United Stales
Environmental Protection EPA/600/4-89/027
Agency June198S
^EPA Research and
Development
AN ENCLOSED AQUATIC MULTISPECIES
TEST SYSTEM FOR TESTING MICROBIAL
PEST CONTROL AGENTS WITH NON-
TARGET SPECIES
RESEARCH PROJECT REPORT
Prepared by
Environmental Research
Laboratory
Gulf Breeze, FL 32561
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AM ENCLOSED AQUATIC MULTISPECIES TEST SYSTEM
FOR TESTING MICROBIAL PEST CONTROL AGENTS
WITH NON-TARGET SPECIES
by
Donald V. Lightner1, R.B. Thurman2
and Bronven Trumpery-
Environmental Research Laboratory
University of Arizona
2 601 East Airport Drive
Tucson, AZ 85706
Contract No.: CR-81 41 75-01-0
Project Officers
Dr. John A. Couch and Dr. John w. Fournie
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, FL 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FL 32561
1 Present address: Department of Veterinary Science, College
of Agriculture, University of Arizona, Tucson, AZ 85721
2 Aquinas College, Ballart, Victoria, Australia 3350
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DISCLAIMER
The information presented in this document has been funded in
part by the United States Environmental Protection Agency under
contract number CR-81 41 75-01-0. It has been subject to the
Agency's peer and administrative review, and it has been approved
for publication as an EPA document. Mention of trade names or
commercial products does not necessarily constitute endorsement
or recommendation for use.
wz-Atsiii
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ABSTRACT
An enclosed test system was developed in which multiple
species of aquatic animals and plants were tested experimentally
for adverse non-target effects of wild-type and genetically
altered microbial pest control agents (MPCAs). The test system
consisted of components that were inexpensive and readily
available from aquaculture supply companies or from retail pet
shops that carry tropical fish aquaria and supplies. A variety
of marine and freshwater non-target animal and plant species
(NTOs), representing diverse phylogenetic taxa and trophic
levels, were collected from wild populations or purchased from
commercial suppliers.
Four different types of model MPCAs were tested in the
multispecies system. These included two different strains of the
mosquito pathogen Bacillus sohaericus. a strain of Pseudomonas
putida (used as a model for the genus), and the insect
baculovirus AcMNPV. The fate, persistence, and infectivity of
these model MPCAs were evaluated using traditional ...
microbiological and histological methods, as well as specific
microbiological assays for model MPCAs that were altered by
addition of a unique genetic marker. For two of the model MPCAs,
gene probes were used as a detection method to track the MPCA in
the test system water and NTOs.
iii
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ACKNOWLEDGMENTS
This study was supported in part by grant CR-81 41 75-01-0
from the U.S. Environmental Protection Agency. Of the staff of
the Environmental Research Laboratory of the University of
Arizona, we thank R.M. Redman for preparation of histological
materials; R.R. Williams, Dr. B. Dehdashti, B. Grim, and S. Berry
for collection and maintenance of the animal colonies; M. Jackson
and L. Steiner-Bruce for media preparation; K. Wood for
photographic illustrations; Dr. R. Frye for assistance in
statistical evaluation; D. Cross for assistance in culture of
Trichoplusia ni for baculovirus production; and T.A. Bell for
assistance in graphic preparations.
Also acknowledged for their assistance in providing model
MPCAs or related materials and information are: Dr. A. Youston,
Virginia Polytechnic Institute and State University, VA; Dr. W.F.
Burke and L.D. Taylor, Arizona State University, Tempe, AZ; Dr.
-E. Genthner, U.S. E.P.A., Gulf Breeze, FL; Dr. Max Summers, Texas
A&M University, College Station, TX; Dr. P. Vail (U.S.D.A.,
-Riverside, -CA; and Dr. Tom Henneberry (U.S.D.A., Western Cotton
Research Laboratory, Phoenix, AZ).
iv
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TABLE OF CONTENTS
ABSTRACT iii
ACKNOWLEDGMENTS iv
TABLE OF CONTENTS v
FIGURES LIST vi
TABLES Vii
INTRODUCTION 1
MATERIALS, METHODS, AND RESULTS 1
TEST SYSTEMS 1
Test System No. 1 2
Test System No. 2 2
Test System No. 3 2
Test System No. 4 4
Test System No. 5 4
MPCA TESTS AND NON-TARGET TEST ORGANISMS 10
MPCA Tests with Marine NTOs 10
MPCA Tests with Freshwater NTOs 10
Mass Culture of NTO Test Species 10
Feeds and Feeding Methods for NTOs 11
MODEL MPCAS TESTED 15
Spore-Forming Bacillus sohaericus 15
Vegetative Cells of Bacillus sohaericus 15
Pseudomonas putida with Genetic Markers 18
The Nuclear Polyhedrosis Virus AcMNPV 18
CONTAINMENT OF MPCAs IN THE TEST SYSTEMS 19
GENERAL METHODS FOR MPCA TESTS 20
Sampling Methods 21
Water 21
Tissues 21
Histological Samples 22
Method for Obtaining Plasmid DNA for Gene Probes 22
TRIALS WITH MODEL MPCAs 22
Background Studies 22
General Sampling Scheme 22
Trial 1: Bacillus sohaericus 2362 with pLT103; Spores 26
Trial 2: Bacillus sohaericus 1593 with pL117;
Vegetative Cells 27
Trial 3: Baculovirus AcMNPV; Inclusion Bodies 27
Trial 4: Pseudomonas putida PP0200 + pEPA74 34
Trial 5: Bacillus sphaericus 2362? Spores 36
Trial 6: Pseudomonas putida PPO200 + pEPA74 37
Trial 7: Baculovirus AcMNPV; Occlusion Bodies 38
DISCUSSION 45
TEST SYSTEMS 45
NON-TARGET SPECIES 45
MPCAs AND DETECTION METHODS 46
REFERENCES 49
v
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FIGURES
ynTn'bgr Page
1 Schematic of the multispecies test system No. 2 3
2 Schematic of the multispecies test system No. 3 3
3 Schematic of the multispecies test system No. 4 5
4 Photograph of the multispecies test system No. 5
used in the saltwater MPCA trials 5
5 Schematics of the multispecies test system No. 5
used in:
