  is my understanding that we'll have to
substitute something for V-8 juice in this medium if we are to commer-
cialize this fungus.   It also requires relatively high aeration for
good spore production.

    In our field tests, after establishing a fairly good population of
the legume weed, the treated plot has 100 percent control of the joint-
vetch 10 days after application of spores without damage to the rice or
the grass weed.

    The following table shows some representative data from our field
tests.  We have been testing it in the field since 1969 but we haven't
found much difference  in the effects of spore concentration (from 2 to
15 x 106 spores/ml) or plant height.  In fact, we haven't found any
situation in the field where it would not effectively control  this weed,
Last year we increased our test to a total of 80 acres using commercial
equipment for application.  It was effective in a variety of field
situations including dried rice fields, flooded rice fields, young
weeds, old weeds, etc., giving excellent control in every case.

               Fungus on Northern Jointvetch, Field 1970
                 _.,  .  ,  .  Lj_     % Control at indicated
                 Plan' h*'9ht         spores/ml x 106
                     (cm)          2   P  3     6	Av?

               Spray applied at 1300 L/ha.

    We feel this fungus has commercial  possibilities and it is our hope
that with the help of the Upjohn Company we will be able to produce
enough spores for testing on 500 acres this coming season, if we can
satisfy Dr. Engler and the others of EPA about  its safety to the envi-
ronment and its toxicological safety.  We are using around 15 ml of
spore paste per acre as it is spun down from the culture medium and
washed, versus 1500 ml of 2,4,5-T required to control the weed.  Also,
compared with levels of Baai-llus thuring-iensis used it is quite low.
Fifteen milliliters of spores in 10 gallons of water applied to an acre
containing a 6-inch flood without any interception by rice plants would
amount to about 125 spores per ml.  And since we are only adding to the
native level of this fungus already present in this environment, I  don't
feel we are inducing a very significant perturbation of the environment.

    Finally,  I would like to say that every weed plant has from one to

 one hundred  or  more diseases of  various  types.  We feel  that development
 of certain ones of  these diseases as  bioherbicides is a  viable alterna-
 tive to many chemical  herbicides in cultivated crops.

 JOHN BRIGGS:   It seems  that problems  of  formulation, application,  regis-
 tration, and identification are  similar  in  plant  pathology as well  as
 insect  pathology.   I  trust we will be able  to maintain a community of
 plant and insect pathologists and identify  this community with particu-
 lar reference to those  areas of  safety,  application, mass propagation,
 and application of  pathogens of  insects  and weeds.  From what Dr.  Allen
 and the principal speaker said,  we have  in  these  integrated programs
 one of  those magnificent opportunities  in biology which  has never  been
 done satisfactorily in  an educational institution—to bring together
 plant and animal sciences.

 IGNOFFO: This  was  one  of the ideas that we attempted to foster here.
 The problems are going  to be common,  and I  don't  think we have to  re-
 strict  ourselves to potential biocontrol of weeds and those of insects.
 There are pest  systems  all over  the world that we can address ourselves
 to, if  we just  broaden  our horizons a little.  I   think many of these
 problems are very similar.

 MARSHALL LAIRD:   We're  tending to label  the beautiful water hyacinth
 with an unfortunate image, due to the fact that in considerable parts
 of the  world  Eiehhomia has become a  thundering nuisance.  But in  some
 areas,  specifically Malaysia and Singapore, it's common  practice for
 Chinese smallholders  to maintain a pond  alongside the house in which
 water hyacinth  is grown and fed  to the pigs.  And I'm wondering whether,
 when we're considering  the massive destruction of Eiohhorn-ia avassipes,
 we might have devoted  little thought  to  replacement organisms which, in
 places  like  Southeast Asia for example,  might be able to achieve the
 same job as  pig  feed  that Eichhomia arassipes achieves  now.

 ALLEN:   It's  interesting that water hyacinth is uti1ized extensively as
 pig  feed  in  Southeast Asia.  But no good is accomplished by doing  this,
 because  water hyacinths are about 92-93% water and are poor fodder

 CHARUDATTAN:   I  made a  point in  my talk  that none of  these target aquatic
weeds which we are  attempting to control are used as  fresh food  for man
or  his animals.  This is a generalization based on the  fact  that  Eich-
hornia crassipes, Hydrilla and the others are not  food  plants.   There
are  instances,  I believe, in Louisiana,  where cows are  allowed  to  graze
on alligator weed and water hyacinth.   However,  these are not usual
pasture  plants and  have only limited  value  as  food for  animals.

J.  E. ZAJIC:   If you're going  to remove  an  aquatic weed can't you  expect
something to replace it?  What  would  be  the replacement system?  You're


dealing with an ecological problem,  in which there is a delicate bal-
ance.  The weed may not be what we want, but unless you have something
to  replace  it,  I can't see what the value is going to be to remove it.
When we're growing crop plants, as in rice fields, I  see a real  value
 in  weed control.

CHARUDATTAN:  You are concerned about filling in the ecological  niche,
once you  remove water hyacinth, or hydrilla.  Probably we'll have a
problem with one kind of weed or another during all our lives,  and per-
haps even our grandchildren's.  We know, in our test systems, in small
ponds and in aquarium tanks, once leafy hydrophytes,  like hydrilla or
water hyacinth, are removed, algae come in.   Perhaps  if we could accom-
plish something like aquatic weed management we could try to introduce
some of our native plant species which always seem to be confined to
their particular ecological niches.

JOHN PASCHKE:  One of the classic examples of weed control  using insects
was with  Klamath weed in California and other western states.  Two spe-
cies of beetles were introduced against the Klamath weed which  led to a
balance between the weed and the beetles.   The Klamath weed is  still
there but large acreages of grazing land have been reclaimed.  In Aus-
tralia, cactus, Opunt-La spp., is still present as a weed but in balance
with its biocontrols, the insect and disease.  The latter occur as a
result of the attack of the 1epidopterous  insects that bore into the
cactus pad.   So, the biological  control  does not usually result in the
total elimination of a target host.

ALLEN:   I  might make one comment here in reference to Dr. Zajic's ques-
tion.  All the plants we're dealing with in the aquatic environment of
the southeastern United States are exotic  species.  Furthermore, we will
never remove the environment of those plants that are here.  With alli-
gator weed,  we do have some success,  but some of this weed will  always
be  there.   At this reduced level it may be beneficial for fish.  We need
to  realize that the reason we have these problems  is that the reproduci-
bility of  hydrilla,  Pistia and Eiohhornia  is tremendous.  For instance,
within one summer one plant of water hyacinth can be expected to vege-
tatively give rise to 5,000 offspring.  These plants reproduce vegeta-
tively and sexually but more so vegetatively-  We're always going to
have these plants in Florida.  All  we're trying to do is to coordinate
our efforts  in reducing the populations of these plants to where we can
1ive wi th them.

E.  I. HAZARD:  I have to address this to Dr. Allen.  Two questions:
First,  I  wonder if you'd comment on the role that the manatee is play-
ing, if any, in the biological control of  water hyacinth.  And  second,
I recall  a study which pointed out that water hyacinth was beneficial,
being capable of trapping a lot of the excess nutrients which would  have
led to eutrophication, algal bloom, and depletion of oxygen.  I  just


 wonder  if  you'd comment on the possible beneficial effects,  if any, of
 the water  hyacinth.

 ALLEN:  The manatee  is native to Florida and one of the first control
 agents  we  thought of.  But its requirements for reproduction are so
 fine--it needs certain water temperature and so forth—that  it will
 never be feasible.
     In  reference to  the beneficial part, hydrilla, water hyacinth and
 most of these aquatic plants are able to take up and store tremendous
 amounts of nutrients, up to 3-]Q% times more than they need.  If we
 could remove the plants easily, we could put them in enriched waters,
 remove  the nutrients, and clear the plants once the water is clean.
 Water hyacinths have been found to be very useful in this respect.
 We're going to use water hyacinths in a sewage plant where they would
 be very beneficial in removing nutrients from the sewage.

 D. W. ANTHONY:  I  just want to make one comment on the manatee.   This
 animal  was tried in  the Chagres River in Panama, as I  understand, for
 about 3 or 4 years before it was finally given up.  It just didn't work
 at all.

 BRIGGS:  There were a couple of comments with respect to metabolic pro-
 ducts of mircoorganisms, their possible use as insecticidal  or herbi-
 cidal materials, and about their possible degradation.  I wonder, Dr.
 Singer, if you would tell  us if microbial  metabolites are easily de-

 SINGER:   We don't  know much about these toxins; there haven't been many
 studies.  The only way to establish the toxicity of a substance is to
 biologically assay it.  With antibiotics,  like penicillin, there are
 cases in which some organisms split the penicillin part.  This is prob-
 ably true of most  antibiotics and microbial  products,  most of which
 probably are biodegradable.   As with microorganisms used as control
 agents,  we have to consider them in relation to total  population.  Pre-
 sumably we will  be putting them into competition with natural popula-
 tions.   Hopefully,  they will  be viable enough to maintain this feedback
 situation--as the  host increases, they will  attack it  and so forth.

 IGNOFFO:  There's  a very excellent example of exotoxin in which  the  re-
moval  of the phosphate radical  leads to its  complete deactivation.
There are several  other examples of such degradation of  compounds in
    I  want  to close with a few remarks.   There's  a series  of  publica-
tions  that  might be helpful  in this area.   One is  the  WHO  Report,  No.
531,  available through Dr. Arata, on the use of viruses  for  control  of
 insect  pests  and disease vectors.  It goes  into some of  the  problems,
safety  requirements,  registration,  etc., for development  of  viruses.


There's another WHO publication by Ray Smith, called "Considerations on
the Safety of Certain Biological Agents for Arthropod Control."  More
recently, there's been a publication in the Proceedings of the New York
Academy of Science which covers this whole area.  Dr. Singer referred
to a book, edited by Burgess and Hussey, on microbial insect control.
These may be more helpful to those people  interested in this area to
see how  it might relate, and solve some of the problems they may be ex-
posed to.

    The area of biological control is a very exciting field with great
potential.  We need to find other ways, but not to the total exclusion
of chemical pesticides.  We should be able to prudently use these chemi'
cals  in  integrated systems to control aquatic pests.  The major concern
is that microbial controls will mutate or  infect non-target organisms,
or will  eventually get  into an ecosystem and affect other animals.



                  OF  CRUDE  OIL  IN  AN OIL TANKER
                   DURING  ITS BALLAST  VOYAGE

   E.  Rosenberg, E.  Englander, A.  Horowitz  and  D.  Gutnick*

      A compartment of an oil  tanker,  containing  107 m3 oily ballast
water, was supplemented with 7-6 mM  urea, 0.57 mM KaHPOi* and 3 m3 air
per min.  After k days, oil  dispersion became evident and bacterial con-
centration reached over 107  cells/ml.  When the treated water was dis-
charged, no sign of oil was  detected  in  the ship's wake.  A non-aerated
control compartment, containing 121  m3 oily ballast water, yielded 10s
cells/ml and no oil dispersion.  In  order to understand the mechanism of
bacterial-induced cleaning of  oil  tanks, the emulsifying agent produced
by RAG-1 , an Arthrobaater sp., has been  partially purified and charac-
terized.  The purified agent,  ERAQ,  forms stable emulsions with over 200
times  its weight of crude oil, gas-oil or hexadecane,  ER/\Q is resistant
to 100 C, excess KIOi* and pronase; the half-life of ERAQ was 5 min in
1 N HC1 at 100 C and 2 hr in 1 N NaOH  at 100 C.  RAG-1 induced oil emul-
sions were toxic to developing sea urchin embryos.  However, emulsions
formed with ER/\Q showed only slight  toxicity which could be removed
with small dilutions into sea  water.   An oi1-degrading bacterium, re-
ferred to as UP-2, has been  isolated by  enrichment culture with RAG-1
depleted oil as the nutrient.   UP-2  has a broader substrate specificity
than strain RAG-1.

     There have been a  large number  of  reports  (e.g., 1, 3~5, 8, 10, 11-
13) describing the properties of  pure and mixed bacterial cultures
capable of dispersing and  degrading  crude oil  in supplemented sea water.
In each instance, the ability of  the bacteria  to significantly degrade
oil was entirely dependent on an  exogenous source of nitrogen and phos-
phorus.  This requirement  for supplemental nitrogen and phosphorus com-
pounds most likely accounts for the  very slow  breakdown of oil in the
open sea,  and suggested  to us that the  utilization of microorganisms in
the treatment of oil  pollution can be more easily accomplished in situ-
ations in  which the polluting oil  is confined,  One such environment is
in the cargo compartment of an oil tanker.
     ^Department of Microbiology,  Tel Aviv University, Tel Aviv,
Israel .


     After delivering  its cargo of crude oil, a petroleum tanker must
 take aboard  large quantities  of ballast water before putting  to sea.
 This ballast water mixes with residual oil  in the cargo compartment  (be-
 tween   0.1 and  1.0%).   Disposal of this heavily contaminated  oily  bal-
 last water  is a major  contributor  to  chronic  sea pollution.   In^addition,
 cargo  compartments of  oil  tankers  must be  cleaned periodically  in  order
 to  prevent clogging  and sludge accumulation which would otherwise  reduce
 the ship's cargo capacity—and inhibit discharge of crude oil.   Pres-
 ently, cargo compartments  of  tankers  are  usually cleaned  with high pres-
 sure jets of sea water; the combined  ballast  and wash  waters are then
 either a) discharged at sea,  b) transferred to separator  tanks on  shore
 where  harbor facilities permit, or c) transferred  to small  settling
 tanks  on deck  ("slop" tanks)  in which the water at the bottom is removed
 and additional  oily ballast water is  loaded on top (load-on-top tech-
 nique).  Prior to dry dock, additional  scrubbing by hand, or removal of
 fine particles with the aid of vacuum cleaners is required.

      As part of a general  program on degradation and dispersion of crude
 oil by bacteria, we decided  to explore the possibility of utilizing bac-
 teria  for cleaning cargo compartments of oil  tankers.   We report here
 a)  the growth of bacteria on supplemented oily ballast water within a
 cargo  compartment during its ballast voyage, b) certain chemical  and
 biological properties of a bacterial-produced dispersing agent, and
 c)  the isolation of oi1-degrading bacteria by sequential  enrichment cul-
 ture procedures.

                           THE SHIP EXPERIMENT

      Prior to the experimental voyage,  several  experiments  were conducted
 (mostly on a 10-liter scale)  under a  variety  of conditions  that might be
 encountered  during  the voyage.   The following general  conclusions  were
 reached:   a)  Using  0.3% crude oil  in  aerated  sea water, maximum oil dis-
 persion and  bacterial  growth  yields were achieved with 7.6  mM urea and
 0.57 mM KaHPO,,.  b)  In the  early experiments, ammonium sulfate  was used
 as  the nitrogen source,  resulting  in  a final pH of  5-6.   This acidic pH
 is  inhibitory for further development of bacteria and could  be detrimen-
 tal  to certain  metal  components in ship tanks.  By using  urea  instead of
 ammonium  sulfate, we were able  to maintain  the pH between 7.6 and  8.1
 independent of  the temperature and oil concentrations chosen  for growth.
 c)  Similar kinetics of oil  dispersion and bacterial  growth occur over a'
 rather  wide  range of temperatures, 20-37 C.

     In order to perform the ship experiment,  we were provided with the
 use of  the two  "slop" tanks on a 120,000-ton oil  tanker.   During the
 ballast voyage  prior to the experimental  trip,  a  device'for  providina
 air was installed in the starboard  "slop"  tank (Tank A).   Both tank
 subsequently filled  with Agha  Jari  light  crude oil  (2).   The aer t'
 system  consisted of  segments of polyethylene hose,  32 mm  in  diameter"1
connected by polypropylene  fittings such  that  the hose  covered th«  k -
 surface of Tank A.   Fifty holes, 2  
was connected to a valve on deck by a long hose of the same diameter
(32 mm).

     We boarded the oil tanker which was in the process of discharging
its cargo in Eilat.  The following day, after discharge was completed
and the ship had left port, ballast water was added to each of the two
tanks, 2.48 meters in Tank A, and 2.70 meters in the port "slop" tank
(Tank B).  The total  volume of liquid in Tank A was 107,000 liters and
that in Tank B, 121,000 liters.  Twenty kilograms of urea and 1  kg
K2HP0lt were dissolved in sea water and added to each tank.  Air  was then
introduced into Tank A at the rate of 3,000 liters/min.  Duplicate
samples were removed from each tank at time "zero."  Subsequently dup-
licate samples were taken 3 times daily from Tank A and once daily from
Tank B for the following 157 hr.  In addition, duplicate samples were
withdrawn from the full oily ballast tank No. 4 at 137 hr.  Measurements
of oil dispersion and viable cell number (10) as well  as microscopic
observations of the samples were made in the Deck Office which served
as a laboratory during the voyage.
     At the beginning of the experiment, a thick layer of oil  could be
seen floating on the surface of the water in the tanks.  The oil layer
in Tank B remained in this form throughout the course of the experiment.
Starting at 93 hr, the appearance of the oil surface in Tank A began to
change.  The oil began to coagulate, and streaks could be seen on the
surface.  The oil in the samples taken from Tank A started to change
dramatically at about 100 hr.  In these samples the oil appeared very
"mushy" (probably a water-in-oil emulsion) with the consistency  of a
pudding.  With increased time, more and more of the oil was found to
disperse into the water phase  (Fig. 1);  In sharp contrast to what was
observed during the early sampling periods, when the oil adhered very
tightly to the walls of the sample bottle, by the end of the experiment
a slight shaking of the container resulted in a complete disappearance
of oil from the walls of the flask.  No such dispersion and cleaning
was observed in any of the samples from Tank B or Tank No. 4.
     Figure 2 summarizes data on viable cell number during the course
of the experiment.  These are minimum values since only those bacteria
which can form colonies on nutrient agar are measured.  The initial
concentrations of bacteria in Tanks A and B, as well as in the unsup-
plemented control Tank No. k, were more than 10 times higher than in
sea water sampled alongside the ship just prior to taking on ballast
water.  The probable source of these bacteria was the residual oil  in
the tanks.   After a lag period of about 22 hr, the bacteria concentra-
tions increased during the subsequent 72 hr, about tenfold in  Tank B
and 1000-fold in Tank A, yielding about 1  x 10s and 2 x 107 viable
cells/ml,  respectively.  Total  cell  number, as determined by use of a
Petroff-Hauser counting chamber, on formaldehyde fixed samples gave
values 5-10 higher.  The protein concentration in Tank A at 156  hr was
95 mg/liter as determined by the Lowry method (6) on cells harvested at
15,000 x g  for 30 min.  This value is also minimal, because some pro-
tein was detected in  the supernatant fluid following the centrifugation.

         100 h-
                          50           100
                              TIME (hrs)
FIGURE 1.  Oil  dispersion inside the cargo compartment of an oil  tanker
  during its ballast voyage.   The oil  ballast water in Tanks A (107,000
  liters) and B (121,000 liters) were each supplemented with 20 kg urea
  and 1  kg potassium phosphate.   Tank A was aerated at the rate of 3,000
  liters/min.  Tank B served  as  a non-aerated control.  Oil  dispersion
  was estimated from the turbidity of the oil-water suspension after
  allowing 2 min for the non-dispersed oil to separate (see ref.  10 for
  details of this method).   Time zero corresponds to the time of  addition
  of ballast water and supplements to the residual  oil in the tanks.

                          50           100
                            TIME ( hrs)
FIGURE 2.   Growth of bacteria  inside the  cargo  compartment of an oil
  tanker during its ballast voyage.   The  conditions are  those described
  in Figure 1.   The ordinate indicates  the  viable cell number as deter-
  mined by plating an appropriate dilution  onto Nutrient Agar.  Tank k,
  a  full normal ballast compartment, served as  an unsupplemented con-
  trol.  Sea water sampled  from alongside the ship just  prior to taking
  on ballast water is shown for comparison.

     Throughout the experiment, the temperature remained between 23 and
25 C and the pH between 7 and 8, in both the experimental  and control

     Tank A was emptied after  156 hr, and during that procedure no mate-
rial could be observed which darkened the appearance of the white foam
in  the wake of  the  ship.  When  Tank  B was subsequently  emptied, we ob-
served the expulsion  of a thick black material  followed by a yellow oil
slick which appeared  in  the  wake.  After Tank  A was vented, we  entered
the tank with  the  Chief  Mate.   The bottom and  lower 2.5 meters  of  the
walls of the  tank  were found to be completely  free of  the thick layer
of  sludge which had accumulated on the  ladder, platforms, and  upper por-
tion of  the walls  of the tank.  There were  almost no residual  oil  stains
along the  bottom surface.


      In order to better understand the mechanism of  bacterial  cleaning
 (oil dispersion) of oil  tankers, we have investigated the production of
extracellular dispersing agent(s) produced  by  the Avtlvcobaotep sp. strain
RAG-1  (1)  growing on hexadecane as the sole source of carbon and  energy.
The evidence  indicates that bacterial growth and production of dispers-
ants are not  necessarily correlated.  Maximum  oil dispersion and  produc-
tion of  the  extracel lular .d ispers ing agent occur 1-2 days after the bac-
terium  reaches stationary phase.  Furthermore, RAG-1  grows well,  but
does not  produce any measurable dispersing  agent, on several nutrients,
such as  glucose,  succinate,  hexadecanol and hexadecanoic acid.  Optimum
production of dispersing agent occurs when  the cells  are grown on
straight chain paraffins  from pentadecane to nonadecane.   We do not yet
know if  the paraffins are  necessary  substrates for biosynthesis,  or play
a  regulatory  function leading  to the production of dispersants,
      The oil-dispersing  agent  has  been  partially  purified from station-
ary phase cultures  of RAG-1  growing  on  0.1%  hexadecane, 0.058  mM  K2HPOn
and 7-5 mM urea  in  sea water.   After  removal of cells  by  centr ifugation
at  10,000 x g  for  15  min, the  clear  supernatant fluid  was dialized for
I8.hr against  distilled water,  concentrated in vaeuo and  then  precipi-
tated with k vol acetone containing  U  Lid.   The  precipitate was dis-
solved  in 0.01 M phosphate buffer, PH 7.2, and  passed  through a Sephadex
G-200 column.  The  void volume  which contained  all of  the emulsifying
activity was dialyzed extensively against distilled water  and  lyophilized
to  yield the partially purified dispersing agent ERAG .  An amount of
250  mg dry white powder per  liter culture fluid was obtained.

     Purified ERAG  rapidly forms stable emulsions with over 200 times
its  weight of crude oil, gas-oil or hexadecane.  Although  Epar  is eluted
in  the void volume following chromatography  on  Sephadex G-200   it can be
dissociated into active dialysable subunits.    In dilute aqueous medi
ERAG appears  microscopically as micelles.   There is no measurable
in activity of ERAG by the  following  treatments:  (1)  1 00 r  i  h
(2)  1 mg/ml  Pronase, 37 C,  18 hr; (3) excess  KIO,.  The half-life'of


ERAG was 5 min in 1  N HC1 at 100 C and 2 hr in 1  N NaOH at 100 C.  A
report on the chemical and spectral analyses must await a demonstration
that the material is homogeneous.

     The toxicity of crude oil  and dispersions of crude oil to develop-
ing sea urchin embryos are shown in Table 1.  The developing sea urchin
embryo system was utilized because of the possibility of subsequently
examining the biochemical basis of the toxicity.   The data clearly indi-
cate that bacterial  emulsions of crude oil are over 100 times more toxic
than the crude oil itself.  The toxicity of the bacterial-induced emul-
sions can be decreased over 20-fold by dialysis against sea water.
Table 2 indicates that the purified ER/\Q is not toxic whereas emulsions
formed by ER/\Q are slightly toxic.  When the ER/\Q-O! 1 emulsion was di-
luted 1:5 into sea water, no toxicity was detectable.  It should be
pointed out that these measurements may differ from previous determina-
tions of toxicity of marine organisms (7) or inhibition of chemotaxis


     Like many oil-degrading bacteria, RAG-1 accomplished only a partial
breakdown of crude oil.  Under conditions of limiting oil concentration,
approximately 50% of the oil is converted by RAG-1 into a form which is
no longer extractable by organic solvents (10).   In order to isolate
additional oil-degrading bacteria, the residual oil (RAG-1 depleted) was
recovered and added to sea water supplemented with urea and KzHPOit.
This medium was then utilized for a second enrichment culture procedure.
The dominant microorganism, referred to as UP-2,  appearing in the secon-
dary enrichment culture was isolated in pure culture for further study.
     Table 3 shows the growth of RAG-1 and UP-2 on different carbon
sources.  Both RAG-1  and UP-2 grew well  on crude oil  or straight chain
paraffins derived from crude oil.  Neither strain was capable of growth
on the asphaltene fraction of oil.  Whereas RAG-1 grew poorly on branch
chain paraffins or oil that had been depleted by prior growth of RAG-1
or UP-2, the strain  UP-2 grew relatively well  on branch chain paraffins
and RAG-1  depleted oil, but poorly on UP-2 depleted oil.  These data
indicate that UP-2 has a broader range of substrate specificity than
RAG-1.  These preliminary experiments indicate the potential of utiliz-
ing "bacteria-depleted oil" as substrates for the  isolation of new
strains of oil-degrading bacteria.  It should be emphasized that UP-2
never appeared in our initial  enrichment cultures on crude oil.

TABLE  1.   Toxicity  of Crude  Oil  and  Bacterial  Dispersions of Crude Oil  to Developing Sea  Urchin
Additions to
the suspension
of embryos
1 .


Crude oil U

Bacterial emulsion
of 1.0% oil,
diluted: 1:4 1
Dialyzed emulsion
diluted: 1:4
Microscopic observations at the
1 hr
3 hr
24 hr
following stages
48 hr
72 hr
late pluteus
few pluteus
rupture of embryo
never reaches
801 gastrula
motile embryo
10% prism

no pluteus
late pluteus
but never reaches gastrula
no pluteus
late pluteus
Normal development
SI ight toxicity
Very toxic
Very toxic
Non- toxic
      Four-day culture of RAG-1 grown on 1.01 crude oil in supplemented sea water  was diluted into  suspension of
       as indicated.

      RAG-1  induced emulsions were dialyzed  extensively against  sea water prior  to dilution.
embryos as indicated.


TABLE 2.  Toxicity of Bacterial  Emulsifier (and the  Emulsions) to Developing  Sea Urchin Embryos



Additions to
the suspension
of embryos
0.25 mg/ml
Emulsion formed
fay ERAG

Microscopic observations at the following stages
1 hr
2 cells

2 cells

1 cell
2 cells

2 cells
3 hr
8 cells

8 cells

2-k cells
8 cells

8 cells
2k hr 48 hr 72 hr
gastrula prism late pluteus

gastrula prism late pluteus

motile but deformed embryos
gastrula never reaches prism

gastrula prism pluteus
Normal development

Non- toxic

Tox i c-devel opmen ta 1
       ERAG was purified as described in the text.

          TABLE 3.   Growth of RAG-1  and  UP-2  as  a  Function  of
                    Oil  Fraction
Nutrient (0.
Stra i
depl eted
ght chain
Branch paraffi
1 tenes


RAG-1 /ml b

X 1
x 1
x 1
x 1
x 1

2 x
7 x
2 x
1 x
3 x

               aln addition  to  the  nutrient,  the  sterile sea
          water contained  0.058  mM  I^HPOit  and  7.6  mM  urea.

                Total  cell  number was  determined with a  Petroff-
          Hauser counting  chamber.

                Depleted oil  was prepared  by growing  the  bacterium
          under conditions  in which the  crude  oil  was growth  limit-
          ing.   The residual  oil was then  extracted and concen-

     We thank Zim Lines and Transasiatic Shipping Co. for making the
ship experiment possible.  Captain U. Svirsky and D. Frumer were espe-
cially helpful  in making the necessary arrangements for the voyage.
Aboard ship, we received extraordinary cooperation from Captain M.
Chinsky, Chief Mate H. Kuypers and the rest of the crew.  The work was
supported,  in part, by the National Council of Research and Develop-
ment , Israel.
                           LITERATURE CITED

1. Atlas, R. M., and R. Bartha.  1972.  Degradation and mineralization
       of petroleum in sea water:  Limitation by nitrogen and phos-
       phorus.  Biotechnol. Bioeng. 14:309-318.
2. Fallah, A. A., M. Badakhshan, M. Shabab, and A.  Maanoosi.  1972.
       Correlated data of  Iranian crude oils.  J. Inst. Petroleum 58:75
3. Floodgate, G. D.  1972.  Microbial  degradation of oil.  Mar.  Poll.
       Bui 1 . 3:41-43.

 4.  Foster,  J.  W.   1962.   Hydrocarbons  as  substrates  for  microorganisms.
        Antonie van Leeuwenhoek J.  Microbiol.  Serol.  28:243-274.

 5.  Jobson,  A., F.  D.  Cook,  and D.  W.  S. Westlake.   1972.   Microbial  uti-
        lization of crude oil.   Appl.  Microbiol.  23:1082-1089.

 6.  Lowry,  0.  H.,  N.  J.  Rosebrough, A.  L.  Farr, and  R.  J.  Randall.   1951.
        Protein measurement  with the Folin phenol  reagent.   J.  Biol.
        Chem.  193:265-275.

 7.  Nelson-Smith,  A.   1968.   Effects of oil  pollution and  emulsifier
        cleansing  on shore life in  South-West  Britain.  J.  Appl.  Ecol.
 8.  Mateles, R. I., and  S. K.  Chian.   1969-   Kinetics of  substrate up-
        take in pure and  mixed  cultures.   Environ.  Sci, Technol.

 9.  Mitchell,  R.,  S.  Fogel,  and I.  Chet.   1972.   Bacterial  chemorecep-
        tion:   An  important  ecological  phenomenon  inhibited by  hydro-
        carbons.  Water  Res. 6:1137-1140.

10.  Reisfeld,  A.,  E.  Rosenberg, and D.  Gutnick.   1972.  Microbial degra-
        dation  of  crude  oil:  Factors  affecting the dispersion  in sea-
        water  by mixed and pure cultures.   Appl. Microbiol.  24:363-368.

11.  Soli,  G.,  and  E.  M.  Bens.   1972.   Bacteria which  attack petroleum
        hydrocarbons in-a saline medium.   Biotechnol.  Bioeng.  14:319"330.

12.  Stone,  R.  W.,  M.  R.  Fenske, and A,  G,  C, White.   1942.   Bacteria
        attacking  petroleum  and oil fractions. J.  Bacteriol.  44:169-178.

13.  Zajic,  J.  E.,  and B.  Supplisson.   1972.   Emulsification and degrada-
        tion of "Bunker  C" fuel oil by microorganisms.  Biotechnol.
        Bioeng. 14:331-343-


                 J.  E.  Zajic  and  A.  J.  Daugulls*


     The existence of  hydrocarbon-oriented  continuous enrichment systems
in nature is not unusual.   This is  substantiated by  the additional one
or two orders of magnitude of hydrocarbon-oxidizing  microorganisms pres-
ent per unit volume in chronically  oil-polluted waters.  The varying
complexity of crude oils,  as well as the  presence of  incomplete and co-
oxidative metabolic intermediates,  promotes the ubiquity of mixed cul-
tures with diverse enzymatic capabilities.   An enrichment approach re-
quires evaluation of hydrocarbons,  toxic  hydrocarbons and high molecular
weight hydrocarbons in terms of their potential as energy for microbes
and how they impose contraints  during biodegradation.  Specific examples
are given to demonstrate the ability of the selective enrichment process
to resolve "organic pollution problems."

                         SELECTIVE  ENRICHMENT

     Recently a considerable amount of attention and  research has fo-
cused on the microbial degradation  of oil pollutants  in terms of artifi-
cially seeding oil spills.  In  this regard, many studies have been con-
ducted to determine the factors affecting biodegradation of hydrocarbons.
The factors, including oxygen concentration,  nutrients, temperature,
turbulence,  etc., were evaluated primarily  in terms  of their effect on
rates of mineralization and decomposition,  components of the hydrocarbon
or oil attacked, and the extent to  which  these constituents were de-
graded.  Many of the factors affecting hydrocarbon biodegradation have
been considered elsewhere  (1, 2, k, 6, 25,  29).
     In order to fully evaluate artificial  seeding techniques, a number
of other, perhaps more basic, considerations  must be  examined.  What
types of microorganisms should  be used, which ones will predominate be-
cause of selective enrichment,  what other artificial  materials, i.e.,
nutrients, emulsifiers, etc., must  be added to spills and, most impor-
tant, what effect will all of this  have on  the naturally-occurring
flora and fauna?
     "Faculty of Engineering  Science,  The  University of Western On-
tario, London, Ontario N6A 3K7  CANADA.

     It is generally agreed (A, 7, 21, 26,  36)  that one genus or even
one class of microorganisms cannot degrade all  the components of crude
oil completely.  This is due to the complex composition of the crude
oil, as well as the numerous physical  and chemical constraints, i.e.,
temperature, toxic intermediates, salinity requirements, etc., that
render a single type of microbe ineffective.  The seeding of oil spills
can be viewed as an artificial  implantation of  a widely mixed microbial
population under probably non-ideal conditions  on a highly variable
system.  Thus, it is likely that continuous selective enrichment condi-
tion will be important in determining  the outcome.  The more highly
toxic the waste, the greater the need  for an enrichment process, since
this permits selection of mutants with a greater tolerance to the toxic
compound and an increased ability to degrade the toxic component.

     The concept of natural selection  in the microbial world was formu-
lated in the classic paper by C.  B. van Neil (32).  This somewhat Dar-
winian approach suggested that  it is the environmental conditions that
determine which biological forms can best compete in the struggle for
survival.  Single cell organisms are especially sensitive to environmen-
tal conditions because changes  in temperature or composition of the
microenvironment can cause related conditions within the microbe to
change rapidly and often significantly.  For example, it has been calcu-
lated (12) that a spherical particle,  one micron in diameter, will
reach the temperature of its environment within milliseconds even with
the assumption of unrealistically low  thermal conductivities.  The large
surface area to volume ratios and minute diffusional path lengths typi-
cal of the microbial world result in high metabolic rates.  In addition,
adaptation and natural mutation rates  are generally high, and generation
times are typically minutes or  hours as compared to days or weeks for
more complex life forms.

     Microorganisms in pure or  in mixed cultures are dependent upon such
factors as composition and concentration of substrates, actual and  in-
duction enrichments, temperatures, availabi1ity of nutrients, etc.   With
mixed culture systems, however, factors such as the production of com-
pounds toxic or inhibitory in very low concentrations to potential  micro-
bial competitors, or differences in growth  rates, play important roles
in determining generic composition and sequence of predominating orga-
nisms.  Furthermore, in mixed populations gradual modification of the
environment brought about by proliferation  of one or more organisms in-
evitably makes the altered environment more favorable for development of
other microbes.  Selective enrichment  considerations, then, are important
in evaluating the effect of artificially-seeded mixed populations on oil
spills in terms of types and degree of component decomposition, effect
of degradation-enhancing materials, microbial species interactions  and
ultimately, possible impact on  the environment.

                          COMPOSITION OF OIL

     One of the most important factors affecting generic composition of
hydrocarbonoclastic microorganisms in oil spills, as well as the pos-
sible extent of biodegradation, is the composition of the crude oil.  In
a recent study of the bacterial seeding of oil slicks (21) it was sug-
gested that the type and quantity of crude oil used, rather than nutrient
concentration or inoculum density, determined the effectiveness of arti-
ficial seeding.  Fractions of crude oil are often categorized as either
n-saturated, aromatic, asphaltene or NSO (nitrogen-sulfur-oxygen); fur-
ther classification to the percent of individual components in each
fraction is sometimes determined as well.
      It is generally agreed that the n-saturate fraction of crude oil
is most readily attacked by hydrocarbonoclastic microbes (3,  10, 13, 18,
31, 35).  Generally, the monoic acid pathway  (alkane to primary alcohol
to aldehyde to carboxylic acid) is the primary mechanism of alkane oxi-
dation; dioic acid formation by di-terminal oxidation is thought, at
best, to be only a minor pathway (31).  It has been suggested (23) that
degradation of individual n-alkanes does not occur at a uniform rate
and that a polyauxie phenomenon may be active.  This condition, however,
is less likely to occur when mixed populations of microbes are used
     Although it has been shown that some relatively long-chain alkyl-
substituted cyclic hydrocarbons, such as n-nonylbenzene and n-dodecyl-
benzene, can support microbial growth and form cyclic acids (11), the
large proportion of aIkyl-substituted cyclic hydrocarbons are only par-
tially oxidized (co-oxidized) in the presence of n-alkanes (11, 16, 2k,
31).  Consequently,  phenyl alkanes tend to be  incompletely oxidized and
may accumulate as phenyl-substituted fatty acids (11, 31).  Microbial
attack invariably is concentrated on the alkyl chain rather than on the
phenyl or cycloalkyl ring (31); co-oxidation of the alkyl substituent is
followed by 3-oxidation  (11).  Further degradation of cyclic  acids with
even numbers of carbon atoms in the fatty acid chain (phenylacetic,
cyclohexaneacetic, cyclohexanebutyric) has been shown to be difficult,
whereas cyclic acids with odd numbers of carbons in the fat acid portion
(benzole, phenylpropionic, cyclohexanepropionic) are readily degraded
further (11).
     The microbial  degradation of aromatics has been reported in sev-
eral  summary papers (16, 31), yet detailed research into the exact
mechanisms has been severely limited by the extremely labile nature of
the enzymes that catalyze the initial incorporation of oxygen into aro-
matic hydrocarbons  (16).  Most available evidence indicates that cate-
chol  or an alkylated form of catechol is an intermediate of aromatic
hydrocarbon biodegradation.  For example, catechol  has been shown to be
a metabolic intermediate in the biodegradation of benzene and toluene;
following the formation of catechol, the aromatic ring is split to pro-
duce a dicarboxylic acid.  Naphthalene, anthracene and phenanthrene
degradation proceed to catechol via salicylic acid (16, 31).


     Considerable work has been done to examine the biodegradabi1ity
and microbial metabolic pathways of hydrocarbon fractions, or more  com-
monly, the degradation of specific components of hydrocarbon fractions
by hydrocarbonoclastic isolates.  This has led to generalizations,  i.e.,
if a microbe is capable of utilizing one or more aromatic hydrocarbons,
it will, in all likelihood, be able to utilize aliphatic hydrocarbons
(10, 29); and,  a microbe grown on a particular hydrocarbon will, in
general, attack related but structurally more complicated hydrocarbons
immediately, but without complete oxidation (31).  However, the extent
and relative rates of utilization by mixed populations of microbes  has
not been fully investigated to date.  Such research hopefully would
answer some of the questions implicit in the many investigations of
hydrocarbon biodegradation by pure cultures and/or with pure substrates:
What effect would the toxic or non-biodegradable hydrocarbpns have  on
utilization of other fractions?  How would accumulation of certain  in-
termediates  (cyclic acids) affect microbial  attack of other hydrocar-
bons?  To what extent can the polyauxie condition of paraffins be ap-
plied to the remaining fractions of the crude oil?  Would microbial
species  interactions influence oil biodegradation?  Which microbial
species would predominate, and for what length of time?

     A number of studies of biodegradation of crude oils by mixed popu-
lations of microorganisms have been conducted recently (2, 3, 17,  18,
33).  Westlake and his co-workers (17, 33) determined that the composi-
tion of the crude oil  substrate, as well  as the composition of the  en-
richment oil, determined not only the generic composition of the micro-
bial population but also the growth characteristics (see Table 1).
Crude oils were classified from a biodegradabi1ity standpoint as being
of high or low quality depending on the compositional relationship  be-
tween saturates and aromatics.  The studies determined that biodegrada-
tion depended not only on the metabolic capabilities of the microbes
present and composition and amount of the paraffinic fraction, but  also
on the chemical composition of the asphaltenes and NSO fractions.   Nor-
mal or high levels of n-saturates did not necessarily assure biodegrada-
bility.  Kator (18) found that the rate of total  n-paraffin utilization
was proportional  to the amount of this fraction present in the crude.
It was found (17, 33)  that microbial populations  enriched on a poor
quality oil readily attacked a high quality oil  but that microorganisms
enriched on high quality crude oil were invariably incapable of effec-
tively metabolizing a low quality oil.  Polar,  non-hydrocarbon NSO' s
and asphaltenes apparently are produced as a result of microbial  action
on crude oils,  resulting in a low-wax, lower API  gravity crude oil  (3,
33)-  Atlas and Barth (2) demonstrated the probable existence of a
volatile toxic fraction in crude oils that had to be removed before
effective biodegradation could occur.  The reduction of a biodegradation
lag period for "preweathered" crude oil  samples versus fresh crude  oil
was demonstrated.

     The preceding investigations indicate the wealth of relevant data
obtainable from studies of the mixed population biodegradation of crude
oil; further research in this area is certainly desirable.


TABLE  1.   Change in Generic Composition  of Microbial  Populations
           at 30 C as Affected by Crude Oil  Compositionb
Percent of Population

Aahpomobaaten> sp.
Aeinetobaoter sp.
Flavobaeterium sp.
and Cytophaga
Aainetobaater sp.
Artkrobacter sp.
Xanthomonas sp.
Unidentified Gram
negative rodsc
Unidentified Gram
negative rods0
Unidentified Gram
negative rods0
Aahfomobaater sp.
Aloaligenes sp.
Arthrobacter sp.
Ps.eudornonas sp.
Unidentified Gram
negative rods0
Unidentified Gram
negative rodsc
Ackromobacter sp.
Alcaligenes sp.
Xan thomonas s p .
Enrichment Oil
Lost Horse
Norman Wei Is

Norman Wei 1 s
Norman Wei Is
Norman Wei Is
Test Oil
Lost Horse
Lost Horse

x Point
Lost Horse
    After 4  days growth of the fourth transfer at 30 C.

    Adapted  from West lake et al.,  1974 (33).
    °Differing at least in one character from  the designated genera.

                          TEMPERATURE EFFECTS

     With the increased interest in tapping the large oil  reserves in
northern Canada and Alaska, the ecological  impact of a possible oil
spill in Arctic waters has received widespread attention and contro-
versy.  Cold ocean waters are not,  however, unique to the Arctic areas
since greater than 3Q%, by volume,  of the world's seas are less than
5 C  (37).  It has been suggested (14) that  the majority of marine micro-
organisms are probably psychrophiles, which has,  in all likelihood,
prompted several of the studies of  hydrocarbon degradation at low tem-
peratures (2, 10, 17,  26, 29, 33,  37, 38).
      In studies by Westlake and his co-workers (17, 33) it was  found
that the temperature used during enrichment of hydrocarbonoclastic
microbes had a significant effect  on the generic  composition obtained.
Enrichments performed  on the same  oil, but  at A C and 30 C,  yielded
substantially dissimilar microbial  distributions  (see Table 2).  Cell
yields and growth rates at k C were only slightly less than those at
30 C.  This is perhaps in contrast  with studies by Atlas and Bartha  (2),
Cundel1 and Traxler (10) and ZoBell  (37) in which lower water tempera-
tures resulted not only in slower  degradation rates but also in longer
lag  periods prior to the onset of  detectable biodegradation.  Studies
of psychrophi1ic, or at least psychrotolerant, microbes revealed that
most oil fractions (except isoprenoids [33] and some cycl ics and aro-
matics  [10]) biodegradable at mesophilic temperatures also were readily
degraded at the lower  temperatures.   Microbial populations enriched
under psychrophi1ic temperatures were found to readily metabolize oils
of similar quality under mesophilic temperatures,  whereas  mesophi1ically-
enriched populations exhibited limited activity on similar quality oils
under psychrophi1ic conditions (17,  33).

     An interesting interpretation  was recently proposed (2, k) to ac-
count for the notably long lag periods preceding  biodegradation of oil
under psychrophi1ic conditions.  Besides the fairly common lowering of
biochemical  activity at reduced temperatures, slow evaporation  of the
aforementioned volatile petroleum  components, inhibitory to oil-
degrading microorganisms, was credited with retarding biodegradation.
At higher (mesophilic) temperatures, the evaporation rate of the inhibi-
tory component(s) was  significantly increased, resulting in notably
shorter acclimatization periods.  "Preweathered"  crudes showed  little
or no lag period.

      Induction enrichment temperatures as well as growth temperatures
have a marked effect on the generic composition of hydrocarbonoclastic
microbes as well as on the degree  of biodegradation that can be ex-
pected.  Enrichment temperatures must be chosen wisely, not only be-
cause of their pronounced effect in determining the make-up of  the mi-
crobial population but also because of the  probable wash-out that would
occur should the microbes be seeded under incompatible temperatures,
i.e., temperatures adverse to enrichment.  Although significantly more
kinds of microbes grow at mesophilic than at psychrophi1ic temperatures,
the necessity of cultivating psychrophi1 ic  or at  least psychrotolerant


microbes  is  substantiated by their ability  to degrade most hydrocarbons
at higher temperatures.   The reverse  is not necessarily true.  Species
must be isolated,  however, that will be effective  against the more re-
sistant components already mentioned.

        TABLE  2.   Effect of Temperature on Generic Composition
             of Microbial Populations Obtained by Enrichment
           Culture on Oils of Different Chemical Composition
Achramobaater sp.
Aainetobactep sp.
Alaaligenes sp.
Artkrobaater sp.
Flavobaateriwn and
Cytophaga sp.
Pseudamonas sp.
Xanthomonasc' sp.
Unidentified Gram
negative rods
Unidentified Gram
negative cocci''

Prudhoe Bay
4 C
30 C

4 C
0 i
30 C
i 1
4 C

30 C

4 C

Horse Hill
30 C
       aAfter 4 days growth during the fourth transfer.
       bAdapted from Westlake et al., 1974 (33).
       cAlthough these bacteria keyed out as Xanthomonas  this genus is reserved for
   plant pathogens; no attempt was made to determine their pathogenicity.
       ''Differing in at least one character from designated genera.
                        AVAILABILITY OF NUTRIENTS

     Potentially  a  significant drawback  in artificially  seeding oil
spills  is  the  nutrient deficiency of seawater, especially in nitrogen
and phosphorus  (1,  A,  6, 21, 25, 26).  Fairly  typical  values of total
nitrogen and phosphorus in seawater are  980 and 70  yg/1  respectively
(1).  Optimal  concentrations of these nutrients for mixed cultures of
hydrocarbon degraders  were found to be 10 mM nitrate and 0.35 mM phos-
phate  (25).  Presumably, these values correspond  to the  cell surface
concentrations of 10"7 M nitrogen and 10~8 M phosphorus  thought to be
adequate for microbial growth (26).  The preceding  figures must be
viewed, however,  in light of data from artificial  seeding experiments


conducted by Miget (21).   It was estimated that at least 100 to 1000
times more bacterial  cells were concentrated at the oil-water interface
than in the seawater  itself.  The implication is that nutrient salt con-
centrations at the interface should perhaps be 100 to 1000 times greater
than the values typically reported.  Methods of maintaining adequate
nutrient salt concentrations by periodic spraying and wax encapsulation
(21), and application of  oleophilic nitrogen and phosphorus "fertilizers"
have been considered  (A).  Although the previously-mentioned techniques
have achieved some degree of success, technology for the cheap and ef-
fective large-scale addition of phosphorus and nitrogen "fertilizers"
must still be developed.
     Some comment should  be made about not only the addition of nutrient
salts to oil spills to facilitate the growth of hydrocarbon degraders
but, more generally,  the  introduction per se of large quantities of mi-
crobial cultures into the environment.  It is probably fairly safe to
say that very few investigations have been conducted to determine the
potential pathogenic!ty of hydrocarbon degraders, or their metabolic
intermediates, to humans, animals or to the organisms occurring natur-
ally in the aquatic environment.  High concentrations of mixed microbial
cultures with presently poorly defined biological activities should be
treated with utmost care.  Crude oil components (28) and metabolic in-
termediates and by-products (5, 8) have been shown to adversely affect
or even limit naturally-occurring aquatic organisms.  To our knowledge,
however, the effect of mixed populations of oil  degraders on the environ-
ment has not been determined.   Should artificial  seeding of oil  spills
be judged operationally feasible, screening of the seed organisms should
be performed scrupulously.   Addition of nitrogen and phosphorus supple-
ments to oil spills will  not only enhance the growth of hydrocarbon de-
graders but also other competitive forms, including pathogenic organisms.
Without further information on microbial ecology and microbial species
interactions, large-scale addition of nutrient salts should be viewed
as a potential and not as a real benefit.

                          CONCENTRATION OF OIL

     Due to the slight solubility of most petroleum hydrocarbons in
water,  typical concentrations  used in investigations of biodegradation
have been between 0.2 and 2.0%.  Despite the fact that massive concen-
trations of hydrocarbons  may exist in at least local  areas of oil  spills,
the influence of high hydrocarbon concentrations  on microbial  kinetics
and metabolism has been poorly documented.  The emulsification of oil
could lead to the exclusion of the aqueous medium and local  concentra-
tions of oil around the microbes could increase by a factor of several
million (26).  Qualitatively it has been suggested (36)  that at high
hydrocarbon concentrations  fractions most susceptible to microbial  at-
tack would be preferentially degraded, whereas in relatively low concen-
trations most fractions would  undergo at least some degree of degrada-
tion .

     Although decrease in a physiological  parameter (specific growth


rate, oxygen uptake, etc.) of a microbe at high substrate concentrations
has generally been associated with substrates not limited by solubility,
some hydrocarbon degraders show similar patterns  (11, 30, 3*0 .  Several
mechanisms of substrate inhibition have been summarized by Edwards (12)
yet no single inhibition mechanism has been assigned to hydrocarbon de-
graders.  Edwards  (12) suggested that inhibition often involves a multi-
plicity of mechanisms rather than just a single one.  The likelihood of
an oil micro-environment, rather than an aqueous one, increases with
oil concentration and this situation may be the first step leading to-
ward  inhibition.

     Direct physical contact between microbial  cells and the oil sub-
strate probably  is required for growth (13, 19, 26).  Microorganisms
have been shown  to adhere to and completely cover oil droplets  (13, 19)
and even enter and metabolize inside the oil phase, although the degree
of oleophi1icity varies between microbes (26).   A degree of oil emulsi-
fication yielding droplets of comparable size to the cells results in
an unfavorable configuration for substrate transfer.  If small  oil drop-
lets adhere to microbial cells, or if the oil  droplets are sufficiently
large to allow microbial adsorption, growth is  enhanced (13).

      It has been suggested that formation of water-in-oil  emulsions is
more common than formation of oil-5n-water emulsions and that the former
type of emulsion is stabilized by the colloidal asphaltene particles oc-
curring naturally  in crudes (27).  Coty et al.  (9) have conducted fer-
mentations with oil-in-water emulsions and water-in-oil  emulsions.  They
determined that microbes were concentrated in the aqueous phase of water-
in-oil emulsions and that the effect of a hydrocarbon layer between air
and water accelerated rather than retarded oxygen transfer.  The inver-
sion of the oil-phase-continuous condition to one of water-phase-
continuous was accomplished by either a dilution of the surfactant used,
a drop in the temperature, an increase in the pH or through the produc-
tion of metabolic  intermediates.

     The applicability of the preceding reports to seeding of oil  spills
becomes obvious when comparing the susceptibility to wash-out of the
microorganisms trapped in the aqueous phase of  the water-in-oil emulsion
with the microorganisms concentrated at the interface of the water and
oil phases.  It might be suggested that the varied oleophilic nature of
hydrocarbon oxidizers, as well as the genetic capability of producing
extracellular emulsifiers, determine where or why a particular microbe
is concentrated  in a particular type of oil waste or spill.

     The large increase in production of cellular and extracellular
lipids by Acinetobacter sp. as a result of hydrocarbon metabolism has
been demonstrated by Finnerty et a 1. (15).  Although extracellular ex-
cretions are thought to increase oil emulsification (35),  the reason
for this rather dramatic metabolic reaction and the extent to which
other microbes may be influenced in a similar way remain unknown.  The
study by Finnerty et al. (15)  also demonstrated the distorted metabo-
lism and internal structure of Acinetobaoter when grown on hexadecane.
Electron micrographs showed internal hydrocarbon inclusion bodies;


similar changes have been demonstrated for Candida lipolijtioa (23) and
Torulopsis sp. (19) as well.  Aa-inetobacter cells cultivated on hexadec-
ane occasionally grew to A to 10 times their normal  size, becoming
greatly extended and elongated with extensive intracytoplasmic membrane
development.  The development of the hydrocarbon inclusions is signifi-
cant because  it was typical  of hydrocarbonoclastic bacteria as well as
yeasts and filamentous fungi.  In the case of Candida lipolytica, the
organism was unable to distinguish between utilizable and non-uti1 izable
hydrocarbons; both types of  hydrocarbons were found  within the cell (23).

     It is perhaps important to question just how far the included hydro-
carbons may travel in the food chain before disappearing, before being
converted to non-toxic intermediates or before being metabolized to po-
tentially harmful ones.  To  our knowledge, no explanation has been pro-
posed to interpret how or why a microorganism stores hydrophobic hydro-
carbons against a concentration gradient or how or why it undergoes sig-
nificant cellular distortions.  Correlations between high hydrocarbon
concentrations, cellular transformation, hydrocarbon inclusions, and
their subsequent effects on  the aquatic environment  are only tentative
as no definitive studies are available.


     It is quite probable that seawater and the open ocean provides the
impetus for another form of  microbial  selective enrichment,  The saline
nature of seawater is a first consideration,  Many microbes originally
isolated from the sea have obligate salinity requirements which must be
satisfied in preparation of  growth media,   For other microorganisms,
natural seawater is inhibitory or even toxic, the effect of salinity
being only one factor.  A number of factors affect the decline of micro-
organisms, particularly non-marine organisms, in seawater.  These have
been summarized in a review  paper by Mitchell (22),

     Several studies have indicated that a biological heat-labile toxin
produced by marine microbes  was the bactericidal  agent in seawater.
This factor was either diminished or eliminated by boiling, autoclaving,
filtration, pasteurization or chlorination.  A number of species of mar-
ine bacteria and actinomycetes were tested for their antagonism toward
non-marine microbes.  A substantial proportion (9 of 58)  were antagonis-
tic to non-marine microorganisms and it was thought  that the antagonistic
material was an antibiotic.   It was also found that  some marine bacteria
were able to  lyse the cell wall  of non-marine microbes.   Other naturally-
occurring sea organisms were active against microbes either through a
parasitic action or directly through predatory activity.   The native
marine microflora was also found to competitively displace non-marine
microorganisms under the growth-limiting conditions  in the sea.   The
removal of microbes by physical  means such as adsorption, flocculation
and sedimentation was also thought to be prevalent.

     The possibility of oil  spills occurring in either salt or fresh
water areas probably requires that enrichment cultures of either marine


or non-marine types of microorganisms be available.  Salinity per se is
not the only factor that affects the decline of the non-autochthonous
(non-native) microorganisms in the sea but that certain biological,
chemical and physical characteristics of the oceans are important fac-
tors in the decline of certain microbial populations,


     An oil spill acts as a selective enrichment system to continuously
select for microorganisms capable of causing degradation and eventual
break-up of the oil.  In some spills, seeding with microorganisms may
not be necessary because of the presence of indigenous hydrocarbonoclas-
tic microbes.  Data would indicate that indigenous populations can re-
move paraffinics, whereas the more complex hydrocarbons are removed by
highly specific microbes.  However, dictates for seeding are probably
more appropriately governed by:  (1) the harshness of the environment,
e.g., saline condition of the ocean, and temperatures as low as k-S C;
(2) highly toxic components present in oil, e.g., phenols, aromatics,
carcinogenic compounds, etc.; (3) nutritional condition of the environ-
ment, i.e., adequate inorganic nitrogen and phosphorus to support growth
of either  indigenous populations or special seed cultures.  If the en-
vironment per se imposes constraints, the application of specially
adapted and mutated cultures is necessary,  Likewise, if the oil  spill
contains toxic components which are normally inhibitory to most microbes,
specially adapted and mutated cultures may be far more beneficial  than
the indigenous populations.

     Selective continuous enrichment systems are easily studied in the
laboratory.  This technique is adaptable for selecting mixed microbial
populations which can compete under adverse chemical and physical  condi-
tions of the environment.  Thus, data presented herein suggest that mi-
crobial seeding of oil spills in aqueous environments may be beneficial.
Nevertheless, adequate studies to establish limitations and instances of
where biotreatment would be most successful are required.

                           LITERATURE CITED

1. Atlas, R. M., and R. Bartha.  1972.  Degradation and mineralization
       of petroleum in sea water:  Limitation by nitrogen and phos-
       phorus.  Biotechnol. Bioeng. 14:309-318.

2. Atlas, R. M., and R. Bartha.  1972.  Biodegradation of petroleum in
       seawater at low temperatures.  Can. J. Microbiol. 18:1851-1855.

3. Bailey, N. J. L., H. R. Krouse, C, R. Evans, and M. A.  Rogers.  1973.
       Alteration of crude oil  by waters and bacteria--evidence from
       geochemical  and isotope studies.  AAPG Bull. 57:1276-1290.

4. Bartha, R., and R. M. Atlas.  1973-  Biodegradation of oil in sea-
       water:  Limiting factors and artificial stimulation.  In D. G.
       Ahearn and S. P. Meyers (ed.), The Microbial Degradation of Oil
       Pollutants.   Louisiana State Univ., Center for Wetland Resources,
       Pubn. LSU-SG-73-01.

 5.  Brown,  L.  R. ,  and  G.  S.  Pabst.   1969.   Microbial  degradation  of  pe-
        troleum in aquatic  and  marine  environments.   Bacteriol.  Proc.,
        p.  62.

 6.  Cerniglia,  C.  E.,  T.  J.  Hughes,  and  J.  J.  Perry.   1971.   Microbial
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12.  Edwards, V. H.  1970.  The  influence of high substrate concentrations
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15-  Finnerty,  W.  R.,  R.  S.  Kennedy,  P, Lockwood, B.  0. Spurlock,  and R. A.
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17.  Jobson,  A., F. D.  Cook,  and D. W.  S. Westlake,   1973.  Relationship
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27. Supplisson, B.  1973.   Microbial degradation and  emulsification  of
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28. Takahashi, F. T., and  J.  S. Kittredge.  1973.   Sublethal  effects of
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29. Traxler, R. W.  1973.   Bacterial degradation of petroleum materials
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32. van Neil,  C. B.   1955-  Natural  selection in  the microbial  world.
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33- Westlake,  D. W.  S.,  A. Jobson, R.  Phillipe, and F.  D.  Cook.   1974.
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34. Zajic, J.  E.  1964.   Biochemical  reactions  in  hydrocarbon metabolism.
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35- Zajic, J.  E., and B. Supplisson.   1972.   Emulsification and degrada-
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36. ZoBell,  C. E.  1973a.   Microbial  degradation of oil:   Present  status,
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37. ZoBell,  C. E.  1973b.   Bacterial  degradation of mineral oils at  low
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                R. M.  Atlas* and  E.  A. Schofield**


      Alaskan Arctic waters from Prudhoe Bay were found  to  contain  in-
digenous microbial populations, capable of degrading  petroleum  hydro-
carbons, in concentrations similar to those found in  our prior  studies
of temperate Atlantic coastal  waters.   The microorganisms were  capable
of degrading Prudhoe crude oil at 5 C and showed  greater oil  emulsifica-
tion than previously studied organisms.  Southern Alaskan waters  from
Port Valdez also contained oi1-degrading microorganisms, but  these  de-
graded Prudhoe crude at much slower rates than the Arctic organisms and
did not show the same ability to emulsify oil.  In situ  tests  in  Prudhoe
Bay revealed higher oil biodegradation rates  when a N and P fertilizer
was added.   Increases of several  orders of magnitude  in  the populations
of oil degraders, especially Pseudomonas species, were found underlying
miniature oil  slicks in Prudhoe Bay.   These increases were  accompanied
by a similar,  although slightly less  extensive,  increase of Staphylo-
aoeeus epidevmidis, a non-oil  degrading mesophile. Several oil degrad-
ing bacteria and yeasts associated with naturally-occurring seepages,
or artificially-introduced oil spills, in the Arctic  were isolated.
These organisms, including members of  the genera  Rhodotamila, and  Pseudo-
monas, are being tested as possible seed inocula  to enhance oil biodeg-
radat ion in the Arctic.


      The discovery and plans  for utilization of  Alaska's north slope
petroleum resources has raised questions of concern over the fate of
accidentally spilt oil.  The adverse  environmental conditions that  char-
acterize the Arctic usually preclude  physical  removal  of the oil, leav-
ing any spilt  oil to natural  degradative and  dispersal processes, in-
cluding emulsification, evaporation of the volatile fraction, microbial
degradation,  and deposition of the undegraded residue.

      The non-biological  weathering and dispersal  of  petroleum is  largely
a function  of  its chemical nature and  environmental factors such  as
temperature, wind,  and wave action.   The natural  biodegradative rates of
      "Department of Biology,  University of  Louisville,  Louisville,
      **Environmenta1  Assessments Unit,  Ohio Department  of  Natural  Re-
sources, Columbus, Ohio.


petroleum in the sea are usually limited by low numbers  of  microorga-
nisms, scarcity of essential  nutrients,  and low temperature (7)•   Low
concentrations of nitrogen and phosphorus in sea water have been  shown
to prevent extensive petroleum biodegradation (2).   In the  Arctic, tem-
perature  is expected to have a profound  limiting influence  on microbial
oil-degrading activities.  In addition to reducing  rates of microbial
metabolism, low temperatures limit evaporation of toxic  components of at
least one crude oil, delaying its biodegradation (5).   Nevertheless, some
microorganisms certainly are capable of degrading petroleum at low tem-
peratures  (3, 1,  16, 13, 10).  One might expect such organisms to be ab-
sent or  in very low concentrations in pristine Alaskan coastal waters.
Robertson et al.  (10)  reported, however, that such organisms are widely
distributed  in the Gulf of Alaska and are present, but more difficult to
isolate,  in the Arctic.  ZoBel1  (16) also has reported that microorga-
nisms from the Arctic  can extensively degrade petroleum hydrocarbons and
crude oils at sub-zero temperatures,  including  up to 61% of Prudhoe
crude within  10 weeks  at -1.1 C.  The distribution and  in situ activity
of such  organisms  in the Arctic,  however,  has not been determined pre-
      The present  study was  undertaken  to  determine natural rates of pe-
troleum  degradation  in Alaskan  coastal  waters,  to elucidate the factors
that  limit these  degradative processes,  and  to  explore  methods for over-
coming these  limitations.  The  first aim was  to determine what would
happen if Prudhoe  crude oil  accidentally entered these  waters; whether
there were sufficient  microorganisms, adequate  nutrient  levels, and
suitable temperatures  to permit  rapid microbial  degradation of this oil.
The next objective was to examine possible ways of accelerating these
degradation rates and  possible  benefits  of  "seeding"  and fertilizing the
oil slicks with suitable nutrients and  organisms.  To this end we tested
the effects of addition of an oleophilic fertilizer previously developed
 (6).  We also isolated a number of potential  "seed" organisms associated
with  natural Arctic oil seepages  or that were enriched  for underlying
artificially  introduced slicks of Prudhoe crude.  It was felt that
autochthonous organisms would be  best for  seeding, providing  that the
numbers  of extant oi1-degrading microorganisms  was a  limiting factor.
                         MATERIALS AND METHODS
      Water  samples were collected from Prudhoe Bay and Port Valdez, the
 proposed  terminals of the Trans-Alaskan pipeline (Fig. 1).  Prudhoe Bay
 is  very  shallow, with an average depth of 0.61-1.22 m  (15), and  is com-
 pletely  frozen over most of the year, being ice-free for only 1-2 months.
 Port  Valdez  is much deeper, with an average depth of over 30 m and is
 open  year  round  (14).  Surface samples were collected  in sterile Erlen-
 meyer flasks.  Bottom samples were collected Using a Van Dorn Water
 Sampler  (Hydro Products, San Diego, Calif.).  The bottle was rinsed
 several  times with bottom water before a collection.  The samples were

stored on ice and returned to the  laboratory for analysis,
                            opo Simpoofl
FIGURE 1.  Map of Alaska showing proposed Trans-Alaskan Pipeline route.
whirl-pack bags.
along a traverse
      Water, soil, and oil samples were also collected from natural oil
seepages at Cape Simpson, seep 2  (11) and Umiat  (8), using sterile
                  Samples from the Cape Simpson  seep were collected
            _._- from one end of  the seep downhill to a lake where the
seep ended.  Samples were taken from the surfaces, both inside the seep,
including oil, tar and overlying  pools of water, and from the adjacent
soil and water.  Samples from the Umiat seep were obtained from soil at
the surface overlying the seep at several depths down to the perma-
frost, k6 cm.


      Water temperature and salinity were measured at each sampling site
using a model RS-5 salinometer (Beckman Instruments, Inc.).  Salinity
also was checked on samples returned to the laboratory using a model
RS-7a inductive salinometer (Beckman Instruments, Inc.).  Oxygen concen-
tration was measured with a dissolved oxygen meter (Yellow Springs In-
strument Co., Yellow Springs, Ohio),  The pH was measured with a pH
meter (Beckman  Instruments, Inc.).  Nitrogen, in the form of ammonium
ions or nitrate plus nitrite  ions, total phosphate, and iron concentra-
tions were determined using a Hach water tester  (Hach Co., Ames,  Iowa).
Nitrate and ammonium ion concentrations were also measured using a Tech-
nicon Autoanalyser  (analyses  courtesy of Dr. R.  Homer).


      Total viable counts were performed using either marine agar 2216
(Difco) for  Bay and sea water samples,  or  nutrient  agar  (Difco) for
coastal pond  and  soil samples.   For  fungi,  Dextrose-Sabouraud agar
(Difco) was  used.  All  samples were  stored  on  ice between  collection and
time of plating,  a period  not exceeding 24  hr.   Serial  dilutions  of
water or soil  samples were  surface-spread  onto  the  agar  in petri  plates
using a Drigalsky spatula,  and  incubated at 5  or 25 C,   Colonies  that
developed were  enumerated at  7 and  14 days  using a  New  Brunswick  Scien-
tific Co, colony  counter  (12).   Oi1-degrading microorganisms were  tabu-
lated by passing  100 ml water samples  through a  0,45 Vim porosity  mem-
brane filter  (Millipore Filter Corp.),  the  latter than  plated on  an oil-
agar medium,  as previously  described  (5).   Colonies  that developed after
incubation at either 5 or 25  C for  14 or 28 days were counted and  iso-
lated for  later identification and determination of  their  hydrocarbono-
clastic abi1i ty.


      One  hundred mill Miter aliquots of water samples  from Port  Valdez
or  Prudhoe Bay  were placed  in a  gas  train arrangement similar to  one
previously described (4), but the flasks were not shaken.  One mi 11 Miter
of  either  Prudhoe crude oil (gift of Atlantic Richfield Co.) or n-hexa-
decane  (Aldrich Chemical Co.) and one ml of  a stock  solution of phos-
phate and  nitrate were added, in final  concentrations of 0.5 mM and 10 mM,
respectively.   Incubation was at 25 C with  constant  aeration with C02-
free air.  C02  evolution from hydrocarbon degradation was  cumulatively
monitored.  After 42 days of  incubation, the residual oil  of n-hexadecane
was recovered by  continuous extraction  for  12 hr with diethyl ether  in a
liquid-liquid extractor (Kontes  Glass Co.).

      Fifty mi 11 Miter aliquots  of the  water samples were  also placed
 into  biometer flasks (Bellco Glass Co.).  One-half milliliter of  either
Prudhoe crude oil or n-hexadecane and 0.5 ml of  the  nitrate and phos-
phate stock  solution described above were added, with incubation at 5 or
25  C.  C02 evolution was monitored cumulatively.  After 35 days   the


residual hydrocarbon was extracted as described above.  The amount of
residual crude oil or n-hexadecane was determined gravimetrically.

    For isolated microorganisms, a A8-hr culture from either marine
broth 2216 or nutrient broth was centrifuged (3500 g for 15 min), washed
twice in and resuspended in Bushnel1 Haas broth.  All broths used were
Difco products,  One mill{liter containing 1,0 mg protein was inoculated
into 250 ml Erlenmeyer flasks containing 99 ml  sterile Bushnel1 Haas
broth and one ml of either n-hexadecane, pristane, Sweden crude (Sun Oil
Co.) or Prudhoe crude (Atlantic Richfield Co.).  The flasks were incu-
bated at 5 C with rotary shaking, 21*0 rpm for 30 days,  The residual
hydrocarbons were recovered by extraction with two 50 ml portions of d i -
ethyl ether.  The extracts were analyzed gravimetrically and by quanti-
tative gas liquid chromatography using a Hewlett Packard model 5700
flame ionization detector gas chromatograph with dual 3 m O.D. by 1.8 mm
long columns packed with 10% Apieyon L on 60/80 mesh Chromosorb (A).


    A styrofoam float containing Plexiglas cylinders, similar to one
previously described  (6), was anchored in Prudhoe Bay at the mouth of
the Putuligayuk River (Fig. 2, site 2).  The Plexiglas cylinders were
5 cm in diameter and A6 cm in length.  One mill filter Prudhoe crude oil
was added to each cylinder.  To some of these miniature oil slicks an
oleophilic nitrogen and phosphorus fertilizer  (6) also was added.  For
other control slicks, the bottoms of the cylinders were stoppered and
]% HgCla added.  The float was set out in mid-June but constant shifting
of the sea ice pushed the apparatus onshore or upset the tubes and it
was early July before the float was continuously deployed.  A second
float set out near the Prudhoe dock  (Fig. 2, site 1) was totally de-
stroyed by the ice.  Replicate cylinders with their crude oil contents
were removed periodically until mid-August,  The microbial populations
in the water columns underlying the oil slicks were enumerated as de-
scribed earlier.  Selected microorganisms were isolated and their oil
biodegradation potential determined.  The oil was recovered from the
removed cylinders by continuous extraction for 2k hrs using liquid-
liquid extraction and diethyl ether as solvent.  The residual oil  was
measured gravimetrically.
    The properties of the water samples collected in mid-June at two
locations in Port Valdez are shown in Table 1.  Site one was adjacent
to the proposed Trans-Alaskan pipeline terminus; site two was at Valdez
Narrows.  The surface waters were relatively warm and well aerated.
Both sites had comparable nutrient concentrations although the salinity
at site two was significantly higher (Table 1).

   FIGURE 2.  Map of Prudhoe Bay showing sampling locations,
Sal ini ty
Oxygen concentration
N as Nht
N as NO! + N02
Loca t i on
Proposed southern
pipel ine terminus
11 C
0. 14 ppm
1 . 0 ppm
0.08 ppm
0 . 1 ppm

Valdez Narrows
12 C
0.11 ppm
1 .3 ppm
0.08 ppm
0.08 ppm

    Water samples also were collected at several sites in Prudhoe Bay
from late June to early August.  Three sites were sampled (Fig. 2), one
about 0.8 km west of Prudhoe dock (site 1), a second at the mouth of the
Putuligayuk River (site 2), and a third about 3.2 km north of the river
mouth, where the water depth was 2.13 m (site 3).  The properties of
these water samples are shown  in Table 2.   As noted, until July 15 the
Bay was highly stratified, with a bottom layer of cold, saline, nutrient-
deficient water.  The thermocline was at about the 1.82 km level in the
deeper portions of the Bay.  Until late July, there was considerable ice
in the Bay and water derived from its melting would be nutrient-deficient
and of low salinity.  The constant sunlight probably explains the rela-
tively high surface water temperature.  It should be noted that, between
the July 15 and July 22 samplings, a storm passed through Prudhoe Bay,
apparently turning over the Bay, as evidenced by the lack of a thermo-
cline on the July 22 sample shown by the similar surface and bottom tem-
peratures and salinities on that date.

    The microbial populations  in the Port Valdez and Prudhoe Bay samples
are shown in Table 3.  The Prudhoe Bay populations were somewhat higher
than those found in Port Valdez.  A notable population was capable of
growth at 5 C, especially at Prudhoe Bay.   The total populations in
Prudhoe Bay showed a general decline until after the Bay had turned over
and the ice had disappeared.   Enumeration of fungi at 25 C showed con-
centrations varying between 0 and 10 viable units/ml.  No fungi were
isolated when incubation was at 5 C.
    The numbers of oil degraders in the surface waters, enumerated at
25 C, were about 100 cells/L at Valdez and 700 cells/L in Prudhoe Bay.
The microbial oil-degrading population in Prudhoe Bay showed only minor
fluctuations in size over the summer.  The oil-degrading population in
Prudhoe Bay enumerated at 5 C was only slightly lower, about 600 cells/L.

    The Cape Simpson oil  seep No. 2 is an  extensive natural  seep con-
sisting of older black asphaltic material  and fresher soft red-white-
brown oil.  At the time of sampling, the temperature of the  soil and
water associated with the seep was about 10 C and the pH 5.7.  The soft
oil-water emulsion contained only fungi, one pink-pigmented  and one
cream-colored yeast and one filamentous green-black imperfect fungus
tentatively identified as a Rhodotorula sp., a Candida sp. and a Mucor
sp.,  respectively.   The size of this fungal population was 4 x 106
viable units/g and 1 x 103 viable units/g  enumerated at 25 and 5 C, re-
spectively.   The adjacent soil  had 3 x 106 bacteria/g and k  x 101*
fungi/g enumerated  at 25 C.  An adjacent lake not directly in contact
with oil  had 2 x 105 bacteria/ml and 3 x 102 fungi/ml.  The  lack of
bacteria  in the seep is unknown, but the low pH may be a contributing


Temperature (C)
Salinity (%o)
Oxygen concentration
PO; (ppm)
N as NHt (ppm)
N as NOa + NOa (ppm)
— a0.9l4 m

Site 1 surface
b2.13 rr

L o c
a t i
o n
2 surface
D a
t e
2 bottom3


3 surface


3 bottomb
not done


fi/i i

a /a
Pi pel ine Site 1
terminus Narrows surface
5C 25 C 5C 25 C 5C 25 C
Number of

3.6 510
	 4.0 23
	 53 61

5 C


: 2
25 C

Prudhoe Bay
Site 2 Site 3
bottom surface
5 C 25 C 5 C 25 C
(in hundreds/ml)

17.1 18 7.0 20
11,2 132 29 69

Site 3
5 C 25 C

10 70
33 73

    The seep at Umiat was much smaller than the Cape Simpson seep.  The
surface soil was largely root mat with a temperature of IOC.  Just be-
low the organic root mat was a clay layer 1 C at the top and 0 C at
30.5-A6 cm.  The microbial concentrations at Umiat were much higher than
in the more northern locations.  There were 3 x 109 bacteria/g in the
surface soil and 2 x 1O8 bacteria/g in the subsurface soil  at 30.5 cm,
enumerated at 25 C, and 5 x 108 bacteria/g surface soil and k x 108
bacteria/g subsurface soi1 enumerated at 5 C.  The fungal  populations
were 2 x 106/g and k x 1 oVg for the surface and 5 x 103/g  and 6 x 102/g
for the subsurface (30.5 cm) at 25 and 5 C, respectively.


    The potential of the indigenous microbial populations  of Port Valdez
and Prudhoe Bay to degrade hydrocarbons was determined in  several  experi-
ments.  When Prudhoe crude oil or n-hexadecane was incubated in gas train
experiments at 25 C, the Prudhoe Bay water showed a higher  rate of miner-
al ization than the Valdez water with a greater C02 production from n-
hexadecane than from Prudhoe crude (Fig. 3).  The rates of  C02 evolution
were diminished when incubation was in biometer flasks where there was
no aeration or agitation, but the same relationship existed between
Prudhoe Bay vs. Port Valdez water and n-hexadecane vs. Prudhoe crude
(Figs, k and 5).  The rates of hydrocarbon mineralization  (C02 produc-
tion) were reduced at 5 C but no extensive lag periods were noted  before
the onset of mineralization,  The percent biodegradation after 31  days
of incubation was consistent with the relative amounts of  C02 evolved
(Table A).  The non-biological losses, determined with sterile controls,
were 1 and 5% at 5 C, and 11 and 23% at 25 C of the added n-hexadecane
and Prudhoe crude oil,  respectively.  There was also a qualitative dif-
ference between the biodegradation of Prudhoe crude by the  microorga-
nisms in Port Valdez and Prudhoe Bay.  The entire water column turned
brown with Prudhoe Bay water, indicating extensive emulsification  which
did not occur with the Valdez water.  The chemical  explanation for this
observation is not yet known.

                                Percent oil  biodegraded

Prudhoe crude
5 C
25 C
Prudhoe Bay
5 C 25 C
21 39
*»1 57

     FIGURE 3.   Mineralization of  petroleum hydrocarbons  by indige-
       nous microbial  populations  of  Alaskan coastal  waters at 25 C
       with aeration.   A = Port Valdez—Prudhoe crude.
       B = Port Valdez—n-hexadecane.   C  = Prudhoe Bay--Prudhoe
       crude.   D = Prudhoe Bay--n-hexadecane.
    The ability of the isolated  potential  seed  organisms to degrade hy-
drocarbons varied from none to extensive.   Apparently some organisms as-
sociated with crude oil  enrichment  develop via  a  commensal  relationship.
Two Pseudomonas species,  one isolated  from the  Umiat seep and the other
from Prudhoe Bay, showed  the greatest  oil  degradation potential.  Both
organisms were able to degrade about 0.5 g Prudhoe crude oil  at 5 C
within k weeks, and were  able to attack resistant components  such as
pristane as well as the more easily degraded  n-paraffins.  The fungi iso-
lated from the Cape Simpson seep showed only  limited crude oil  degrada-
tion, the range of hydrocarbons  subject to attack being  limited to n-
paraffins.  Even this latter activity  was negligible at  low temperatures.


                                      TIME  (Days)

FIGURE k.  Mineralization of Prudhoe crude without aeration,
  A = Port Valdez--5 C.   B = Port Valdez~25 C.
  C = Prudhoe Bay--5 C.   D = Prudhoe Bay--25 C.

    The percent oil  lost from miniature slicks floated  in  Prudhoe  Bay
 is shown in Fig. 5.   The float was located at Prudhoe Bay  site  2.
 Tables 2 and 3 show the physico-chemical  and microbiological  properties
of the water.  The greatest extent of biodegradation  occurred when  the
oleophilic N and P fertilizer was added.   The apparent  biodegradation
 losses may be somewhat inflated due to extensive emulsification which
was observed in the  in vitro studies.  The microbial  populations in the
water columns underlying the slicks are shown in Table  5-   There was a
great increase in the populations underlying the active slicks, with
 the largest increase under the fertilized slicks.   The  elevated micro-
bial  biomass reflects the increase of a non-pigmented,  gram negative,


short, rod-shaped bacteria and  a  gram  positive coccus.  The normal di-
verse population, including highly pigmented organisms, appeared  to  re-
main  in the same total numbers  as in the water column adjacent  to the
float.  The organisms showing the large population  increases^have been
identified as a Pseudomonas sp. and Staphylocoaeus  epidermidis.   When
tested for their ability  to degrade petroleum hydrocarbons,  only  the
Pseudomonas sp. gave  positive results.  This organism was able to exten-
sively degrade  Prudhoe crude at  both  5 and 25 C.  The Staphylocooous was
unable to attack hydrocarbons at either  5 or 25 C and failed to grow at
5  C.   It  is not yet  known whether  this  bacterium can grow at the^expense
of intermediary hydrocarbon metabolites  which would explain its  increase.
                                       TIME  (Days)
       FIGURE 5-  Mineralization of n-hexadecane without aeration.
         A = Port Valdez--5 C.   B = Port Valdez--25 C.
         C = Prudhoe Bay--5 C.   D = Prudhoe Bay—25 C.

Pert 11ized
   si ick
water column
                Number of viable cells  (in hundreds/ml)

    6/28          1,100          1,100          1,100

    7/8             690            760            500

    7/15            370          5,300             21

    7/22            kOO         20,000
          At 25 C.
                            20           30
                              TIME (Days)
FIGURE 6.  In situ losses of Prudhoe crude oil  in Prudhoe Bay.
  A = non-biological  (poisoned).    B = natural.
  C = stimulated (fertilized)


    Prudhoe crude, the type of crude oil found in the Prudhoe Bay re-
gion, is a relatively low aromatic, high paraffinic oil  with a moderate
sulfur content, about 0.9%, and a notable lack of light hydrocarbons,
Ca-Ce (9).  Since this is the type of crude which is likely to acciden-
tally enter the environment at the Trans-Alaskan pipeline terminals or
along the shipping routes from Prudhoe  Bay to the conterminous states,
the fate of this crude was chosen for study.  Similarly, Prudhoe Bay and
Port Valdez, the most likely  locations  for spillages, were  selected for
intensive study.  Two rivers, the Putuligayuk and  the Sagavanirktok,
flow  into Prudhoe Bay and conceivably could carry oil spilt  inland  into
the Bay.

    The surface temperatures  during  the summer  in  both  Prudhoe Bay and
Port Valdez were  sufficiently warm for  microbial oil degradation.  The
laboratory experiments showed slower rates at 5  C  compared  with 25 C,
nevertheless,  an active  microbial oil-degrading  population  was present
at the  lower temperature.   No extensive lag periods  were found before
the onset of Prudhoe crude  biodegradation, as had  been  observed earlier
for Sweden crude  (3),  indicating a  lack of a  volatile toxic  fraction in
the Prudhoe oil.  The missing light  fraction  in  Prudhoe crude possibly
explains this  observation and eliminates the  need  to consider removal
of this fraction by measures  such as  ignition in order  to  stimulate bio-

    Prudhoe Bay was highly  stratified,  indicating  little mixing even
through the shallow water column.  This lack of  mixing, coupled with
the relatively low nutrient concentrations, suggests that  fertilization
probably would be essential for extensive biodegradation.   In situ  oil
biodegradation experiments, however, did show high  losses  from the  un-
fertilized slicks.   It is not known  whether all  of  these  losses repre-
sent  emulsified but undegraded oil.  The latter  is a likely possibility
 in view of the extensive emulsification observed in  enclosed  laboratory
studies using  Prudhoe crude and Prudhoe Bay water.   Fertilization with
an oleophilic  nitrogen and  phosphorus source clearly enhanced biodegra-
dation  but the losses from  the fertilized slicks may also  be  inflated,
particularly  in light of the  lower degradation rates in laboratory  ex-
periments compared with  those in the field,

     In  spite of the harsh environmental  conditions,  the microbial popu-
 lations  in Prudhoe Bay were high, being surprisingly higher  than  in  Port
Valdez, and comparable to those found in temperate waters  such as Rari-
 tan  Bay, New Jersey  (7).  Thus, there was an adequate oi1-degrading mi-
 crobial population in these Alaskan  waters.  The size of this microbial
 population  increased when exposed to Prudhoe crude,  especially under nu-
 tritionally favorable conditions.   Introduction  of  these oi1-degrading
organisms  in high numbers,  by "seeding," may enhance oil removal  by
eliminating the lag period  necessary for the population to  naturally  in-
crease  to concentrations capable of  extensive oil biodegradation.   Such
 seeding with oi1-degrading microorganisms might  be especially useful  in


Port Valdez where the microorganisms are not adapted to Prudhoe crude
and were found in our studies to be less capable of degrading oil  pollu-
tants.  Such seeding would shorten the time course of natural degrada-
tion and would not be expected to alter the extent of oil degradation.
Re-inoculation with the same organisms that would naturally "bloom" fol-
lowing an oil spillage should not result in any additional environmental

    Several possible seed organisms were isolated from Prudhoe Bay and
natural Arctic oil seeps.  Natural oil seepages especially were examined
for oil utilizers.  Effective oil degraders also were isolated from
Prudhoe Bay.  The potential advantage of seeding with these organisms
must be demonstrated in future field experiments.  Also to be examined
is the fate of oil spilt during the winter, when Prudhoe Bay is frozen,
and whether oil biodegradation can be stimulated under the harsh condi-
tions of the Arctic winter.

    This work was supported by the Office of Naval  Research.  It was
conducted  in part while the authors were Resident Research Associates
at the Jet Propulsion Laboratory,  The work was aided by the skillful
assistance of F. A, Morel 1i.  Logistic support in the Arctic was gra-
ciously supplied by the Naval Arctic Research Lab and the U.S. Tundra
Biome Program.
                           LITERATURE CITED

1. Agosti, J., and T, Agosti.  1972.  The oxidation of certain Prudhoe
       Bay hydrocarbons by microorganisms indigenous to a natural  oil
       seep at Umiat, Alaska.  (Presented at 23rd Alaska Science Confer-
       ence.  Fairbanks.  August 17, 1972.)
2. Atlas, R. M., and R. Bartha.  1972.   Degradation and mineralization
       of petroleum  in sea water:  Limitation by nitrogen and phosphorus.
       Biotechnol. Bioeng. 14:309-318.
3. Atlas, R. M., and R. Bartha.  1972.   Biodegradation of petroleum in
       sea water at  low temperatures.  Can. J. Microbiol. 18(12):1851-
4. Atlas, R. M., and R. Bartha.  1972.   Degradation and mineralization
       of petroleum by two bacteria  isolated from coastal waters.   Bio-
       technol. Bioeng. 14:297-308.

5. Atlas, R. M., and R. Bartha.  1973-   Abundance, distribution and oil
       biodegradation potential of microorganisms in Raritan Bay.
       Environ. Poll. 4:291-300.
6. Atlas, R. M., and R. Bartha.  1973.   Stimulated biodegradation of
       oil slicks using oleophilic fertilizers.  Environ. Sci. Technol.


7. Atlas,  R. M., and  R.  Bartha.   1973.  Fate and effects of polluting
        petroleum  in  the  marine  environment.  Residue Rev. k3:^3~^-
8. Collins,  F.  R.   1958.  Test  wells,  Umiat area Alaska.  U.S.  Geol.
        Survey  Professional  Paper  305-B.   U.S.  Govt. Printing Office,
        Washington,  D.C.

 9. Rickwood, F. K.   1970.  The  Prudhoe Bay field.   In  Proc. Geological
        Seminar on the North Slope of Alaska.   Pacific  Section,  American
        Association of Petroleum Geologists.   Los Angeles,  Calif.

10.  Robertson,  B., S. Arhelger,  P. J. Kinney, and D. K. Button.   1973.
        Hydrocarbon biodegradation in Alaskan waters.   In D.  G,  Ahearn
        and S.  P.  Meyers  (ed.),  The Microbial Degradation of  Oil Pollu-
        tants.   Louisiana State Univ., Center for Wetland Resources,
        Pubn. LSU-SG-73-01.  p.  171-l8*».

11.  Robinson, F. M.   1964.  Core  tests Simpson area, Alaska.   U.S.  Geol.
        Survey  Professional Paper 305-L.  U.S. Govt. Printing  Office,
        Washington, D.C.

12.  Standard Methods for  the Examination of Water and Waste Water,   1971.
        APHA, AWWA, WPCF, Publications Office American Public  Health As-
        sociation, Washington, D.C.

13.  Traxler, R. W.  1973-  Bacterial degradation of petroleum  materials
        in low temperature marine environments.  In D. G. Ahearn and S. P.
        Meyers   (ed.), The Microbial Degradation of Oil Pollutants.   Lou-
        isiana  State Univ., Center for Wetland Resources, Pubn.  LSU-SG-
        73-01.   p.  163-170.
14.  U.S. Department of Commerce, NOAA, National Ocean  Survey.    1972.
        Port Fidalgo and  Valdez  Arm C and  GS 8519.   Washington,  D.C.

15.  U.S. Department of the Interior Geological  Survey.   1970.   Beechey
        Point B-3  SE Quadrangle  Alaska.  Fairbanks,  Alaska.

16.  ZoBell, C.  E.   1973-   Bacterial  degradation of mineral  oils  at  low
        temperatures.   In D.  G.  Ahearn and S.  P.  Meyers (ed.),  The  Micro-
        bial  Degradation  of Oil  Pollutants.   Louisiana  State Univ., Cen-
        ter for Wetland Resources, Pubn.  LSU-SG-73-01.   p.  153-161.


   Nancy  H.  Berner,  Donald  G. Ahearn,  and  Warren  L,  Cook**

    The use of hydrocarbonoclastic  microorganisms has been recommended
for facilitating the removal  of residual  hydrocarbons from shipboard
and harbor bunkers and from natural  sites  polluted with oils.  The de-
gree of microbial  emulsification and  utilization of oil varies markedly
with the type of oil and  fluctuating  environmental conditions.  Mixed
or pure cultures able to  utilize significant amounts of the asphaltene
fractions of oil have not been  obtained.   Moreover, microorganisms with
the capacity to emulsify  appreciable  amounts of oil have yet to be
proven of practical value under field conditions.  Only cursory atten-
tion has been given to the potential  toxicity of the organisms, includ-
ing the by-products of growth on oil  and  the possible pathogenic!ty of
the cultures for flora or fauna in  the environment.

    Two hydrocarbonoclastic yeasts  with emulsifying properties, Candida
lipolytiaa 37-1 and C. subtropioalis  Rk2,  were seeded into fresh water
estuarine environments inundated with a light, high-paraffinic Louisi-
ana crude oil.  Significantly higher  populations of predator species
(i.e., various protozoa and nematodes and,  in the estuarine site,  gas-
tropoda) were observed at yeast-seeded sites.  Candida lipolytiaa  was
not recovered from seeded sites after 3 to  5 months, whereas C. sub-
tropiealis persisted for  over a year  in fresh water systems  and over
seven months in estuarine systems.  At the  estuarine site, the seeded
yeasts eventually were replaced by  indigenous flora, some of which after
continued exposure to crude oil  apparently  acquired the ability to uti-
1 ize hydrocarbons.
     *This research was  supported  in  part by Office of Naval Research
Contract N0001 4-71 -C-OUfS.

    **Department  of Biology,  Georgia  State University, Atlanta,
Georgia 30303.



    The capacity to metabolize various hydrocarbons has been demonstra-
ted for a diversity of microorganisms.  One or more species of seventy
genera, including twenty-eight bacteria, thirty filamentous fungi, and
twelve yeasts, have been shown to oxidize one or more kinds of hydrocar-
bons (^7) •  Hydrocarbon-metabolizing microorganisms have been isolated
from air, soil, water, animal feces, food products, and other sources
(A, 15, 16, 18, 2k, AO) , but they have been found  in greater numbers in
environments associated with oils (7, 26, 27).
    The n-alkane fraction of petroleum has, in  laboratory experiments,
been biodegraded most completely.  Al kanes  in the  range of  kerosene,
Cio to Cie, are utilized more readily than  those  in the range of gaso-
line, C5  to C9  (20).  Crude oils or refinery distillates with a high
asphaltic content are less biodegradable than those with an abundance
of aliphatic hydrocarbons, and generally are assimilated by a limited
number of microorganisms  (7).
    Numerous organic compounds formed as a  result  of direct or indirect
biological action  (classified as alkanes, cycloalkanes or naphthenes,
aromatics, asphalts, and combinations of these) are components of crude
oils.  Asphaltic compounds in petroleum contain oxygen  (0), sulfur  (S),
and nitrogen  (N) , and hence are not true hydrocarbons.  The principal
complexes of these compounds are asphaltenes and  resins, both of which
contain a  large proportion of aromatics.  For example, Pennsylvania
crude oil contains nearly 100% hydrocarbons (i.e., 85% carbon and 13-5%
hydrogen) with  the bulk as n-alkanes, whereas midcontinent  and gulf
oils range from 90 to 35% hydrocarbons and  California and Mexico oils
 (including Tia  Juana crude) are composed of approximately 50% hydrocar-
bons and  50% asphaltics including heavy distillate fractions.  South
Louisiana crude oil contains approximately  30% gasoline, 10% kerosene,
15% light distillate oil, 25% heavy distillate oil, and 20% asphaltic
residuum.  This crude oil is of low viscosity and contains  natural  sur-
factants  which  promote  spreading over water.  In contrast,  Venezuela
crude oil, a high viscosity, low surfactant oil, consists of approxi-
mately  10% gasoline, 5% kerosene, 20% light and 30% heavy distillate
oils, and 35% asphaltic residuum.  Mississippi crude oil has over 50%
asphaltic  residuum, and bunker C or No. 6 fuel oils, which  constitute
a  major  portion of transported refinery products, are the heaviest  dis-
tillate  fractions of petroleum (20, 25, 30,
     When  oil  spills occur,  the  paramount consideration  is  the  rapid re-
 moval  of  the  oil.  Since most mishaps have occurred on  large bodies of
 water,  relatively  little consideration has been given to terrestrial
 spills, yet numerous  petroleum  transport systems do involve terrestrial
 transfer.  The  trans-Alaskan pipeline will traverse more than  800 miles
 of  ecologically  sensitive terrain.   In such an environment, physical
 and  mechanical clean-up procedures following an oil spill  could cause
 more damage than the  oil itself  (25).  Disasters with far-reaching and
 deleterious environmental effects, such as the tanker Torrey Canyon,


Santa Barbara channel, and Gulf of Mexico spills are most likely to oc-
cur in coastal and estuarine areas (12).  The coastal marshes of south
Louisiana are adjacent to offshore oil fields; the effects of a major
oil spill disaster on such a broad, shallow, salt-marsh estuary are
largely speculative since studied oil spills have occurred mostly in
coastal areas characterized by cliffs and pocket beaches  (37).  The
need to ship large volumes of hydrocarbon products by sea from the mid-
east and South America to the industrial centers of western Europe, Ja-
pan, and the United States and the development of offshore oil fields
will perpetuate the possibility of oil-spill disasters for as long as
the world's oil reserves last.

    The removal of oil from/a body of water involves the collection and
physical extraction of gross quantities of the spilled oil (\k, 31, 39)-
However, a substantial amount of oil  remains after mechanical  or chemi-
cal techniques are used.  Chemical dispersants in extremely large amounts
have been employed at most major oil-spill sites.  The toxicity of these
chemical dispersants has been a major concern, as has the toxicity of
the dispersed oil  itself (17, 22, 41).
    The ultimate clean-up of an oil spill requires biological  oxidative
activity (k, 7, 10, 21).  When oil is left on the sea or  in marshes,
harbors, rivers, or lakes, stimulation of the rate of normal  biodegrada-
tion may reduce damage to the environment.  Microbial seeding, enriching
oil spills with nutrients, or both, may be used since unaided  biodegra-
dation does not provide effective relief against massive disaster (k, 7,
10, 28).  No single microorganism  or combination of microorganisms has
completely degraded any crude oil.  Microorganisms with enzymatic prop-
erties for rapid attack on a broad spectrum of hydrocarbons may be un-
common .
    Most research on the microbial degradation of oily pollutants has
centered on bacteria or mixtures of unidentified microorganisms (47).
Fungi, due to their greater osmotic flexibility and enzymatic  capaci-
ties, may prove more practical.  Ahearn et al. (7) reported that yeasts
in oil-bearing regions frequently utilized hydrocarbons as substrates
for growth, suggesting in-situ oil biodegradation.  Oil  enrichment of
estuarine and marine environments was shown to initiate increased densi-
ties of certain yeasts (4).  Further, yeasts from aquatic sites chroni-
cally polluted with oil demonstrated  higher rates of degradation and
emu Is ification of crude oil than did  some common marine-occurring yeasts
(such as isolates of KhodotoTula, Debaryomyoes, and members of the Can-
dida parapsilosis complex)  from apparently oil-free habitats.

    Although numerous yeasts demonstrably utilize hydrocarbons, at
least to some degree, strains of Candida tropiealis and C. lipolytiaa
are particularly active (20,  29, 33,  38).  In a series of reports (k,
7, 3*0, isolates of C.  tropicalis and C.  lipolytiaa were found  to grow
readily on Louisiana crude oil and a  variety of its refinery products.
Seeding of yeasts to facilitate biodegradation was suggested by Ahearn
et al.  (7).  Definitive studies of the rates of oil degradation by
yeasts  or of the effects of microbial seeding on the ecology of an oil-


spill  site have not been undertaken.
    This study examines the ability of selected fungi, particularly
strains generally identified with the genus Candida, to grow and survive
in oil-enriched fresh and marine waters was investigated.
                         MATERIALS AND METHODS
    Fungi were obtained from the culture collection of the Department of
Biology, Georgia State University, from the Department of Food Science,
Louisiana State University, Baton Rouge, Louisiana, and by direct isola-
tion from sites polluted with crude oil or hydrocarbon distillates.  The
cultures included  isolates from oil-polluted marine and fresh waters and
contaminated aircraft fuel systems (Table 1).
                      TABLE  1.  Sources of Fungi
Organ! sm
Candida lipolytica 37"!
Candida subtropiaalis AJ4476
Candida subtropiealis R42
Candida tropioalis NB2
Candida tropicalis W12B
Candida tropicalis 231
Candida tvopicalis 6418
Cladosporium resinae SA300
Cladosporiim resinae SA8
TvichospoTon fermentans SA1 00
Candida parapsilosis GM181
Triahosporon cutaneum GM180
Rhodosporidiwi toruloides GM183
Itersonilia sp. JK29
Pichia ofaneri LSU215
Pichia spartinae FST119
Pichia saitoi MENA
Kluyveromyces drosophilarum FST125
Kluyveromyees dobzhanskii N-l
Pichia spartinae N-l 8
Frankfurter (6)
Air (38)
Asphalt refinery (A3)
Asphalt refinery (43)
Asphalt refinery (A3)
Jet fuel
Jet fuel
Jet fuel
Oil slick, Gulf of Mexico (35)
Oil slick, Gulf of Mexico (35)
Oil slick, Gulf of Mexico (35)
Eye cosmetic (46)
Barataria Bay, marsh (35)
Barataria Bay, marsh (35)
Barataria Bay, marsh (35)
Barataria Bay, marsh (35)
Barataria Bay, oiled plots
Barataria Bay, oiled plots
 Direct  isolations were made by streaking oily materials onto selected
 media.   These  included yeast nitrogen base  (YNB, Difco) agar with 0.5
 g/1  chloramphenicol; Mycosel (BBL) and Mycobiotic  (Difco) agars which
 contain  0.4 g/1 cycloheximide and 0.05 g/1 chloramphenicol; casein agar


(6) prepared with and without 0.5 9/1 chloramphenicol and 0.4 g/1 cyclo-
heximide; Mycological (Difco) agar acidified to pH 4.5 with lactic acid;
and diamalt agar (5).  Media were prepared in both distilled water and


    Oil assimilation capacity was assessed by visual determination of
fungal growth in 4.8 ml  YNB medium (45) supplemented either with ap-
proximately 0.2 ml of Louisiana crude oil  (a slight residue remained on
the inside surface of the pipettes) or with the weight of the more vis-
cous oils  (Tia Juana, Venezuela, or Mississippi) equal to the weight of
Louisiana crude used.  Cells for inoculum were grown  in YNB broth with
0.01% glucose for 24 to 48 hr to accomplish carbohydrate depletion.  Of
this growth, 0.05 ml was used to inoculate each hydrocarbon utilization
test.  All cultures were incubated at 22 to 26 C on a roller drum set
at an angle of 80° and a speed of 40 to 50 rpm.  The hydrocarbons were
not sterilized; uninoculated controls were incubated to assess any pos-
sible contamination.  Visual determination of growth was recorded using
a 0-3 scale; zero indicated the absence of growth and three indicated
maximal growth as compared to a glucose control.  The cultures were ex-
amined microscopically at various stages during the incubation period.


    Oxygen consumption was determined by a modification of the tech-
nique of Tool (42) employing a Hach manometric biochemical oxygen de-
mand (BOD) apparatus, Model 2173.  Inocula for the respiration studies
contained 4 x 107 to 6 x 107 cells from 24-hr YNB broth cultures with
0.5? glucose.  Respiration bottles contained approximately ].0% hydro-
carbon (v/v) and 0.01% yeast extract (Difco) in distilled water or sea-
water.  Net oxygen consumption was determined after 72 hr at 6, 20, and
30 C.  Although usual BOD procedures require no mixing of bottle con-
tents during the incubation period (8), the procedure used required
constant agitation with stirring initiated before the addition of hydro-
carbon.  Upon completion of the experiment, oily samples from the res-
piration bottles were streaked on isolation agars to check for culture
purity.  Control respiration bottles contained either hydrocarbon and
yeast extract, inoculum and yeast extract, hydrocarbon only, inoculum
only, or yeast extract only.  Net oxygen consumption  (mg/1) with 1.01
(v/v) crude oil  as substrate was determined by subtracting both endoge-
nous respiration and oxygen consumed in auto-oxidation.


    Tests were conducted to compare the emulsification of Mississippi
and Louisiana crude oils by the yeasts C.  subtropiaalis R42 and C.
lipolytica 37-1-  Live cells, dead cells (autoclaved), and lyophilized
cells in concentrations  of 1 x 1010, 1  x 1012, 1 x 10 13, 1 x 101"*, and


1  x 1015 were added to test tubes  (18 mm) containing 9,9 ml of sterile
tap water and 0.1 ml of crude oil.  The various yeast-oil mixtures and
control tubes were agitated at kk  rpm on a roller drum at 25 C for one
month.  Visual observations were made weekly to record the degree of
oil emulsification.  All tests were run  in triplicate.  Samples were
taken from  tubes which displayed the best emulsification of oil after a
period of one month.  The diameters of 25 oil droplets from each tube
were measured microscopically.  Additional observations were made on
aquaria separately enriched with the two crude oils, with and without
the same two yeasts.  Adhesiveness of the oil to  the glass walls and to
wooden paddles was noted as was the visible emulsification of the oil
in the various aquaria.  The degrees of  emulsification and adhesiveness
were compared with results from aquaria  containing a commercial disper-
sant, Polycomplex A-ll  (Guardian Chemical Corp.,  Hauppauge, New York).


    Selected fungi were examined for their ability to survive and multi-
ply in  simulated oil spills.  Water from an asphalt refinery holding
pond  (heavily oil polluted; for description of this site see Turner and
Ahearn  [43]) was used as diluent for approximately k%  (v/v) Louisiana
crude oil or bunker C fuel oil.  Buckets, containing a total volume of
five  liters, were employed as testing containers.  Each was equipped
with a  cover to  prevent addition of water by precipitation.  One bucket
in each test series was enriched with 25 g of (NHit^SOi*.  The test
buckets were placed in the open in the vicinity of Atlanta, Georgia, be-
tween December 1970 and May 1971.  A mixture of nine fungi was used as
inocula for the  containers.  The cell crop for each organism, after 48
hr growth  in five ml of YNB broth with Q.5% glucose, was obtained; the
inoculation mixture was prepared by pooling the resultant broth cultures.
Samples from test and control buckets were taken with sterile dacron
swabs at 3  to 5  week  intervals over a period of five months.  Isolation
agars were  surface streaked with a swab and incubated at room tempera-
ture.   Developing fungi were isolated and identified as previously de-
scribed  (4).

     In  a separate series of field  tests, water from a non-oil polluted
lake was enriched with approximately 4%  (v/v) Louisiana crude oil; all
tests employed a final volume of five liters.  The  inoculum consisted of
a mixture of six yeasts and one filamentous fungus  (1.0 ml each from 2k
hr cultures grown on YNB broth with Q.5% glucose).  Samples from test
and control buckets were taken over a period of twelve months (August
1971 to August 1972) and the fungi were  isolated and  identified.


    Water,  sediment, and vegetation (Spartina alterniflora) samples were
taken from  the northwestern region of Barataria Bay, Louisiana, at in-
tervals between  August 1971 and July 1973, and examined for their fungal


flora.  Detailed descriptions of the collection area and the isolation
and identification procedures have been published (35, 36).  Mainte-
nance of the seed plots in the estuary was facilitated by the use of
50-gal (189-liter) drums with both ends removed and with lateral holes
below the water line.  These barrels were positioned in the estuarine
waters and inoculated with a mixture of either growing cells or lyo-
philized cells of six fungi, including five yeast and one filamentous
isolate.  Louisiana crude oil was added to all barrels at 3 to 5 week
intervals over an eight-month test period.  The mixed inoculum of grow-
ing cells consisted of a total  of 30 ml from 24-hr broth cultures (2.0%
glucose, 1.0% peptone, and 0.$% yeast extract broth prepared with sea-
water).  The cell crops obtained from 48-hr cultures of each of the
same six fungi  (grown on medium of the above formulation with 2.0%
agar) were lyophilized and used as inocula for additional field tests.
    In subsequent seed culture tests in the marsh, only Candida lipo-
lytioa 37-1 and C. subtropicalis R42 were used for inoculation of sedi-
ment plots.  One hundred ml of Louisiana crude oil was applied to the
plots prior to  inoculation and then periodically over an eleven-month
test period.  Duplicate samples from the estuarine barrels were fil-
tered through cellulose-ester membranes (Millipore Filter Corp.) of
0.45  ym porosity, and the membranes were implanted aseptically onto
distilled and seawater isolation media.  Sediment and vegetation sam-
ples from the oiled plots were taken with sterile utensils and plated
directly onto isolation media.   Occasional samples were kept on ice for
5 to 10 hr prior to culturing.   Part of each sediment sample was di-
luted with sterile seawater, vigorously agitated, and the supernatant
was filtered through cellulose-ester membranes; the membranes were then
aseptically implanted onto isolation media.  Control  samples were taken
from the natural bay waters, sediments, flora, and from oil-seeded,  un-
inoculated barrels and marshland plots.  Representative yeasts and fila-
mentous fungi  were selected from the isolation plates on the basis of
colony morphology.  Identification procedures for yeasts followed the
methods of Wickerham (45) and Lodder (32).
    Hydrocarbonoclastic fungi grew readily when oily residues from the
environment were streaked on YNB agar without carbon enrichment.   Yeast
and fungal colonies were selected from the isolation plates,  usually
after incubation for 72 or more hr.  Isolation of yeasts was  facili-
tated when the antibiotics chloramphenicol and cycloheximide  were
added to the medium.

    The latter compound, a di-ketone, is inhibitory to numerous sapro-
phytic fungi, but two of the most active hydrocarbonoclastic  yeasts
(Candida lipolytiaa 37~1 and C.  subtropiaalis R42 and AJ4476) were re-
sistant to 0.4 g/1  cycloheximide.  Most strains of C. tropiaalis


(including clinical isolates) which hydrolyzed and utilized  starch and
gave fair to good growth on crude oil, were also resistant to  cyclohexi-
mide.  Other starch-utilizing strains of C. tropicalis  (e.g.,  6418,
Table 1) were susceptible to Q.k g/1 cycloheximide and  gave  latent
growth on crude oil.  Certain yeasts (when  isolated from oil-soaked
plots) proved resistant to cycloheximide, yet representative clinical
isolates of the same species were susceptible to this antibiotic  (e.g.,
species of Cvyptooooous [1]).  Candida lipolytioa 37~1  produced signifi-
cant amounts of extracellular proteinase as indicated by the development
of a clear zone in casein agar (6).  This property of C. lipolytioa,
along with the resistance of both C. lipolytioa and C.  subtropioalis to
cycloheximide and their inability to hydrolyze starch,  permitted  a ready
distinction of these yeasts.


     Most yeast isolates grew within three days with Louisiana  crude oil
as the  sole source of carbon (Table 2).  The heaviest growth on Louisi-
ana  crude oil after three days incubation was by strains of  C. lipolytioa,
C. subtropicalis, C. tropiaalis, and Triohosporon feymentans.   In con-
trast to most yeasts tested, both C. subtropioalis R*»2  and Mkkj6 and C.
lipolytioa 37~1 produced weak growth on Tia Juana crude oil  by ten days.
Generally, growth of yeasts with either Tia Juana, Venezuela, or Missis-
sippi crude oil as substrate was minimal and evident only after twenty
days  incubation.  Candida subtropioalis Rk2 and C.  lipolytioa  37~1 , in
particular, emulsified the crude oils; both the cell-free, spent-culture
broths  and whole cells showed emulsifying properties.

     The mean diameters of oil droplets of both Louisiana and Mississippi
crude oils generally decreased with an increased concentration of yeast
cells (Table 3)-   In general, live yeast cells enhanced the  formation
of smaller oil droplets than did either dead or lyophilized  cells.  The
adhesion of crude oil  to glass test tubes or to wood paddles decreased
immediately upon addition of live, dead, or lyophilized yeast  cells.
This dispersive effect was enhanced by increased concentrations of cells.
Emulsification of the crude oil  in test tubes inoculated with  live
yeasts, as determined by weekly visual observations, increased with time
and  yeast growth over a one-month period.  This same effect  was noted in
the  aquaria which were enriched with either of the two  crude oils and
live yeasts.  Cells of C.  lipolytioa 37-1 were more effective  dispersants
of Louisiana crude oil, whereas cells of C. subtTopioalis R^2  gave vi-
sual evidence of greater emulsification of the more viscous  Mississippi
crude oi1.  In general, Mississippi crude was not as d ispersed as the
Louisiana crude, although the average size of measurable oil droplets
of Mississippi crude oil was smaller than those of Louisiana crude.
Many more large, hardened globules above one millimeter in diameter were
present in the tests involving Mississippi crude than in those with
Louisiana crude.

     TABLE 2.  Relative Growths of Fungi  with Louisiana Crude Oil
                          as a Carbon Source
UP ya ii i sin
Candida lipolytica 37~1
Candida subtropiaalis Mkkj6
Candida subtropioalis Rk2
Candida tropioalis NB2
Candida tropiaalis W12B
Candida tropiaalis 231
Candida tropioalis 6418
Cladosporium resinae SA300
Cladosporium resinae SA8
Triahosporon fermentans SA100
Candida parapsilosis GM181
Triahosporon autaneum GM180
Rhodosporidium toruloides GM183
Itersonilia sp. JK29
Piahia ohmeri LSU215
Piahia spartinae FST119
Piahia saitoi MENA
Kluyveromyces drosophilarum FST1 25
Kluyveromyaes dobzhanskii N- 1
Piahia spartinae N-18
3 days
10 days
          aGrowth on YNB medium with k% (v/v)  Louisiana  crude oil.
     Visual determination of growth:  0 negligible,  3  maximal.
          bGrowth after 20 days incubation;  cells aberrant.

    The mixture of either crude oil  with either yeast  was  more effec-
tive in reducing the adhesiveness of the oil  to glass  than was the  mix-
ture of Polycomplex A-ll dispersant  with either oil.   This effect was
noted in both test tubes and aquaria.  Microscopic examination of agi-
tated cultures showed that these yeasts coated the subsurface oil
globules with budding cells, whereas in surface slicks in  non-agitated
systems, mats of hyphae were formed  within and upon  the  oil  layers.
Similar growth results were obtained from media prepared with either
fresh water or seawater.  Rhodosporidium toruloides  GM183  and numerous
other red yeasts (species of Rhodosporidium and Rhodotorula)  isolated
from oil-polluted areas utilized components  of crude oil for  growth and
energy, but never with amounts of growth or  emulsification comparable
to those of the species of Candida.   Pichia  spartinae  FST119, P.  saitoi
MENA, and Kluyveromyaes drosophilarwn FST125 from relatively  oil-free
estuarine marsh sites failed to grow with oil  as a sole  carbon source
even after ten days incubation.

            TABLE 3.   Average Diameter of  Crude  Oil  Droplets
                          Emulsified by Yeastsa
Louisiana Crude
Cel 1 type
Lyophi 1 ized
Control (no yeast)
C. tropiaalis
Diam'5 Range0
C. lipolytiaa
Diam Range


Mississippi Crude
C. trop-iaalis
Diam Range
C. Hpolytiea
Diam Range

1 5-230

      Measurements in micrometers by ocular micrometer; sample from suspended material
  after agitation for 25 days.
      Mean diameter, 25 globules; globules larger than 1  um not considered.
      cSize range of globules; globules larger than 1 ym not considered.  Adapted from
  Cook et al. (19).

    A  mixture  of eight fungi  (C.  tropicalis NB2 and  W12B,  C.  lipolytioa
 37-1,  C.  paraps-ilosis GM181,  Triohosporon fermentans SA100, Cladosporim
 resinae SA300, Tr-iohosporon cutaneum GM180, Rhodosporidium toruloides
 GM183, and Itersonilia sp.  JK29)  was introduced  into fresh water ob-
 tained from  an asphalt refinery drainage system.   The water was enriched
 with Louisiana crude or bunker C  oil.  The most  noticeable effects of
 the fungi on the oiled water  were partial disruption of  the surface
 slick, development of a more  evident oil-in-water  emulsion, and the for-
 mation of matted, irregular hyphal layers at the  oil-water interphase.
 Such phenomena were much less evident in the control  buckets.   Periodic
 samplings of the test systems followed by isolation  and  identification
 of yeasts over a five-month period demonstrated a  gradual  decrease in
 certain populations, the disappearance of a few species, and  the estab-
 lishment  of  wild fungi in both test and control systems  (Table 4).  Ap-
 proximately  the  same numbers  of fungi were obtained  from test  buckets
 with and  without enrichment with  25 g of (NHiJaSOi,.   Less  microbial  ac-
 tivity was evident in the bunker  C fuel oil  systems  than  in the Louisi-
 ana crude.   Species of Cladosporium which gave negligible  evidence of
 growth on crude  oil  in pure culture systems were  the predominant iso-
 lates from the bunker C field tests (Table k).  Bacteria were  present
 in all  test  systems,  particularly in those enriched  with bunker C fuel
oil.  Neither oil  was completely  degraded during  the five-month test
 per iod.

    A second series of seed-culture tests involved fresh water from a
non-oil polluted  lake; the  water  was enriched with k%  (v/v)  Louisiana
crude oil.   Containers were inoculated with a mixture of seven fungi


     TABLE A.  Predominant  Fungi  Recovered  from Asphalt Refinery Lake
               Waters Enriched with  ^%  (v/v)  Hydrocarbons
Louisiana crude oil

CQ,Ylu,1rQ£L ifX?OTpTfGCilr1rS
Candida lipolytioa^
Candida parapsilosis^
Triahosporon fermentans^
Red yeast
Filamentous fungi :
Cladospofiim sp.'3
Cephalospoviwn sp.
Fusariian sp.
Penioillium sp.
Alternaria sp .
Aspergillue sp.
Triohoderma vvnde
1 mo.


3 mo. 5 mo.

30 40
25 20
/ 10
20 10
20 13

/ 7
5 5
/ /
/ /
/ /
/ /
' '
1 mo.



3 mo.


5 mo.



Bunker C fuel oil3
1 mo. 3 mo.

10 5
10 5
75 15
5 5

/ 50
/ 5
/ 5
/ 5
/ /
/ /
/ 5
1 mo.


3 mo.


   aBacterial growth predominant  in all  bunker C buckets.
    Organisms seeded into inoculated buckets.
   °Percent of total fungal  isolates.
   d/ indicates not isolated.
 (Cladosporium resinae SA300,  Candida parapsilosis GM181, Pichia olmevi
 LSU215, C.  tropicalis NB2, C.  subtropiealis R^2, Triahosporon fermentans
 SA100, and  C. lipolytica 37-1).   Samples from both test and control sys-
 tems were taken  periodically  for twelve months.  Within three months,
 the  integrity of  the  initial  oil layer (approximately one centimeter) in
 each inoculated  bucket  was disrupted, and there was noticeable evapora-
 tion of water.   At  one  year,  a film of crude oil was no longer present
 in the inoculated buckets; instead a thick, spongy pellicle covered the
 water surface; the  pellicle had  a black, hardened, asphalt-like upper
 surface and a brown,  spongy underside with much stringy  mycelial growth.
 Thick hyphal strands  extended down into the turbid water;  many strands
 sank to the bottom  of the containers.  Only about 500 ml of water re-
 mained.  The oil  film in the  control  container was reasonably unaltered,
 and the water volume  remaining was approximately four liters at the end
 of one year.  The uninoculated control  (water from the non-oil polluted
 lake) showed little evidence  of  microbial  activity.  Of the seven spe-
 cies of fungi introduced into the test systems, four species  (Tricho-
spopon fermentans,  Cladospovium  resinae, Candida parapsilosis, and
Piahia ohmeri) were never recovered.   Candida lipolytioa was recovered
at the end of the first three weeks but not thereafter.  Candida sub-
 tropicalis was obtained on each  sampling throughout the year.  During
this time both control  and inoculated buckets were populated with nu-
merous fungi, mainly  species  of  Alternaria, Cephalosporiwn, Fusariian,
and Penicillium.


    Prior  to the  introduction of  the  seed  cultures  into estuarine waters
species of Piahia, Kluyvevomyces, Rhodotorula,  and  Cryptocooous were
found  to predominate at  the  test  site.   No isolates of  these species
produced strong growth on  hydrocarbons  before  exposure  of the marsh site
to  oil.  A mixture of six  hydrocarbonoclastic  fungi  (Triahosporon fer-
mentans SA100, Cladosporiwn  resinae SA300, Candida  tropiaalis NB2, C.
subtropicalis Rk2, C. lipolytica  37~1,  and C. parapsilosis GM181) was
 introduced  into estuarine  waters  in Barataria  Bay,  Louisiana.  Colony-
forming units  (cfu) of C.  subtropiealis  and  C,  lipolytica constituted
approximately 35% of all microorganisms  recovered from  water samples
taken  2k hr after  inoculation.  A few red  yeasts were also evident.  No
Trichosporon or Cladospovium species  were  recovered.

    After  the first sampling, high tides in  the fall of the year com-
 pletely  inundated  the test barrels; both the seeded  crude oil and the
 top stratum of water were  washed  away.   Samples for  fungal identifica-
tion were  taken and Louisiana crude oil  again was applied to the plots,
but the plots were not reinoculated with yeasts.  Analysis of the sam-
ples  indicated that the  indigenous fungi  had returned and the seeded
Candida species were lost.   Of  the fungi  isolated,  approximately kO% of
 the cfu were species of  Piahia, 20% Kluyveromyces,  20%  Cryptoaoaaus, ]Q%
red yeasts, and 10% filamentous fungi.   Cladosporiwn species were rarely
 isolated;  those isolates examined were morphologically  distinct from the
"kerosene  fungus" Cladosporiwn vesinae  (40).  Almost eight months after
the original inoculation,  high  spring tides  washed  some of the barrels
out to sea.  Essentially the same isolations of fungi as those reported
earlier were made.  Additions of oil  during  the interim period had not
encouraged evident growth  of organisms other than the  indigenous fungi.


    The yeasts Candida lipolytiaa 37~1 and C. subtropiaalis R^2 were
introduced  into marshland  sediments;  the test  plots  were enriched with
Louisiana  crude oil both prior  to inoculation of the yeasts and periodi-
cally  thereafter.

    Samples from the plots were taken before inoculation and after two
months, three months, seven  months, and  eleven  months  (Table 5).   Al-
though the yeasts C.  subtropiaalis Rk2 and C.  lipolytiaa 37~1 were not
found  in Barataria Bay prior to their inoculation as seed cultures,  they
persisted for over seven months  in oiled plots  in the marsh.  Candida
subtropiaalis was recovered  in greater densities from all  samples of the
oil-soaked plots.   Neither yeast was  isolated from  adjacent non-oiled
sites  throughout the entire  study.  The  seed yeasts  survived the seasons
from late fall  to late spring or  early summer.  Representatives of
Pichia spartinae with the  ability to  utilize and emulsify  crude oil


            TABLE 5.   Fungi  Isolated from Oiled  Sediments,  Barataria  Bay,  Louisiana
Prior to Time after inoculation of seed cultures
inoculation 2 months 3 months 7 months
Sites 1 E2 Sitel Site 2 Control3 Site 1 Site 2 Control Sitel Site 2 Control
Seed Cultures:
Candida subtropiaalis RA2 Ob + + Oc + + 0 + + 0
C. lipolytiaa 37-1 0 + + 0 + + 0 + + 0
Indigenous Fungi :
Yeast- like:
Rhodotorula-Rhodospoi>idium sp. + + + + + + + + + +
Cryptoaocaus sp. + + + ++ + ++ + +
(C. albidus and lawcentii )
Kluyveromyces sp. + + + + + + + + + +
(K. drosophilaman and dobzhanskii)
Piahia sp. + +4++ + ++ + +
(P. spartinae, saitoi, and ofmeri)
Trichosporon sp. + +0+ +0+ +++
(mostly T. cutaneum)
Aureobasidium sp. + 00+ 00+ 00 +
Fi lamentous:
Cladosporium sp. + 00+ +++ 00+
Penicilliwn sp. + 0 + ++ + +00 +
Cephalosporium sp. + + + + + + + + + +
Fusarium sp. + + + ++ + ++ + +
Alternaria sp. ++0000+00 +
Trichoderma viride 0 0 + + 000 00 +
Aspergillus sp. 0 000000000
1 1 months
Sitel Site 2 Control
C + +
00 +
+ 0 +
0 + +
00 +
+ + 0

 Control plots, periodically oiled, no  inoculum.        0 indicates not isolated, + present.

°Seed cultures introduced in non-oiled  sediments were not recovered after 72 hr.

were isolated after the plots were subjected to oil for at least several
months.  Microscopic examination of the growth on oil revealed that the
yeast produced short chains of aberrant, pseudo-hyphal cells  inter-
spersed with numerous chlamydospores.  Of the major species  indigenous
to the area, no other hydrocarbonoclastic isolates were found.  Filamen-
tous fungi with weak hydrocarbonoclastic properties,  including species
of Trichosporon (mainly T. outanewn), Aureobasidiwn, Penicillium, and
Cephalosporium were obtained throughout the study.


    Oxygen consumption by Candida lipolytioa 37~1 at 20 C with various
crude oils as substrate was compared  in both distilled water and fil-
tered  seawater  (Table 6).  Significant oxygen consumption within a 72-hr
period was obtained only with Louisiana or Tia Juana crude oil as sub-
strate.   No essential difference in results obtained with distilled
water or  seawater was noted.
        TABLE 6.  Oxygen Consumption by Candida lipolytiaa 37-1
          after 72 Hours at 20 C with Crude Oil as Substrate3
Crude oi 1
Lou is iana
Tia Juana
M i ss iss i ppi
Distil led water
Filtered seawater
             a!.0% crude oil with 0.01% yeast extract added.
             bMg oxygen/1, average of 3 repeat tests.

     The oxygen consumption at 72 hr with 1.0% Louisiana crude oil as
 substrate for C. lipolytica 37-1 and C. subtropioalis RA2 was deter-
 mined at 6, 20, and 30 C  (Table 7).  Both yeasts had scant oxygen up-
 take at 6 and highest activity at 20 C.  The maximum growth  temperature
 of  C. subtropiaalis R42 on Mycological agar was about 42 C;  this yeast
 did  not grow at 45 C.  Candida lipolytioa 37~1 , unlike most  isolates of
 this species (32) grew readily at 30 C and produced only slight growth
 at  37 C.

        TABLE 7.  Oxygen Consumption by Candida lipolytiea 37 "1
             and Candida subtvopiaalis Rk2 after 72 Hours
                 with Louisiana Crude Oil as Substrate3


Un inoculated
(auto-oxidat ion)
    a1.0% crude oil with 0.01% yeast extract added, distilled water.
    °ln 0.01% yeast extract, distilled water, no crude oil.
    cMg oxygen/1, average of 3 repeat tests; ±25 mg Oa/1  at  20 and 30 C,
±5 at 6 C.

    No adverse ecological conditions were observed which could be at-
tributed to addition of yeasts to water containers or sediment plots.
In microscopic examinations of fresh water material, a variety of pro-
tozoa was observed.  These included members of the genera Phaaus, Sty-
lonyahia, Parameoiwn, and Amoeba, some of which were seen feeding on
yeast eel Is.
    In marine plots, the gastropod Nerita, a grazing herbivore, appar-
ently infiltrated oil sites.   In the sediments, some nematodes and pro-
tozoa were observed.  These populations in the oiled plots, seeded with
yeasts, appeared to be equivalent to or higher than those in control

    The indigenous yeast flora of pristine fresh water and estuarine
environments demonstrated limited capacities to utilize crude oils as
contrasted with yeasts from sites enriched with petroleum.  Only after
about a year of exposure to oil did a few representatives of the indi-
genous flora show hydrocarbonoclastic activity.  Hydrocarbonoclastic
fungi introduced into oi1-soaked habitats did not all survive.  Of the
fungi used, isolates of C.  subtropiaalis and C. lipolytiea persisted in
both fresh water and estuarine environments enriched with oil for vary-
ing periods of time without apparent adverse effects on the ecology of
the study sites.  Moreover, these yeasts seemed to be localized at the
oiled plots.  Candida subtropiaalis persisted the longest at both fresh
water and estuarine sites.


    Ahearn (3) reported that C. tropicalis was found generally in pol-
luted waters which had a BOD in excess of 2 mg/1.   This report made no
reference to C.  subtvopiaalis.   Nakase et al. (38) differentiated C.
tropiealis from C. subtropicalis using properties such as starch utili-
zation, hydrocarbon assimilation, maximum growth temperature, DNA base
composition, and antigenic distinctions.  Culture R42 and the type
strain of C. subtropioalis (AJA476) both were unable to utilize starch,
did not grow well at temperatures over 42 C, and gave rapid growth with
hydrocarbons as sole carbon sources.  In addition, both cultures were
resistant to 0.4 g/1 cycloheximide.  The natural surfactant of C.  sub-
tropicalis R42 more effectively dispersed Mississippi crude oil  (which
contains over 50% asphaltics) than did the yeast C.  lipolytica.   Guire
et al. (23) identified the peptidolipid surfactant of C. petrophilum
ATCC20226  (synonym C.  lipolytiaa) and reported it relatively nontoxic
for the water flea Daphnia magna, stable to high temperatures, func-
tional over a wide pH range,  and susceptible to hydrolytic enzymes.
Analysis of the surfactant from C. subtropicalis has not been reported.

    The most obvious emulsification effect noted was the decrease in
adhesiveness of both crude oils to glass walls (test tube and aquaria)
and wood paddles when cells of  either yeast were added.  Lyophilized
and live yeast cells had similar emulsification effects on both Louisi-
ana and Mississippi crude oils.  The differences in degrees of emulsi-
fication noted in comparison tests of the two oils could be attributed
in part to their content of short chain hydrocarbons and their viscosi-
ties.  Generally, the greater the number of yeast cells present, the
smaller the average size of emulsified oil droplets.  The natural  sur-
factant material of both yeasts (C. subtropiaalis R42 and C. lipolytiaa
37-1) had only negligible toxic effects on guppies (19).  These same
yeast surfactants when added to oil films in beakers acted as oil
herders, concentrating the oil  in the center of each beaker.  Micro-
scopic observations indicated that an increase in the number of oil
droplets present as well as a decrease in droplet diameter accompanied
the decrease  in adhesiveness as cell numbers increased.  Such an effect
should have far-reaching environmental significance when the removal of
pollutant oils from an area is  considered.  In the natural biodegrada-
tion of oils, the amount of surface area available to form an oil-water
interface may be rate limiting.  An increase in surface area accom-
plished by the presence of very small droplets should enhance microbial
degradation of the oils.

    Candida subtropicalis  R42 was able to degrade a broad range of al-
kanes and alkenes, grew and multiplied in both fresh water and seawater,
remained stable under adverse conditions, emulsified oils, and most  im-
portantly, once established in  oil-seeded marine plots did not spread
into the environment.   No  literature reports on the pathogenic!ty of C.
subtropiealis for man or animals were found.  All  of these properties
are important for strains  used  as seed cultures to facilitate the bio-
degradation of pollutant oils.   Candida subtropiaalis possesses charac-
teristics which warrant further research into its  practical  applications
in combatting oil pollution.


    Numerous technical and practical considerations suggest that hydro-
carbonoclastic seed cultures will have restricted applications.  Ahearn
(2) stated that further research was needed as only cursory information
was available on the  immediate and long-term effects of the microbial
seeding of oil  spills.  Bartha and Atlas (11) indicated that nitrogen
and phosphorus are limiting factors for oil degradation in seawater.  In
a subsequent report, these researchers suggested that an oleophilic fer-
tilizer (e.g., a combination of octylphosphate and paraffinized urea)
could be employed to  increase the rate of natural degradation (10).  Ac-
tual field tests of this process have not been reported.

    In compiling the weathering history of two light paraffinic crude
oils which stranded on Martha's Vineyard, Massachusetts, and on Bermuda,
Blumer et al. (13) noted the great persistence of spilled oil,  and con-
cluded that its half  life must be measured in years.  Our studies sug-
gest that fertilization techniques for open environments may not be
readily effective in regions normally free of oil and thus lacking an
indigenous hydrocarbonoclastic flora.   Jobson et al. (26)  and Anderes
(9) also found that non-oil polluted sites lacked significant popula-
tions of organisms capable of crude oil utilization.  Moreover,  the fer-
tilization of an open aquatic system could result in eutrophication with
the development of unwanted species.  Therefore, seed systems may find
their primary application  in refinery waste treatment systems or in fa-
cilitating the removal of sludge oils  from shipboard installations.  In
such systems, growth conditions could  be partially controlled,  and if
necessary, nutrient enrichment or natural surfactants could be  used.
Although complete utilization of crude oil  has not been achieved by any
culture system, a microbial seed system which emulsified as well as uti-
lized part of the crude oil would still be of practical value in remov-
ing oils from contained situations.
                           LITERATURE CITED

1. Ahearn, D. G.  1970.  Systematics of yeasts of medical  interest.   In-
       ternational Symposium on Mycoses, Proc.  PAHO (Pan-American
       Health Organization) Scientific Pub. No. 205:64-70.

2. Ahearn, D. G.  1973-  Microbial-faci1itated degradation  of oil:  A
       prospectus, p. 1-2.  In D. G. Ahearn and S. P.  Meyers (ed.),  The
       Microbial Degradation of Oil  Pollutants.  Louisiana  State Univer-
       sity, Center for Wetland Resources, Pub. No.  LSU-SG-73-01.

3. Ahearn, D. G.  1973.  Effects of  environmental stress on aquatic
       yeast populations, p. 433-439-  In L- H. Stevenson and R.  R.  Col-
       well  (ed.), Estuarine Microbial Ecology.  Belle W.  Baruch Sympo-
       sium  in Marine Sciences.  University of South Carolina Press,
       Columbi a, S.C.

4. Ahearn, D. G., and S. P. Meyers.   1972.  The role of fungi in the
       decomposition of hydrocarbons in the marine environment,  p. 12-
       18.  In H. A. Walters and D.  G. Hueck van der Plas (ed.),  Bio-


        deterioration of Materials,  vol.  2.   Applied Science Publishers,
        Ltd.,  London.
 5.  Ahearn,  D.  G.,  D. Yarrow,  and  S.  P-  Meyers.   1970.   Piohia spartinae
        sp.  n.  from Louisiana  marshland  habitats.   Antonie van Leeuwen-
        hoek 36:503-508.
 6.  Ahearn,  D.  G.,  S. P. Meyers, and  R.  A.  Nichols.   1968.  Extracellular
        proteinases of yeasts  and  yeastlike fungi.   Appl.  Microbiol.
 7.  Ahearn,  D.  G.,  S. P. Meyers, and  P.  G.  Standard.  1971.   The role of
        yeasts  in the decomposition  of oils  in the  marine  environment.
        Dev. Ind. Microbiol.  12:126-133-
 8.  American Public Health Association,  American Water  Works Association,
        and  Water Pollution Control  Federation.   1971-   Standard Methods
        for  the Examination of Water  and  Wastewater.  13th ed.   American
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 9.  Anderes, E. A.   1973-  Distribution  of  hydrocarbon  oxidizing bacteria
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                     IN  SOUTHEASTERN LOUISIANA

         S.  A.  Crow,*  M.  A.  Hood,**  and S.  P. Meyers**

    Microbial populations in experimental  oiled marsh  plots and from the
site of an oil spill  in the Barataria  Bay  of  Louisiana were monitored
for occurrence and density of various  physiological  types.  Results in-
dicate that oil  stimulates an increase in  hydrocarbonoclastic and pro-
teolytic microorganisms, and a decrease in cellulolytic microorganisms.

    Microbiological  samples collected  along a  transect from near-shore
waters and sediments  to a fresh-water  swamp in southeastern Louisiana
showed similar increase in hydrocarbonoclasts  in areas exposed to oil.
A greater diversity of bacterial  species was also observed  in the oiled

    The ability of numerous microorganisms  to  partially degrade oil
under certain simulated environmental  conditions  is well documented.
However, knowledge of the effects of  crude  oil on microbial processes in
the environment is, in the main,  restricted to several rather broad ob-
servations.  As early as 1922,  Baldwin (2)  noted changes in microbial
activity of a corn field after  its treatment with petroleum.  Such modi-
fications following the introduction  of  oil  included  increased numbers
of heterotrophs, reduction in nitrate production, decreased diversity
of bacterial species, and slightly lowered  ammonia production.  Kincan-
non (5) reported alterations in microbial populations of soil treated
with crude oil  and presented evidence for microbial succession and the
predominance of yeasts in several samples.   More recently  (1973), Cobet
and Guard (3) found no noticeable changes in the microbial  communities
of a beach contaminated by bunker fuel,  especially in diversity of bac-
terial genera with either time  or depth  in  the sand.

    Considerations of the possible effects  of  crude oil intrusion on
microbial  processes are of particular significance in the coastal wet-
lands of Louisiana.  Implications of  such intrusion on the marshland
    *Department of Biology,  Georgia  State  University, Atlanta, Georgia
   **Departments of Marine Science and  Food  Science, respectively,
Louisiana State University,  Baton Rouge, Louisiana 70803.


microbial ecosystem have been discussed elsewhere (6).  Numerous petro-
leum industries are active in these estuaries which are of primary im-
portance to the fishery resources of the state.  The work reported here
is concerned with effects of production of crude oil on extant microbial
processes in the salt-marsh estuary.  Microbial conversion of the pri-
mary plant producer (Spartina alterniflora) to utilizable substrates is
essential to the overall energy flow of the salt-marsh environment.
    An ancillary phase of this work was a study of the distribution of
hydrocarbonoclastic bacteria over a wide spectrum of habitats including
beach zone salt marsh, brackish marsh, fresh marsh and swamp forest of
cypress-tupelo gum.  The total transect involved a distance of approxi-
mately 80 miles.
                         MATERIALS AND METHODS

    Sediment samples were collected at sites as previously described
 (7).  Samples were placed in sterile petri  plates and enclosed in ster-
 ile Whirl-Pak bags.  All  materials were then stored in an ice chest for
 return to the laboratory (within 2k hr),  with final processing within
 2k hr.  Sampling locations were in several  regions according to the ob-
 jectives of each specific phase of the investigation.  Primary sampling
 sites were:
  1) Oil retention plots and control marshes, located near the entrance
     to Airplane Lake (Grand Isle, Louisiana).  The oil  plots were es-
     tablished in November, 1970,  and were  treated with 250 ml of oil
     each month over a nine-month  period.
  2) Martigan Point, a small island, located NW of Grand Isle in the
     Barataria Bay region.  This site received oil following a break in
     an oil pipeline in October, 1972.  Several areas of this island
     were ignited and burned as part of the standard cleanup procedures
     used to remove standing spilled oil.

    The second phase of this study involved collection of material for
 examination along the transect of  a proposed pipeline from near-shore
 marine environments to cypress-tupelo gum fresh-water swamps.  Table 1
 gives a brief description of the environments of the wetlands transect
 sampling si tes.

    For the initial study, standard enumeration techniques were used to
 document populations and activities of 1) total heterotrophic bacteria;
 2) total yeasts; 3) cellulolytic bacteria;  k) cellulolytic fungi, in-
 cluding yeasts and filamentous fungi; 5)  proteolytic bacteria; 6) pro-
 teolytic fungi; 7) hydrocarbonoclastic bacteria; and 8)  hydrocarbono-
 clastic fungi.  For the transect investigation, only total heterotrophic
 bacteria and hydrocarbonoclastic bacteria and fungi were enumerated.
 All  determinations were of aerobic microorganisms only,   Cellulolytic
 populations were evaluated using a modified MPN-method,  with carboxy-
methyl-cellulose (CMC) incorporated as sole carbon source in the medium.
 Inoculated  tubes were compared with uninoculated controls, and those

containing medium with reduced viscosity were recorded as positive.  Hy-
drocarbonoclastic microorganisms were enumerated using the modified MPN-
method described by Gunkel  (A).
                  TABLE 1.  Wetland Transect Stations

   Station                        r  .
    Number                        Environment

      1       Gulf of Mexico:  Offshore from beach (1 m depth) located
             between Belle Pass and Caminada Pass.  A = water sample;
             B = submerged sediment.

      2      Intertidal area of beach Station 1

      3      Fourchon area near Bayou Lafourche.  Salt marsh system.

      k      West end of Fourchon area:  Spartina marsh.

      5      Leeville Oil  Field:  Spartina marsh.

      6      Bayou Sevin,  north of Leeville:  Spartina marsh.

      7      Point au Chien, Louisiana Wildlife and Fisheries Commis-
             sion Wildlife Refuge:  Brackish marsh dominated by
             mixed Spartina alterni flora, S. patens, Disfhchlis

      8      Bayou Blue north of Bully Camp Oil  Field:  transitional
             area between intermediate and fresh marsh:  Dominant
             vegetation, Sagittaria falaata.

      9      Bayou Boeuf,  east of Lake Boeuf.

     10      Bayou Citamon, cypress-tupelo swamp on east side of
             Bayou Lafourche.
    Following enumeration, selected organisms were isolated for further
physiological studies.  Those with cellulolytic or proteolytic ability
were tested for inhibition of growth by crude oil  using oi1-saturated
discs (1 cm) placed on plates seeded with test organisms.  Measurement
of inhibition of enzyme activity was accomplished  in an enriched sea-
water medium containing appropriate substrates, i.e., skim milk or car-
boxymethyl-eellulose.  Growth and activity in media containing 2% Loui-
siana crude oil  was compared to controls without oil  to ascertain any
reduction in activity of the particular enzyme systems.

                        RESULTS AND DISCUSSION


    Table 2 summarizes the densities of proteolytic, eellulolytlc, hy-
drocarbonoclastic bacteria, and total heterotrophic aerobic bacteria.

    The total populations of aerobic heterotrophs from all stations  in
Airplane Lake were comparable.   Addition of oil  increased concentrations
of hydrocarbonoclasts by a factor of 100, slightly increased numbers of
proteolytic forms, but resulted in the reduction in the biomass of
cellulolytic bacteria.  Samples from Martigan Point, collected k months
after an oil spill, showed similar trends.   In both Martigan Point and
Airplane Lake, samples of non-oiled sediments showed cellulolytic popu-
lations greater than the hydrocarbonoclastic portion of the populations.
In sediments exposed to oil, the relationship was reversed, i.e., hydro-
carbonoclast ic organisms were more numerous than cellulolytic species.

    Average heterotrophic and hydrocarbonoclastic populations from six
bimonthly samplings of the ten transect sites are given in Table 3.  The
ratio of hydrocarbonoclasts to heterotrophs is expressed on a percentage
basis.  The higher ratio obtained at Station 5 (Leeville Oil  Field)  is
indicative of enrichment of hydrocarbonoclasts in an environment sub-
jected to chronic low-level oil intrusion.   A ratio of >10 may be used
as an indicator of an oil-stressed environment,  while pristine habitats
regularly have ratios of <10.  The Leeville station consistently demon-
strated the highest number of different species.


    Of 54 isolates studied, ]k (25%) possessed both proteolytic and hy-
drocarbonoclastic ability.  Only k (7%) exhibited both cellulolytic and
proteolytic activity, while 3  (5%) were cellulolytic and hydrocarbono-
clastic.  Nine (16%) of the isolates were proteolytic, and 14 (251) were
hydrocarbonoclastic.  Five (9%) exhibited none of these capabilities and
a similar number utilized all three substrate types.  None of the iso-
lates demonstrated cellulolytic ability alone.

    None of the yeasts examined possessed proteolytic activity, while
only one utilized CMC.  This latter organism, a  Tr-LahospoTon sp., was
also weakly hydrocarbonoclastic.  Hydrocarbon utilization was observed
in 16 (75%) of the yeasts studied.  The almost complete absence of
yeasts with extracellular proteases is not  surprising since few yeasts
possessing such activities have been noted  (1).   Only 3 isolates of
filamentous fungi (Epicocaum, Fusarium, and Alternaria) used both hydro-
carbons and cellulose, whereas none of the  isolates possessed hydrocar-
bonoclastic activity alone.

    The higher frequency of bacteria exhibiting  both proteolytic and
hydrocarbonoclastic activities may explain  the increased biomass of  pro-
teolytic organisms in areas exposed to oil.  Similarly, the lower number


        TABLE  2.   Populations of  Heterotrophic  Bacteria
          in Oiled  and  Non-Oiled Marsh  Sediments,  1973
Concentration of bacteria (log 10/g wet sediment)

Airplane Lake Site
Oil Plot-A
Oil Plot-B
Martigan Point,
Oil Spill Site
Station No. 1
Station No. 2
cell .
hyc .






_ «•






™ ~


















Jul y






cell. = eellulolytic;  prot.
hyc. = hydrocarbonoclastic
= proteolytic;

of isolates with both eellulolytic and hydrocarbonoclastic capacities
may explain the decrease in the percentage of cellulose utilizers in
areas exposed to oil.  However, this does not preclude the possible in-
hibition of cellulolytic microorganisms by oil.
    TABLE 3.  Total  Heterotrophic and Hydrocarbonoclastic Bacteria
                        along Wetland Transect
Stat ion
Ratio of
Average No. Cel ] s/wet wt sampi e Hydrocarbonoclasts
°ta L. Hydrocarbonoclasts to Total Heterotrophs
Heterotrophs (Expressed as percent)
1 .
1 .



     In laboratory studies, none of the proteolytic or cellulolytic orga-
nisms studied were inhibited by crude oil  on a  peptone medium.   More-
over, neither inhibition of caseinolysis nor carboxymethylcellulose uti-
lization was evidenced with incorporation of oil  into the growth medium.

    Although numbers of microorganisms per se are not always absolute
indicators of microbial activity,  use of population levels as  a relative
indication of environmental activity appears justified when differences
in biomass are large.  The reduction of cellulolytic microbial  popula-
tions in areas exposed to oil  suggests that oil  affects the overall nu-
trient turnover in the estuarine-sa1t marsh.  This reduction possibly
may be attributed to unavailability of substrate due to physical coating
of the substrate particles, since crude oil did  not inhibit the carboxy-
methylcel lu lose metabolism of  selected isolates.

     Increased populations of hydrocarbonoclasts  observed at Airplane
Lake  (experimentally-oiled plots), Martigan Point (single spill), and

Leevilleoil  field (low-level  chronic pollution)  indicate that similar
enrichment processes were operative at all  three levels of oil pollu-
tion.  Increased species diversity, i.e., occurrence of different colo-
nial  types, noted at the Leeville oil  field suggests that chronic oil
pollution may induce a different population than that found after single
spill incidents.  This is in contrast with the report of decreased spe-
cies diversity at oil spill  sites (2).  These differences, in all like-
lihood, can be attributed to a range of factors,  including inherent dis-
similarities of the environments and types of oils applied.
    The preliminary findings reported here indicate that crude oil does
affect the overall metabolic activity of microbial populations of the
salt marsh.  Further study is necessary to establish the mechanism of
interference as well as the consequences of the reduction in select
metabolic types.

1. Ahearn, D. G., S. P. Meyers, and R. A.  Nichols.   1968.   Extracellular
       proteinases of yeasts and yeastlike fungi.   Appl .  Microbiol.
2. Baldwin,  I. L.  1922.  Modification of  soil  flora induced by applica-
       tion of crude oil.  Soil Science 1
3. Cobet, A. B., and H. E. Guard.  1973.  Effect of a bunker fuel  on the
       beach bacterial flora, p. 815-819-   In Proc., Joint Conference on
       Prevention and Control of Oil  Spills.  American Petroleum Insti-
       tute, Washington, D.C.
4. Gunkel , W.  1967-  Exper imentel 1-okologische Untersuchungen u'ber die
       1 imi tierenden Factoren des mikrobiellen Olablanes in marinen
       Milieu.  Helgolander Wiss. Meeresunters. 15:210-225.
5. Kincannon, C. B.  1972.  Oily waste disposal by soil  cultivation pro-
       cess.  A report.  Project 12050 EZG.  Office of Research and
       Monitoring, U.S. Environmental Protection Agency, Washington,
6. Meyers, S. P., D. G. Ahearn, S.  Crow, and N. Berner.   1973-  The im-
       pact of oil on marshland microbial  ecosystems, p. 221-228.   In
       D. G. Ahearn and S. P. Meyers (ed.), The Microbial  Degradation of
       Oil Pollutants.  Louisiana State University, Center for Wetland
       Resources, Pub. No. LSU-SG-73-01 .
7. Meyers, S. P., M. L. Nicholson,  J. Rhee, P. Miles, and D. G. Ahearn.
       1970.  Mycological  studies in Barataria Bay, Louisiana, and bio-
       degradation of oyster grass,  Spartina alterni flora.  Louisiana
       State University Coastal Studies Bull. 5:111-124.


S. P. MEYERS:  It occurs to me that maybe  we're pushing our seed culture
concept a little too hard at this time.  A lot of concern about the
Arctic deals with the sea ice itself,  and  the main problem in many of
these polar seas would be the oil getting  under the ice itself.  There
are reports, both from Antarctica as well  as the Arctic, that the sub-
ice contains a very active diatom population as well  as other primary
producers.  These populations contribute considerably to the produc-
tivity of the areas.  I  wonder if we may be throwing  out ideas about
seed cultures, as far as the polar seas  are concerned,  that bear really
no relationship to that environment.  Some of the projections, for in-
stance, are that the dissemination of  any  oil  film, or  spill, under the
ice has about a 7~10 year transport time under the ice  pack.   Possibly
Dr. Atlas would like to speak to this.

ATLAS:  The work that I  addressed myself to today was in terms of the
Arctic summer.  We are contemplating work  when the bay  is frozen over
and  I agree that the fate of under nearshore sea ice  should be studied.
The only person I  know who has done any  work in that  area is  Dr. D. K.
Button of the University of Alaska, who, using radio-labeled  hexadecane,
placed some of the hexadecane under ice  and trapped volatiles, presu-
mably carbon dioxide.  He found apparent degradation  of the hexadecane
under the sea ice during the winter months.  Another  person who has
worked on the interactions of ice and  crude oil  is Dr.  C. E.  ZoBel1,
who found that when you had ice crystals present in a medium you ac-
tually increased the rate of degradation.

LEWIS BROWN:  Showing the laboratory utilization of isolated  hydrocar-
bons does not mean that they'll be used  in crude oil.   For example, we
have used crude oil and related anthracene and added  several  fractions
to it.  With resting cells, if I  remember  correctly,  we had to go to
about 1000 ppm additional material before  it was used in that total sys-
tem  in the presence of the crude oil.  Still,  the organisms will grow
on a pure fraction.  The question in my  mind is whether many  of these
aromatics, or these potential carcinogens, etc., will  be used under the
environmental conditions that we're talking about in  the open ocean.

R. J. MIGET:  I  think that environmental conditions in  the open ocean
are more conducive to oil degradation  than the laboratory flask.  In
the oceans the more soluble low-boiling  aromatic compounds will either
evaporate or be greatly diluted in the water column--essentially frac-
tionating into individual molecules.  This situation  is totally unlike
laboratory studies where the more soluble  hydrocarbons  in crude oils
(low-boiling aromatics)  are forcibly concentrated in  a  relatively small
volume of water within a closed flask.   In this sense,  then,  I think
that studies with particular pure hydrocarbons are quite valid.


    There have been numerous studies  (Boyland and Tripp, 1971, Nature
230:44-1*7; McAuliffe, Chem. Tech., Jan. 1971) which show that the alkyl-
benzenes  I have used are the first ones to get  into the water.   In this
same regard  I would also like to refer to the work of Smith and Macln-
tyre (1971,  Prevention and Control of Oil  Spills, API/EPA/USCG, Washing-
ton, D.C.) on the  initial aging of fuel oil films in the open ocean.
They showed  that a large percentage of compounds in crude oil boiling
below 270 C  were lost in the first six hours due to evaporation.

BROWN:   I was looking at some of the  low-level materials that poten-
tially could be bio-accumulated.  When you have a low level of the ma-
terial you may not stimulate the population to such an extent that they
would degrade.  So, in a natural system I  was questioning whether this
would occur, although you can demonstrate these things rather adequately
in the laboratory  under artificial conditions.  I'm not clear whether
emulsification is  a good or bad phenomenon, because there are disadvan-
tages to both sides.  If the oil is emulsified, our work has shown that
it is much more toxic than if adsorbed to some material.  At first
thought, one might say that this is deleterious, if the concentration
reached  too  high a level.  On the other hand, if it's not emulsified,
the oil  has  a tendency to pick up other materials that are toxic, like
pesticides.  Thus, as a concentrator of other toxicants, it might be
better to emulsify the oil.

D. T. GIBSON:  At  the Atlanta meeting, the point was raised that we
should be considering the higher molecular weight compounds in oil.
I'm a little disappointed that so far we haven't had much discussion
on these.  It seems to me that if we are. talking about anything that's
going to be  practical  in the field, we have to come under federal regu-
lations.  It appears that chemical carcinogenesis by many of the com-
pounds which are found in oil occurs after metabolic activation in the
liver.  The  liver  cells apparently fix one atom of molecular oxygen
into compounds like benzpyrene or benzanthracene to make oxides which
have been implicated as carcinogens.  In a limited number of bacterial
species that we've studied, both atoms of  molecular oxygen are fixed
and there is apparently no oxide intermediate.  Fungi  and yeasts that
attack aromatic hydrocarbons appear to have a system similar to that
found in mammalian liver.  I  don't see that there will  be any approval
to use fungi  and yeasts unless we know a lot more about what some of
these organisms do to some of these higher compounds.

    And the  other  thing is the solubility  of these aromatics.  I  don't
think, as you said in the case of benzanthracene, that there are fewer
organisms that are capable of degrading them.  These compounds have
zero solubility.    If you could find a way  to increase the solubility
you could probably find organisms which would utilize them.
    I  have a  comment in regard to Dr. Zajic's paper.  I  think I'm one
of the men who reported growth of an organism on phenol  above 100 mg
per liter.   In fact,  we had no problem growing the bacterium up to
500 mg per 1iter .


R. J. MIGET:  Dr. Ahearn, do you have any idea of the oxygen concentra-
tion in the sediments you were talking about?

AHEARN:  All the yeasts we're working with are aerobes.  They have the
capacity to ferment, but they are not facultative anaerobes in the sense
of bacteria.  The yeasts will metabolize in an anaerobic environment but
their actual growth is limited.

R. L. RAYMOND:   I know of no valid experimental data that's ever been
published in which oil was utilized without the presence of oxygen.

GERALD BOWER:  A question for Dr. Rosenberg:  What was the minimum
amount of nitrogen and phosphorus needed for RAG-1 dispersion of crude

ROSENBERG:  Approximately 1  mM (NHlt)2S0lt and 0.01 mM KaHPOif.

BOWER:  How much dissolved oxygen did you try to maintain in these

ROSENBERG:  It wasn't measured.  The air was run at 3000 liters/minute
which was the maximum allowed us by the ship's Chief Engineer.

ALLEN LASKIN:  I  have a couple of comments rather than questions on a
few of the points that Dr. Rosenberg made.  I  might take slight issue
with his comment about the fact that microorganisms have had millions
of years to develop oil emulsifiers.  I'm a great believer in the power
of microorganisms and their numbers and the length of time they've been
around.  However, bacteria may not have developed emulsifiers during
these hundreds of millions of years in the same way we'd like to do it.
There are, as you may know, some new chemical  dispersants that are
really quite remarkable in their activity in dispersing oil, the so-
called second-generation, no-mix dispersants.   So we may have a bigger
problem competing with such dispersants if all we want to do is micro-
bially emuls ify oil.

     I'd like to make one other point in relation to toxicity in develop-
ing sea urchins.   This is a very neat system and it may give you quite
a bit of information.  It will tell you how the emulsified oil kills
sea urchin embryos; however, it may not tell you how it kills fish or
brine shrimp or algae.  The toxicity of emulsified oil for gill breath-
ers may well be related to particles of oil clogging the gills.  So al-
though we may know how oil affects nucleic acid synthesis or protein
synthesis in developing sea urchin embryos, it may not give us all the
answers we really need to have.

GIBSON:   I think  it's nice to see a field trial at long  last.   I wasn't
clear how you  inoculated your tank, Dr. Rosenberg.  Did  you use your
isolated organism and, if so, what size of inoculum?  Or did you use
indigenous organisms that were already there?

ROSENBERG:  We planned to inoculate it with 101* RAG-1 per ml.  However,
we ran into technical problems in transporting the inoculum to the ship.
The indications were that we inoculated with only 102 RAG-1 per ml.

GIBSON:   It seems to me from the data there's probably very little deg-
radation of the oil.  I  wonder if we might run into the danger of "out
of sight, out of mind" with this kind of thing at this stage of develop-

ROSENBERG:  That's an excellent point.  In this initial  ship experiment,
we did not degrade more than 10% of the crude oil.  Our  long-range goal,
however,  is more extensive breakdown and recovery of the products on
board.  However,  if you have a present choice of putting dispersed oil
or crude oil that's not dispersed into the open sea I would recommend
dispersed oil.  It has less chance to get to where most of the marine
life  is concentrated:  it has much less chance of aggregating and form-
ing tar on the shore, and most importantly, because of the large dilu-
tion there should be much faster microbial degradation in the sea.

P. A. LaROCK:  First, a comment on your technique.  I think this type
of process has certain industrial applications obviously for cleaning
vessels, but perhaps instead of just releasing this emulsified material
from the holds, it could be passed through an oil-water separator or
held until the vessel got to port.  In this way,  additional oil  sludge
would not be released.  It could be returned to the refinery and pro-
cessed with other oils or other cargoes.
    Secondly, a comment on dispersants in general.  We find that certain
bacterial species are affected by certain aromatics in the ppb range.
What would be the effect of dispersants or emulsifying agents on the
persistence of these aromatics, some of which may normally evaporate?
If you emulsify them and disperse them, you may do more harm to phyto-
plankton production or microbial  activity.

RAYMOND:  I  would like to correct something Dr.  Rosenberg has said
about the emulsification capabilities of his microbes exceeding the
chemical systems.   I  believe that if Vern Coty were here, he'd take a
great exception with you.   I  think we need also to correct the impres-
sion that we have to develop new great strains of microorganisms.
Those of us  in the field for many, many years know that these are
around.  What we have to do is to alter the environment so that the
organisms can grow on the substrate that's available to them.  You are
not going to find  a universal  panacea in any one microorganism for any


one situation in this world.   Just because we have a nutritionally bal-
anced system in the Delaware  River,  say,  or in the Marcus Hook Refinery,
that system doesn't prevail  in Tulsa, Oklahoma, or Los Angeles, Califor-
nia.  Each situation has to be studied on its own merits.  It's fairly
simple, I  think, to compute the amount of nitrogen needed to use almost
any hydrocarbon.  I  take the  universal figure as a "I'm going to have,
for every 100 Ibs of hydrocarbon, about 6 Ibs of nitrogen."  It's got
to come from somewhere.  You  have to have at least a ton of oxygen for
each ton of hydrocarbon to convert to cell material.  There's no way
this can be avoided.  This must be considered.  If you have a body of
water and you have 100 or 1000 tons  of oil, it would take a lot of ni-
trogen to convert all that oil into  cell  material.

ROSENBERG:  From the practical point of view,  in a closed system, nitro-
gen is not the problem.  In other words,  if you compute the cost and
savings, and you talk to people here who  know about this, the major cost
is the air, and how to get the air.   Cheap enough fertilizers are avail-
able.  -In an open system which may require special kinds of nitrogen,
then cost might be excessive.

AHEARN:  One of the interests  in developing seed culture systems is pos-
sibly to increase the rate of  degradation of the entire crude oil  com-
plex, by increasing the surface area.  So strains that have the capa-
bility of emulsifying the substrate  as well  as utilizing a significant
fraction of it would be of extreme value.  In fact, one of the questions
that did come up earlier is,  how common are strains that will  show good
utilization and good emulsification?  One of the problems that we've
had in our work is finding these two characters together in the same
strain.  I would like to ask  Dr. Crow, if he would, to give us a comment
on the percentage of organisms with  both  these properties that he has
found in his surveys.

S. A. CROW:  We've studied approximately  50 isolates that all  utilized
hydrocarbons fairly well.   Emulsification was a fairly rare property in
the organisms we were working  with.   We've done all our work in the es-
tuarine environment and less  than 5% of our isolates showed any emulsi-
fication of crude oil.   I  think this is fairly significant--!t shows
it's not a real  common property.

ZAJIC:  I  only have one comment.  In our  experience most of the orga-
nisms isolated using oil enrichments have an emulsification property.
Thus it must have something to do with how you isolate the cultures.  In
my experience, emulsification  is a very common phenomenon in microbes
degrading oil.  In the early  work completed on kerosene, we used a kero-
sene enrichment.  Eighty-four  cultures were isolated in pure form that
would grow on kerosene and each was  tested for its emulsification capa-
bility on kerosene.   Whereas  many of them emulsified kerosene, only four
produced quantities of an emulsifying agent that would be regarded as

interesting.  Of these, only four had industrial potential.

AHEARN:  I've been trying to find out from different workers the inci-
dence of emulsifying strains.  It seems that when you study chronically
polluted sites, or if you look at strains from oil fields, you more
commonly find organisms which emulsify as well as utilize various frac-
tions of the oil.  Dr. Crow's work has been in an area which has been
remote from direct oil pollution, or chronic oil pollution, and his re-
sults agree with what we have found.  When we have looked at oil pol-
luted sites we have found a fairly high percentage of the isolates of
one or two species which emulsify oil.  But these represent only a mi-
nority of the species isolated.  Our culture systems have used both
enrichment and general isolation procedures.

ZAJIC:   I would make another comment, that  is, we have been using some
of the aromatics for enrichment purposes.   In so doing, a much lower
percentage of microorganisms produce emulsifiers.  So I  think most of
the emulsifiers observed in complex systems are from the paraffinic
hydrocarbons, and the emulsification property will be greatly enhanced
by the presence of paraffins.

RAYMOND:  Dr. Zajic and Dr. Rosenberg both, I  believe, mentioned that
you don't find these organisms prior to seeding.  The technique that is
used in  looking for these organisms must be considered.   If you use
conventional enrichment techniques, for instance, you will  isolate the
organisms.  Numerous production reports mention the problem of emulsi-
fication of oil.  Every producing oil field has probably had to worry
about breaking the emulsions that bacteria cause in these systems.   If
you study a soil which is virgin to oil, you can't use the same isola-
tion techniques that are used when you work in a system that has oil
contamination.  But the organisms are going to be there.  You have to
look at a tenth of a gram of soil instead of 1-10,000 or 1-1,000,000
dilutions and different techniques are necessary.  You don't have to
develop microbial seed systems—they're there.

ROSENBERG:  Both of us were not arguing that these bacteria were not
there.  The gist of the Zajic talk was that if you choose conditions
right, you can by definition of enrichment culture enrich for the bac-
teria you particularly want.  If you want to add it back later in a
closed system or an open system, you can do so.  But I don't think
either of us  intended, in any sense, to infer that these bacteria
aren't there.  The whole definition of the enrichment culture is to
select conditions to enrich for a particular kind of desired microbe.
Certain enrichment cultures turn up more emulsifiers while others turn
up other things.  I  selected one organism on purpose by transferring
the enrichment culture some 8 or 10 times and subjecting it to condi-
tions where I selected the fastest grower under a particular set of
conditions.   I eliminated, purposely, many other bacteria.


ARTHUR KAPLAN:  In our experience with refined products,  JP-A, in a
military fuel distribution system for aircraft, we found  that emulsifi-
cation with the bacteria was quite common.  1  can't recall  an instance
where the fungi gave us any emuIsification and I  don't recall the situ-
ation with the yeasts.

                        CONCLUDING REMARKS
                          Dr. William Upholt
                 U.S. Environmental  Protection Agency
                           Washington, D.C.

    The EPA has the responsibility of regulating all  materials sold or
used to manage pest populations as well  as to protect man and his envi-
ronment from all types of pollutants.  This is the basis for EPA spon-
sorship of  this important conference.   We are especially appreciative
of the insights that the participants in the conference have given us
into these problems of the use of microorganisms as you have discussed
them for the past two days.  We are impressed,  as I'm sure you are as
well, by the difficulty of protecting against a hazard that is actually
unknown.  In the past we have registered some chemical pesticides, only
to discover later on that they created hazards that we had not antici-
pated.  In some of those cases the unanticipated hazards have led us to
cancellations and very expensive and sometimes overly emotional  public
hearings.   We would hope to prevent these types of mistakes as microbial
pesticides are submitted for registration and tolerances or exemptions
from tolerances.  Obviously, it is to everyone's advantage if we can
provide guidelines on the needed information for registration as early
as possible.  We are in the process now of issuing guidelines for regis-
tration of chemical pesticides, but they are hardly adequate for micro-
bials.  As most of you know, we have suggested preliminary guidelines
for nuclear polyhedrosis viruses but even those are of limited value
with other types of viruses and with bacteria,  fungi  and protozoans.
Frankly, it is difficult to write guidelines for submittal of informa-
tion regarding an unknown hazard.  Of course, we, like all of you, hope
that there is no hazard to man*or to other non-target organisms from
these microbial pesticides.  Nevertheless, there are obvious potential
hazards, such as infectivity to man, domestic animals or beneficial  non-
target organisms.  These hazards are doubtless greatest to those non-
target organisms which are most closely related to the target organism.
Information to this effect would be essential for registration.  Beyond
that, discussions during the past two days have suggested possible al-
lergic reactions in man, or even some type of highly atypical infection,
perhaps by a fungus.  Such atypical  reactions are very difficult to
predict, and therefore difficult to evaluate.  As also was pointed out
in several  comments in the last day or so, negative results are ex-
tremely difficult to evaluate.  They are never any better than the ade-
quacy of the tests that produce them.  This is doubtless the reason, or
at least one reason, that editors hate to accept for publication these

negative results.   It's also the reason that we hesitate to accept them
as a basis for registration.  We register on the basis of balancing
benefits against risks.  Unless you know the hazards versus the risks,
it's difficult to  get an objective evaluation of such unknown risks.
In this situation, we will  doubtless make full use of the stepwise pro-
cedures which permit registration for experimental purposes prior to
full-scale commercialization.   During this period and later on, monitor-
ing will be necessary and extremely crucial  to the procedure.  We will
need the full cooperation of all of the experts throughout the country
to design adequate guidelines for registration and monitoring during
and after the experimental  stage, if we are going to successfully pro-
tect our society from the unanticipated hazards from these microbial
pesticides.  We are anxious to have these new materials available for
use but as you know society takes a dim view today of providing 200
million guinea pigs.   Society is demanding more cautious approaches to
all hazards.  We are sure that if we all  work together, we can provide
that needed assurance of safety as well as efficacy within a reasonable
time.  We therefore do appreciate the opportunity to participate in
this conference and we will  be calling on you more and more in the fu-
ture to help us set up the  guidelines and decide what is needed for our
full registration.



                        BACTERIA:   SUMMARY

    Anopheline and Culicine mosquitoes are the target invertebrates in
aquatic environments for which species of Bacillus are considered to be
used as control agents.  The principal entomopathogenic bacterial spe-
cies are:  B. thuringiensis, B. sphaericus, B.  moritai, B. oereus var.
juroi, and varieties within the B. alvei circulans morphological  Group
II.  Of these, only B.  sphaericus  is currently considered specific for
mosquitoes.  Each Bacillus species should be considered as a source for
selection of strains with specific activity for target aquatic inverte-
brate pests.

    Preliminary laboratory evaluations of B.  tluupingiensis (BA068) and B.
sphaericus have been completed in  the United States, the latter also in
Nigeria.  Additional laboratory tests and pilot small plot field  trials
are anticipated for 197^ at locations within and outside the United

    Commercially available preparations of B.  thuringiensis are limited
in their activity to Lepidopteran  insects.  Mice in standardized  labora-
tory safety tests and fish from aquatic environments have not been af-
fected by B. thuringiensis.  B. sphaericus appears to be limited  in  ac-
tivity to culicine and anopheline mosquitoes.   Additional screening  has
not demonstrated activity in the house fly (Musaa domestica)  or a single
species,in each of the orders Lepidoptera and  Coleoptera.

    Documentation is not available  to describe  either the persistence of
the entomopathic bacilli in aquatic environments or the results of di-
rect or indirect introduction of bacilli to aquatic systems.

    A multi-stage review system for biological  agents anticipated by the
vector biology and control  unit (VBC) of the World Health Organization
(WHO) should provide specific information of impact of bacteria in en-
vironments and associated water systems.


1.  Investigate:
   a. persistence of bacilli and their products in aquatic environments.
   b. safety of B.  sphaericus for non-target organisms as a model for
      similar bacilli.
2.  Support programs for detection and development of useful microorga-
   nisms for management of target  invertebrates in aquatic environments.

Summary prepared by:  S. Singer (presiding), J. Briggs, S. R. Dutky,
A.  M. Heimpel, E.  W. Davidson, T.   L. Couch, T.  C. Cheng.

                         VIRUSES:   SUMMARY

    Virus diseases are known to affect  many invertebrate species living
in aquatic habitats.  These include the nuclear polyhedrosis types (NPV),
e.g. Baeulovirus; cytoplasmic polyhedrosis viruses (CPV); entomopoxvirus
(EPV); and non-inclusion viruses (NIV), e.g.  Ividoviruses.   None of
these has been studied sufficiently to  recommend a specific virus as a
control agent for the aquatic insect pests.

    Laboratory and field studies have concentrated on viruses of mos-
quitoes.  The Iridovirus from Aedes taeniorhynehus and the  Baoulovirus
from Aedes so11iaitans have received the most attention.  Both of these
viruses have been studied in the laboratory in attempts to  quantity and
determine levels of infectivity and susceptibility.   Limited host speci-
ficity studies also have been made.   Cross-infection tests  with the mos-
quito  iridescent virus (MIV) from A. taenioTTnynclnus  have been carried
out against approximately 18 species of mosquitoes,  3 species of Lepidop-
tera, and 2-3 additional species of Diptera.   Present testing indicates
that MIV appears to be restricted to the floodwater  genera  of Aedes and
Psopophora.   Safety testing against aquatic species  has not been done
with any viruses from aquatic insects.   Limited studies have been made
with the NPV from A. soiliaitans against mosquito predators such as Gam-
busia and insects of the families Hydrophi1idae, Dyticidae, and the
order Odonata.  Preliminary cross infectivity tests  indicate that this
NPV is restricted to mosquitoes of the  genera Aedes  and Psorophova.

    Studies  on the persistence of virus in the aquatic environment have
not been made.  Similarly,  there is no  available information regarding
possible effect of metabolic by-products from viral  infections on
aquatic organisms.

    Field related studies with MIV from A. taeniorhynahus and NPV from
A. soilioitans (1 test with each virus) have  been reported.  It is ob-
vious from the previous assessment that commercially available virus
preparations for use in control  of aquatic insect pests are not avail-


1. There is  an urgent need  for intensive surveys to  discover additional
   potentially useful  viruses.  Research in this area should be closely
   coordinated at national  and international  levels.

2.  It  is highly  important that research be greatly expanded on the
   presently known viruses.  This research should include biochemical-
   biophysical characterization and identification,  studies on infec-
tivity, host specificity, and safety; and production of viruses for
poss i ble field use.

Summary prepared by:  D. W. Anthony, T. B. Clark, A. M. Heimpel, J. D.
Paschke, M.  D. Summers, J.  N. Couch.

                           FUNGI:   SUMMARY

    At present there are no fungal pathogens of Invertebrates employed
 in wide-scale pest control operations which directly involve aquatic
 habitats.  This could change within the next few years.  Laboratory and
 exploratory outdoor tests for larval mosquito control are now underway
 with Coelomomyoes spp., Lagenidiwn giganteum, Beauveria tenella, and
 Metarvhiziwn anisopliae.  Field observations indicate that some species
 of Entomophfhora offer promise as pathogens of adult Culex.

    The current lack of detailed information on proper timing, dosage,
 and formulation, in addition to generally inadequate host-range data,
 poses serious difficulties in estimating the impact of most  entomogenous
 fungi on aquatic environments.

    None of the above species are commercially available but the tech-
 nology exists for mass production of all except Coelomomyaes.

    Laboratory infections have been readily obtained with all  except
 Coelomomyaes.  Small-scale field studies have been conducted with Coelo-
 momyoes, Lagenidiwn, Beauvevia, and Metarvhizium with promising results.

    Meaningful safety tests of Coelomomyoes are not possible at present,
 because the infective unit of the fungus is unknown.  Field  collections
 indicate these fungi have narrow host ranges, usually one or a few mos-
 quito species.  One isolate of L. giganteum has a wide mosquito host
 range but apparently poses no threat to. other organisms in mosquito
 habitats.  Specific laboratory tests with a wide variety of  aquatic and
 terrestrial organisms were negative.  Other isolates of Lagenidiwn are
 little studied, but at least one is reported as infecting Daphnia.   The
 majority of the EntomophthoTa currently under investigation  appear spe-
 cific to Culex adults.  A wide range of target species are susceptible
 to the B. tenella isolate from mosquitoes;  however, its infectivity for
 non-target species is unknown.  The isolate of M.  anisopliae under con-
 sideration is more virulent for mosquito larvae than most other aquatic
 invertebrates  tested, but certain aquatic  organi'sms were susceptible.
 All major mosquito groups were susceptible.   Toxins are unknown from
 Coelomomyoes and Lagenidiwn and tests on those metabolites produced by
 the other fungi under consideration, although incomplete, have not in-
 dicated any serious problems.
    Coelomomyaes spp., L. giganteum, and possibly B. tenella will  prob-
 ably become established when properly introduced.   M.  anisopliae does
 not produce new spores on submerged cadavers, so it is not likely to
 become established as a self-perpetuating mosquito pathogen  after arti-
 ficial introduction.

    Conidia of Beauveria bassiana are commercially available in the
 USSR for use against orchard and field pests.  Field trials  are being
conducted in France with B.  tenella against subterranean beetle larvae,
and in the United States Hivsutella thompsonii and Entomophthora


thaxteviana are being tested against orchard mites and row crop aphids,
respectively.   The amounts of these pathogens which may find their way
into aquatic environments following application to terrestrial  environ-
ments is unknown.   B. bassiana has a very wide insect-host range, but
testing with aquatic organisms is meager.  Allergic reactions to this
fungus have been reported.  H. fhompsonii and E.  thaxteviana, in limited
safety tests,  were safe for vertebrates.   Field observations indicate
both have relatively narrow insect-host ranges, but neither has been
tested against aquatic invertebrates.

Summary prepared by:  D.  W. Roberts, E. M.  McCray.

                        PROTOZOA:   SUMMARY

    Only one microsporidan, Nosema algerae Vavra and Undeen,  is presently
being considered for field tests against aquatic insect pests.   This mi-
crosporidan has been shown to cause high mortalities in many  species of
Anopheles mosquitoes in laboratory infection studies.   Its ability to
produce mortalities in field populations of Anopheles  aibimanus was
demonstrated early this year by insect pathologists from the  Insects Af-
fecting Man Research Laboratory, USDA-ARS, at Gainesville, Florida, in
cooperation with the Army in the Canal  Zone.  Several  other undescribed
species recently found also have been shown to cause mortalities in mos-
quitoes in laboratory tests, and some of these may be  considered for
field testing in the future.  No microsporidans are presently being
seriously considered in tests against ceratopogonids,  simulids, tabanids,
or other aquatic pests.
    Viable N. algerae spores were fed to numerous non-target  animals by
researchers at the Gainesville Laboratory to determine host specificity
of this microsporidan.  Some animals tested were:  amphipods, aquatic
predaceous hemipterans, cabbage loopers, chickens, chironomids, cock-
roaches, corn earworms, crayfish, damsel flies, dragon flies, dytiscids,
fire ants, fish, fresh-water shrimp, helgramite, house flies, and mice.
Only three (chironomids, corn earworms, and house flies)  were suscep-
tible.  Under natural conditions, however, only the chironomids are
likely to be exposed to Nosema,
    Spores of N. algerae are unstable in water at room temperature and
even lose viability when stored for more than six months  under  refrig-
eration.  Drying immediately kills spores, making it even more  unlikely
that they will infect terrestrial animals.  Accumulation  of N.  algerae
spores in aquatic environments should not be a serious problem.  Spores
not ingested by mosquitoes rapidly lose viability.  Microsporidans do
not produce toxic metabolites.
    Development of N. algerae was obtained in mammalian tissue  cultures
of kidney cells at 26 and 35 C, but development could  not be  completed
at 37 C.  Small numbers of meronts, sporonts, and sporoblasts were ob-
served at the sites where large numbers of N. algerae  spores  were sub-
dermal ly  injected into low temperature regions of mice, i.e., base of
the ears, tail, and hind feet.  Spore-to-spore development was  not con-
firmed, and the protozoan was confined to the injection site.   None of
the stages of Nosema were found at the injection sites 12 days  after
i njection.
    No mice or other vertebrates were ever affected by massive  dosages
of spores per os by by intravenous or intraperitoneal  injection of
spores.  Development was never complete at temperatures at or above
those of warm-blooded animals in tissue cultures.
    The studies indicate that the host range of N. algerae can  be ex-
tended experimentally to include a large range of hosts;  however, under

normal field conditions, these routes of infection would not normally
be avallable.

    There may be a remote possibility that microsporida may enter non-
host warm-blooded animals through injury sites or punctures produced
by mosquitoes or biting insects.   Present information indicates that
these introduced microsporida would  be contained at the site of entry
and would be destroyed.  However, such possibilities should be investi-
gated in conjunction with safety  tests on pathogenic microsporida in
mosquito and biting flies.   All other microsporidans that may be con-
sidered in future field tests should be evaluated for safety to man and
the environment utilizing guidelines produced  by the Environmental  Pro-
tection Agency before they are used  in large-scale field tests.

Summary prepared by:  E. I.  Hazard,  J. E. Henry, J. Maddox, A.  Undeen.


    Knowledge of persistence of insect pathogens in the terrestrial  en-
vironment has increased substantially.  However, that concerning the
aquatic environment (both fresh water and marine) is still  very limited.
This situation demands immediate attention,  because a number of patho-
gens are now showing real promise for control  of aquatic insect pests
and vectors.

    Investigations of persistence should consider both biotic and abiotic
factors of the environment.  Pathogens of not  only aquatic  insects  but
also terrestrial ones with potential  for control of aquatic pests should
be studied in this context.  Major abiotic factors that should be con-
sidered are salinity, temperature, water depth and movement, desiccation,
redox potential, soluble materials, pH, turbidity, and the  sediment  of
the aquatic environment.  In the biotic environment, the primary and
secondary hosts, animal carriers, and other  microorganisms  should be
considered.  This will involve studies on interspecific transmission as
well as on persistence in the digestive tracts and on body  surfaces  of
animal  carriers.  In addition, the interactions of hosts and pathogens
with other aquatic fauna and flora should be investigated.

    Relevant laboratory studies are important, but the ultimate assess-
ment demands aquatic-field studies.  Persistence of pathogens that are
highly mobile, because of buoyancy or motility, require investigation
in both fresh-water and marine environments.   The studies to be con-
ducted  will be determined primarily by the type and nature  of the

    Other speakers have enumerated pathogens of mosquitoes  and black
flies which offer the best potential  for development.  Due  to the ad-
vanced  state of investigations, the following  are suggested as candi-
dates for immediate studies of persistence in  aquatic environments:
nuclear polyhedrosis viruses; Bacillus s-plnaer-ieus, B. thuringiensis,
(var. BA068); Coelomomyoes spp., Lagenidium  giganteim, Beauvevia te-
nella,  Metarvhizium anisopliae; Nosema algerae; Reesimermis nielseni.
Studies on persistence should consider:  control of the target pest,
safety to man and other terrestrial vertebrates, pathogenic!ty for non-
target  aquatic invertebrates and vertebrates,  and pollution from the
standpoints of health and aesthetics.

Summary prepared by:  H. C. Chapman,  R. B. Jaques, M. Laird,  Y.  Tanada.

                   OTHER THAN INSECTS:   SUMMARY

    To our knowledge,  no extensive  research  program  is  currently  under-
way in this country or abroad  to isolate  and develop  microorganisms for
biological control  of  aquatic  pests other than  insects, although  there
are occasional  reports of organisms that  may be  potentially  pathogenic
to aquatic snails that serve as intermediate hosts for  the human-
infecting schistosomes.   Such  organisms,  however, are currently not
being maintained in culture  in any  laboratory.   Bac'illus p-inotti-'i, a
Gram-variable species, was thought at one time to be  useful  for the con-
trol of Biomphalaria gldbrata  in Egypt and Venezuela; however,  its
pathogenicity to this  gastropod vector of Schistosoma mansoni has been
seriously questioned.

    At Lehigh University's Institute for  Pathobiology, a yet unidenti-
fied coccus has been isolated  from  Israel from the desert snail Theba
pisana which is lethal to this gastropod  when the ambient temperature
is lowered to 10-18 C.  The  usefulness of this bacterium against other
species of molluscs, however,  remains to  be ascertained.

    Despite the lack of  rigor  in efforts  to search for biological con-
trol agents for aquatic  pests  other than  insects, the potential useful-
ness of microorganisms for this purpose should not be underrated.  Sup-
port for goal-oriented basic studies that will permit development of a
rational  approach to isolation,  selection, and development of control
agents against  toxic marine  invertebrates .and disease-carrying fresh-
water animals,  especially molluscs, must  be forthcoming from agencies
with vi si on.

Summary prepared by:  T.  C.  Cheng.

                    PLANT  PATHOGENS  FOR CONTROL
                    OF AQUATIC  WEEDS:   SUMMARY

    Studies on the evaluatipn of plant pathogenic biocontrols of water
weeds are in their  infancy.  However, recent explorations on the use of
plant pathogens to control aquatic weeds have been encouraging.  Two re-
lated aquatic systems subject to weed problems have been studied.  In
one, consisting of a total aquatic ecosystem, four exotic aquatic weeds--
water hyacinth, hydrilla, alligator weed, and Eurasian water milfoil--
have been the target of our studies.  The second is a semi-aquatic agro-
ecosystem in which a weed  (Aesohynomene virginiaa)  that occurs in rice
has been the subject of extensive and successful field tests.

    Several  foliar fungal pathogens of water hyacinth have been tested
for pathogenic!ty, host range, and efficacy as biocontrols under labora-
tory conditions.  Of these, Cephalosporiwn zonatum,  which causes zonal
leaf spot of water hyacinth, is the most likely candidate for control  of
this weed in Florida.  When used, its effect on water hyacinth is likely
to be increased due to the presence of two arthropods (Seoohetina e-ich-
hovn'ia.e, weevil; Orthogaltanna tex>ebvanti,s, mite) already in use as bio-
control  agents of weeds in Florida.  CephalospovLim zonatim is present
in Florida and Louisiana on water hyacinths.   Therefore, special  plant
quarantine regulations will not be needed for release of this organism
for field tests.  Laboratory tests on the host range of this pathogen
are in progress.  Numerous crop and non-crop plants  are being screened.
Limited  field tests are underway to determine efficacy of this pathogen
under natural conditions and its effects on non-target hosts.  Studies
have not been done on possible toxicity or pathogenic!ty of Cephalo-
sporiim zonatum to fish, other aquatic animals, terrestrial inverte-
brates and vertebrates; persistence and survival in  the environment;
and impact of its use on the "total ecosystem."

    Available evidence suggests that in the absence of suitable hosts
like water hyacinth, C. zonatum wi11 lose viabi1ity in aquatic habitats.
Long-term persistence of this organism in water is  therefore not antici-
pated .
    Other pathogens of water hyacinth that are currently being evaluated
include Alternaria eiahhorniae, Ceroospora piaropi,  a Helminthosporiwn
sp., (Dreehslera sp.), and Rhizoatonia solan-i.  Several  isolates of
Pen-isilli-um, Aspergillus, and Tviohoderma that are toxic to hydrilla
are also being studied to identify possible host-specific phytotoxins.
Attempts are being made to seek newer pathogens of  water hyacinth, hy-
drilla,  alligator weed, and Eurasian water milfoil.

    The  success in controlling Aesahynomene Divginloa (northern joint-
vetch),  a semi-aquatic weed of the rice fields, with the fungal patho-
gen Colletotr-iahim gloeospoTioides signals the entrance of plant patho-
genic bioherbicides in the weed control scene.  Laboratory and field
tests indicate that this pathogen is specific to Aesohynomene.  Out of

150 plant species screened,  only Aesahynomene was  susceptible.
    Preliminary toxicological  studies  indicate that  this  fungus  is  non
toxic to warm-blooded  animals.   Research  on  this extremely  host-
specific, endemic fungus  has advanced  to  a  stage where its  commercial
production and large-scale aerial application are  being  seriously con-
s idered.

Summary prepared by:   R.  Charudattan,  G.  Allen, G.  E.  Templeton.

                         SUMMARY  COMMENTS
                             C. M.  Ignoffo

    It is rather obvious that the status of aquatic microbial pesticides
is still  in the formative stage.  I  think it far-sighted of EPA to take
the initiative in arranging a conference such as this to document the
baseline now in an attempt to aid future developments and, again, his-
torically to measure the developments that will  undoubtedly occur in the
future.  Now the meeting scope was  limited to aquatic habitats.  This
major theme meant by its very limitation that the successes and develop-
ments of microbial insecticides for  terrestrial  pests could not be cov-
ered.  I  want to put this in its proper perspective now, to give you
some idea of the extent of developments that have occurred in this ter-
restrial  area.

    There are presently about 40 trade-name microbial  insecticides in
the world.   These are based on approximately 12  pathogens, representing
groups of bacteria, fungi,  and viruses.  These materials have been used
on every major continent except one—and I'll  let you guess which one
that might be.   One commercial  bacterial insecticide,  which is used  to
control the Japanese beetle, has been introduced in about dozen states,
in the northeast, and since 1950 has  exerted control  over an area esti-
mated at a quarter of a million square miles.  Another bacterial  insec-
ticide is used  on cabbage,  lettuce and many cold crops in the United
States, and has been used since 1959  at an estimated  rate of about 2-3
million acre treatments per year.  In Russia,  approximately 10 percent
of their total  insecticidal control,  perhaps 45  million hectares, is
devoted to biological control, with at least 50% of this being microbial
insecticide applications.  In Japan,  a commercial  viral insecticide  has
been developed  that is used to control a pest  of pine forests.  Now,
hopefully, these figures and facts will help you to better understand
that microbial  insecticides have been developed, from concept, from
isolation in the field, to commercialization.  Associated with this  de-
velopment has been a vast accumulation of basic  information on pathogen,
host and ecosystem.  Microbial  insecticides are  not just laboratory
curiosities.  They are safe, they are selective, they are efficacious,
they are commercial products currently in use as pesticides.


    The use of microbial  seed  systems  to  facilitate  the  biodegradation
of oil or other hazardous chemicals  is  in  a  primitive  state of develop-
ment.  Indeed, there is  some controversy  that  fertilization to stimulate
the indigenous microorganisms  might  not provide  the  most  efficient  bio-
degradation.  It is  questioned also  if either  procedure  is applicable
to open systems.  Still,  preliminary studies,  as  presented and discussed
here, indicate that  fertilization or seed  systems, or  a  combination of
both, are presently  being, used or explored.   It  is possible that both
types of procedures  may  eventually be  found  useful for varying goals or
ecological conditions.

    Generally, those here agree that there are insufficient data to
fully evaluate the effectiveness of  these  seed or enrichment systems in
degrading a target substrate.   Moreover,  there are few data available
to permit an assessment  of potential  hazards of  seed systems to the en-
vironment.  Guidelines for the control of  seed or enrichment systems
should consider the  relative or potential  hazards of the  process and
target systems.  The present status  of the art requires us to proffer
only general guidelines.   As a committee we  suggest  that  no established
pathogens for man be added to  the environment  to  treat oils and/or re-
lated products.  It  should be  emphasized  that  treatment procedures
being proposed are directed  toward acceleration of natural processes.

Summary prepared by  all  participants of Session  II.


                  Reto Engler, Registration Division
                     Office of Pesticide Programs
          William Roessler, Criteria and Evaluation Division
                     Office of Pesticide Programs
                           William M. Upholt
                 Office of Hazardous Materials Control
           Environmental Protection Agency, Washington, D.C.
    Although there are broad and common phrases used by the Environmen-
tal Protection Agency to state its mission and its goals—such as  to
"protect man and his environment" and to assure that pesticides are used
in a "safe and effective manner"—any regulatory agency also has the
responsibility to encourage the research or effort leading  to these
goals, to fulfill its mission.  Thus, as evidenced by this  symposium-
workshop, the Agency is looking to alternate methods of pest control  to
replace hazardous, toxic or persistent chemical  pesticides.  The Agency
encourages, through support of research under contract, through its own
laboratories and various cooperative efforts with industry, academia,
and other Federal agencies, the development of alternate methods of pest
control and the implementation of the concepts of biologically inte-
grated pest management.  Section 20 of the 1972 Federal Environmental
Pesticide Control Act (FEPCA) points out that priority must be given to
this type of pest management research.
    But we also must remind  everyone  of the regulatory aspects of  the
Agency and the intent of Congress in establishing the Environmental  Pro-
tection Agency in the first place.  We do have regulatory responsibili-
ties under FEPCA, Food, Drug and Cosmetic Act, the Water Acts, etc.—
particularly when a product reaches the open market.  These responsi-
bilities relate to the safe and effective use of products intended  to
destroy, control, or mitigate various pests--some of which  have been the
primary focus of attention at this symposium.  In the past  some chemical
pesticides were registered only to discover later that they created
hazards that had not been anticipated.  In some cases these unantici-
pated hazards have led to cancellations and very expensive  and sometimes
overly emotional  public hearings.  Such mistakes should be  prevented as
microbial pesticides are submitted for registration and tolerances  or
exemptions from the requirement of a tolerance.

    We must ask the question, "What could possibly happen?" when using
microbial pesticides.  But we also should qualify the question by  ask-
ing, "How likely is it to happen?"  The scientific search for answers
to these questions should eventually fill  in the voids that are apparent
in our knowledge about these new generation pesticides.

    It is to everyone's advantage if EPA can provide guidelines on
needed information as early as possible.  We are in the process now of
issuing guidelines for registration of chemical  pesticides but they are
hardly adequate for microbials.   Preliminary guidelines for nuclear
polyhedrosis and granulosis viruses are near ing  completion but even
these are of limited value for other types of viruses,  bacteria, fungi,
and protozoans.  It is difficult to write guidelines for submittal of
information regarding an unknown hazard.  Because of the multitude of
issues involved, experts in the fields of microbiology, epidemiology
and public health must cooperate in order to arrive at  the best possible
guidelines for registration and  tolerance setting as well  as for moni-
toring programs during and after the experimental use.   We hope that
there is no hazard to man or other  non-target organisms from these mi-
crobial pesticides, but that does not seem to be enough to make a final
decision.  There are obvious potential hazards such as  infectivity to
man, domestic animals, or beneficial non-target  organisms.  These haz-
ards are doubtless greatest to those non-target  organisms  most closely
related to the target species.  Beyond that there is a  possibility of
allergic reactions in man from some exo- or endotoxin that has not
been detected, or some type of highly atypical,  unpredictable infec-
tion from a fungus perhaps in lungs or possibly  other organs.

    The eventual full-scale use  of  microbial  pesticides must follow a
carefully planned, stepwise approach and the issuance of experimental
use permits under Section 5 of FEPCA will  be an  important  aspect of
their development, which can be  briefly summarized  as follows:
(i) Identification of the pathogen,  including growth requirements, sta-
bility, bioassay and infectious  process.  (ii) Preliminary determination
of usefulness.  (iii) Effects on non-target organisms,  acute, subacute
and long-term studies.  (iv) Review of aspects of safety.   (v) Small-
scale use to gather data on efficacy as well  as  to  monitor effects on
the environment.   (vi) Second, comprehensive review of  safety aspects
and performance under field conditions.  (vii) Extended use, with con-
tinuous monitoring of environmental  effects.   The monitoring programs
will be of special importance.  Good ecological  baseline data are needed
in order to assess effects of the deliberate use of pathogens, and
humans exposed to the pathogens  during production and use  must be moni-
tored for any adverse reactions.

    Many, if not all, of the safety tests will give negative results
which are extremely difficult to evaluate since  it  is axiomatic that
it is impossible to prove a negative.  The adequacy of  the tests there-
fore must be scrutinized as much as the actual results.  The empirical
tests, although they probably will  never be completely  replaced, should
be complemented at an increasing rate by basic knowledge about the
mechanisms which make an insect  (or plant) pathogen as  selective as we
hope it is.

    It is important that we have addressed these questions today and we
are anxious to have the candidates  available for use.  Society demands
a  cautious approach to all  potential hazards and takes  a dim view of
providing 200 million human guinea  pigs.  Large  commercial endeavors


are expected to occur only in 3~5 years and this time should be used to
gain progressively more knowledge concerning the safety as well as ef-
ficacy of microbial pathogens.  More interest and collaboration in this
area of pest management must be stimulated, and this can best be done
by supporting applied and basic research, by conducting symposia and
workshops but more importantly by cooperative efforts on the national
(USDA-EPA-lndustry) as well  as international (WHO/FAO) level.

                        CONCLUDING REMARKS

                           Dr. John Buckley
        Office of Research and Development, Program Integration
                    Environmental Protection Agency
                            Washington, D.C.

    Really, the only reason I  accepted  this was that it gives me a
chance to say something after  Bill  Upholt.   In all  seriousness, I  sup-
pose  I want to add my thanks but I  want to direct them the other way.
I missed most of this meeting  because I attended another one.  I  heard
enough of the discussions both at the laboratory last  evening and  then
this morning to know that it must have  been an extremely stimulating
time.  I'm sorry indeed that I couldn't have been here for it.  I'd
like to thank each of you, then, for coming and talking and discussing
because it seems to me that several things  seem clear  to me.   One  of
them  is that all of us in the  room, I believe,  have an inherent feeling
that the use of organisms that are already present  in  the environment
to do something that we want done in the environment is likely to  be
safer than the introduction of exotic chemicals to  perhaps do these
same things.  Dr. Upholt touched on this a  bit when talked about the
weighing of benefits and risks.   And the last discussions that just
came up highlighted, again, the  problem of  non-pathogenic or  safe—we
really shouldn't write those words  without  quotes around them.  Patho-
genic to what and with what frequency and under what circumstances?
Or safe,  to what again, to one in a hundred million, to all  but one in
a hundred million,  to half the population?   There are  enormous questions
bound up in this and we try to summarize our thoughts  on it,  we tend to
treat things as absolutes, as  being one way or the  other.  In reality^
the kinds of things we need to deal with are almost all in some kind
of miserable shade of gray that  won't fit neatly into  "safe"  or "unsafe,"
"pathogenic" or "non-pathogenic," or any of these other words we like
to use.   I  was fascinated this morning  by what seemed  to me a very con-
cise state-of-the-art review,  particularly in the insect pathogen  area.
I  know that something is going on in the oil area.   But it seemed  to
me that  discussions of the sort  that you have been  having have been
very worthwhile and let me add my thanks to all that other pile of

                          Sympos iurn-Workshop
                           April 9-11 , 1974
                 Galatea Inn, Pensacola Beach, Florida
Dr. Donald Ahearn
Georgia State University
33 Gilmer St., S.E.
Atlanta, GA  30303
Dr. George Allen
Dept. of Entomology
University of Florida
Gainesville, FL  32601

Dr. Darrel1 Anthony
Insects Affecting Man
  Research Laboratory
P.O. Box 14565
Gainesville, FL  32604

Dr. Ronald M. Atlas
Dept. of Biology
University of Louisville
Louisville, Kentucky 40208

Dr. Andrew Arata
World Health Organization

Mr. J. E. Blair, Manager
Biochemical Corp. of America
Box 808
Salem, VA  24153
Mr. Gerald C. Bower
Bower Industries, Inc.
1601 West Orangewood Ave.
Orange, CA  92668

Dr. John Briggs
Dept. of Entomology
Ohio State University
1735 Neil Avenue
Columbus, OH  43210

Mr. David Brown
University of West Florida
Pensacola, FL 32504
Dr. Lewis R. Brown
Mississippi State University
Box AS
State College, MS 39762

Dr. John Buckley
ORD, Program Integration
U.S. Environmental Protection
Waterside Mall
401 "M" St. S.W.
Washington, DC  20460

Dr. H.  C. Chapman
Avenue J. Chennault
Lake Charles, LA  70601

Dr. R.  Charudattan
Dept.  of Plant Pathology
University of Florida
Gainesville, FL  32601

Dr. Thomas C. Cheng
Dept.  of Biology
Lehigh University
Bethlehem, PA  18015

Mr. Gary ChiIders
Mississippi  State University
State  College,  MS  39762

Mr. T.  B. Clark
Western Insects Affecting Man
  and  Animals Laboratory
5544 Air Terminal Drive
Fresno, CA  93727
Mr.  Wi11 iam Cokee
Nutr i1i te Products,
P.O. Box 98
Lakeview, CA  92353

Mr. Phi 1ip Conklin
University of West Florida
Pensacola, FL  32504

Dr. David Cook
Gulf  Coast Res.  Laboratory
Ocean Springs, MS  39564

Dr. Warren Cook
Georgia  State University
33 Gilmer St., S.E.
Atlanta, GA  30303

Dr. T.  L. Couch
Abbott  Laboratories
Abbott  Park-14
North Chicago, IL  60064

Dr. Sidney Crow
Georgia  State University
33 GiImer St., S.E.
Atlanta, GA  30303
Dr. B. W. Davidson
Division of Agriculture
Arizona  State University
Tempe, AZ  85281

Mr. Rich Dime
University of West Florida
Pensacola, FL  32504

Dr. S. R. Dutky
Insect Physiology Laboratory
Ent.  Bldg. C#467
Beltsville, MD   20705

Dr. A. Emery, Jr.
Office of Naval  Research
800 No.  Quincey
Arlington, VA  22217

Dr. Reto Engler
Environmental Protection Agency
Pesticide Tolerance Division
South Agriculture Bldg.
Washington, DC   20460

Dr. David T.  Gibson
Univ.  of Texas at Austin
Dept.  of Microbiology
Austin,  TX  78712
Dr. A. M. Heimpel
Insect Pathology Laboratory
Room  112, Bldg. A, No. 476
Beltsville, MD  20705

Dr. John E. Henry
Grasshopper Laboratory
Montana State University
Bozeman, MT  59715
Dr. Fred W. Hink
Faculty of Entomology
Ohio  State University
1735  Neil Avenue
Columbus, OH  43210

Dr. Mary Hood
Center for Wetland Resources
Louisiana State University
Baton Rouge, LA  70803
Dr. R. L. Huddleston
Research and Development
Continental Oil  Company
Ponca City, OK  74601

Dr. Carlo Ignoffo
Biological Control of Insects Lab
Box A
Columbia, MO  65201

Dr. Robert B. Jaques
Agriculture Canada
Research Station
Harrow, Ontario NOR  160 CANADA,

Dr. Arthur Kaplan
U.S.  Army Natick Laboratories
Pioneering Research  Labs
Natick, MA  01760

Dr. Donna Kuroda
U.S.  Environmental Protection
ORD,  Program Integration
Room  635 C Waterside Mall
401 "M" St. S.W.
Washington, DC  20460
Dr . Cornel 1  Ladner
Mississippi  State University
State College,  MS  39762

Mr. Marsha]] Laird
Dept. of Biology
Memorial Univ. of Newfoundland
St. John's, Newfoundland CANADA

Dr. P. A. LaRock
Dept. of Oceanography
Florida State University
Tallahassee, FL  32306

Dr. A. I. Laskin
Esso Research & Engrg. Co.
P.O. Box 45
Linden, NJ  07036

Dr. W. W. Leathen
Gulf Research & Development Co.
P.O. Drawer 2038
Pittsburgh, PA  15230

Dr. Morris Levin
National Marine Water
  Quality Laboratory
West Kingston, Rl   02882

Dr. Jul ia Lyle
Gulf Coast Research Lab.
Ocean Springs,  MS   39564

Mr. Joe Maddox
Section of Economic Entomology
Illinois Natural  History Survey
Urbana, IL  61801

Dr. E. M. McCray
Tropical Disease Program
Chamblee, GA  30341

Dr. R. J. Miget
Institute of Marine Science
University of Texas
Port Aransas, TX  78373

Ms. Glennis Mitchel1
Georgia State University
Department of Biology
Atlanta, GA  30303

Dr. Howard Ohr
Biology Control Weeds
Stonevilie, MS 38776
 Dr.  Samuel  P.  Meyers
 Dept. of  Food  Science
 Louisiana  State University
 Baton Rouge, LA   70803

 Dr.  John  Paschke
 Dept. of  Entomology
 Purdue University
 Lafayette,  IN  47904

 Dr.  Walter  H.  Preston
 Watershed  Ecosystems Branch
 R&D  684
 Environmental  Protection Agency
 Washington, DC  20460

 Mr.  R. L.  Raymond
 Sun  Ventures,  Inc.
 1801 Eugene Court
 Wilmington, DE  19810

 Dr.  Roger  Reid
 University of West Florida
 Pensacola, FL  32504

 Dr.  Sammuel F.  Rickard
 Biological Control Insects & Pests
Agricultural Products Division
 Pesticide  Regulation Office
 Upjohn Corporation
 Kalamazoo, Ml  49003
 Dr.  Don Roberts
 Boyce Thompson Institute
 1086 N. Broadway
 Yonkers, NY  10706

 Dr.  Wi11iam G.  Roessler
 Environmental  Protection Agency
 Office of  Pesticide Programs
 Criteria & Evaluation Division
 Room 415
 Washington, DC  20460

 Dr.  Eugene Rosenberg
 Dept.. of Microbiology
 Tel-Aviv University
 Ramat-Av iv
 Tel-Aviv,  ISRAEL

 Dr.  and Mrs. Robert Shapiro
 Eastern Biochemical Laboratories
 159-07 Fourteenth Ave.
 Whitestone, Long  Island, NY 11357

Dr. Sam Singer
Dept. of Biological Sciences
Western Illinois University
Macomb, IL  61455
Mr. Norm Smith
Georgia State University
Department of Biology
Atlanta, GA  30303
Dr. Carl R. Sova
Environmental Protection Agency
Region  IV
1421 Peachtree St., N.E.
Atlanta, GA 30309

Mr. Jack Staton
Biochemical Corp.
Division of Sybron
Salem, VA  24153
Mr. John Sul1ivan
Lehigh University
Bethlehem, PA  18015
Dr. Max Summer
Univ. of Texas at Austin
Austin, TX  78712

Dr. Y. Tanada
Division of Entomology
333 Hilgard Hall
University of California
Berkeley, CA  94720
Ms. Vicki Tayoe
Georgia State University
Department of Biology
Atlanta, GA  30303
Dr. G. E. Templeton
University of Arkansas
Dept. of Plant Pathology
Fayetteville, AR 72701
Dr. Michael F. Terraso
Laboratory Director
Harris Co. Pollution Control
Pasadena, TX  77502

Dr. Richard Traxler
University of Rhode Island
Wakefield, Rl   02881
Dr. Charles P. Truby
Northrup Services,  Inc.
Box 344416
Houston, TX  77034

Mr. Mike Ubanks
U.S. Army Corps of  Engineers
P.O. Box 2288
Mobile, AL  36628

Dr. Al Undeen
Dept. of Zoology
University of 111inois
Urbana, IL  61801

Dr. William Upholt
U.S. Environmental  Protection
Waterside Mall, West Tower,
  Room 1037A
401 "M" Street, S.W.
Washington, DC  20460

Dr. D. D.  Vaishnev
Mississippi State University
State College, MS   29762

Dr. John Walker
Dept. of Biology
University of Maryland
College Park, MD  20742

Dr. Wi11iam Walker
Gulf Coast Research Laboratory
Ocean Springs, MS   39564

Dr. Robert K. Wash!no
Dept. of Entomology
Univ. of California at Davis
Davis, CA  95616

Dr. Douglas Worf
Environmental Protection Agency
Research Triangle Park, NC 27711

Dr. David  C. White
Florida State University
Dept. of Biological Science
Tallahassee, FL  32306

Dr. J. E.  Zajic
Faculty of Engineering Science
The University of Western Ontario
London, Ontario N6A 3K7 CANADA

    U.S. Environmental Protection Agency
Gulf Breeze Environmental  Research Laboratory
           Gulf Breeze, FL  32561
               Dr. Thomas Duke
             Dr. Al  W. Bourquin
             Dr. Del Wayne Nimmo
               Dr. John Couch
               Dr. Pete Schoor
              Dr. Nelson Cooley
             Mr. Terry Hoi 1ister
              Ms. Linda Kiefer
             Ms. Gerta Guernsey
              Mr. Scott Cassidy
             Ms. Chiara Shanika
              Mr. Lee Courtney
               Mr. Gerrit Nudo
                Mr.  Joel  Ivey
              Ms. M. Sandra  Gay

                    1. Report No.
  Title and Subtitle
       Impact of the Use of Microorganisms on the
       Aquatic Environment
                    3. Recipient's Accession
                                                                    5. Report Date
                                                                    December* 1974
                             Samuel P.  Meyers  Donald G.  Ahea
                             Louisiana  State U.   Georgia State 13
. Author(s) Al W.  Bourquin
                   t8fl Performing Organizatio
                      N°'GBERL 235
 . Performing Organization Name and Address
       U. S.  Environmental Protection Agency
       Gulf Breeze Environmental Research Laboratory
       Sabine Island
       Gulf Breeze, Florida 32561	
                                                                    11. Contract/Grant No.
12. Sponsoring Organization Name and Address
                                                                    13. Type of Report & Peri

                                                                    Final-FY 7 4
 15. Supplementary Notes
 Proceedings of Workshop-Symposium held at Pensacola Beach,  FL, April,  1974
     stracts rjjj^g  repOrt contains the  proceedings of a symposium-workshop sponsored
 by the EPA Gulf Breeze, Environmental Research Laboratory  to determine the possible
 impact of artificially introducing  microbial insect control agents or oil-degrad-
 ing agents into the aquatic environment.   The efficacy and safety testing,  especi-
 ally against non-target aquatic organisms, for use of bacteria, viruses, fungi,
 and protozoa to control aquatic insect pests is discusse4 with remarks of panel
 members representing government, academia, and industry.   Special attention is
 given to persistence of pathogens in aquatic environments as well as control of
 aquatic weeds and  other non-insect  pests.
      The use of microorganisms to clean up oil spills in  aquatic environments is
 discussed by industrial, academic,  and governmental scientists.  Special consider-
 ations are given to selection of hydrocarbonoclastic microorganisms and use of
 these microorganisms in special environments—Arctic regions and Louisiana  salt
 marshes.                         	    .	,	
 17. Key Words and Document Analysis. 17a. Descriptors
      Summary papers  are presented for each panel concerned with microbial pesti-
cides and one summary for the  session on microbial  degradation of oil.
bibliographies are presented with each paper and  discussion.

17. Ke.y WoAdf> and Document.
hydAoc.aA.ba.no c£aA£ic.
aActic tn.viA.ome.nt
 17b. Identifiers/Open-Ended Terms
               In&tat ContAol Jin Aquatic. Sy*tm&
     Hydn.ocaA.bon dtgAading
17c. COSATI Field/Group
18. Availability Statement

     Release unlimited
                                                         19. Security Class (This
                                                         20. Security Class (This
                                                            P-. S. GOVERNMENT PRINTING OFFICE: 1975-697-939/88 REGION 10
                                                                               IJSCOM'M- DC