5a. the saltwater MPCA trials 1-5
5b. the freshwater MPCA trials 6-7 6
6 Schematic of the functional arrangement of culture
and MPCA test systems located within the structures
at the University of Arizona's ERL 12
7 Photograph of the culture area in the greenhouse .... 13
8 Photograph of the culture area in the metal building.
The biological filter (far left) supports the round
culture tanks used to rear saltwater NTO species .... 13
9 Light photomicrograph of normal sheepshead minnow
gut showing presumed Bacillus sohaericus spores
and bacilli 31
10 Light photomicrograph of normal snail gut showing
presumed Bacillus sphaericus spores and bacilli 31
11 Light photomicrograph of normal shore fly gut
showing presumed Bacillus sphaericus spores and
bacilli 32
12 Autoradiograph showing the results of the gene
probe assay for the detection of AcMNPV in
seeded and unseeded non-target organism tissues 32
13 Autoradiograph showing the results of the gene
probe assay for the detection of AcJINPV in
non-target organism tissues and tank water for
Trial number 3 33
14 Autoradiograph showing the results of the gene
probe assay for the detection of Pseudomonas
putida in seeded and unseeded non-target organism
tissues and non-target organism tissues from Trial
number 4 35
vi
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FIGURES (continued)
Viimhar Page
15 Graphs representing the microbiological results
for the detection of Pseudomonas putida in
non-target organism tissues from test tanks from
Trial number 6 40
16 Graphs representing the microbiological results
for the detection of Pseudomonas putida in water
samples from the test and control tanks used
in Trial number 6 41
17 Autoradiographs showing the results of the gene
probe assay for the detection of Pseudomonas
putida in non-target organism tissues and tank
water for Trial number 6 42
18 Autoradiographs showing the results of the gene
probe assay for the detection of AcMNPV in
non-target organism tissues and tank water for
Trial number 7 43
vii
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TABLES
Number Page
1. List of marine species of indicator test organisms
used in tests with model MPCAs, their original source,
culturability in the laboratory, and suitability as test
organisms in tests with model MPCAs 7
2. List of freshwater species of indicator test organisms
used in tests with model MPCAs, their original source,
culturability in the laboratory, and suitability as test
organisms in tests with model MPCAs 8
3. List of plant and animal species acquired and tested in
recirculating seawater and freshwater systems as candidate
non-target species in multispecies tests with model MPCAs 9
4. Feeds and feeding methods for non-target test animals .... 14
5. List of model microbial pest control agents (MPCAs) used
in this study 16
6. List of MPCA trials run with marine and freshwater
non-target organisms 17
7. Ranges of environmental parameters in test and control
tanks during each MPCA trial run in seawater 20
8. Ranges of environmental parameters in test and control
tanks during each MPCA trial run in freshwater 21
9. Summary of histological observations of lesions,
parasites, and other anomalies present in the marine
non-target organisms (NTOs) used in MPCA Trials 1-5,
that were unrelated to treatment effects of the MPCAs .... 23
10. Summary of histological observations of lesions,
parasites, and other anomalies present in the freshwater
non-target organisms (NTOs) used in MPCA Trials 1-5, that
were unrelated to treatment effects of the MPCAs 24
11. Dose levels of model MPCAs used in Trials 1-7 25
12. General sampling scheme used in Trails with model MPCAs .. 26
13. Summary of microbiological assays for Bacillus
sphaericus (spores) in Trial 1 28
14. Initial number, observed mortalities, and adjusted percent
survival of selected NTOs following exposure to model MPCAs
in seawater Trials 1-5 and freshwater Trials 6-7 29
15. Summary of observations in which organisms, presumed to
be the MPCA being tested, were observed in histological
sections of marine non-target test organisms 30
16. Summary of microbiological assays for Bacillus
sphaericus (vegetative cells) in Trial 2 34
17. Summary of microbiological assays for Pseudomonas
putida in Trial 4 36
18. Summary of microbiological assays for Bacillus
sphaericus spores in Trial 5 37
19. Summary of microbiological assays for Pseuodmonas
putida in Trial 6 39
20. Summary of observations in which organisms, presumed to
be the MPCA being tested, were observed in histological
sections of freshwater non-target test organisms 44
viii
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PREFACE
Because microbial pest control agents (MPCAs) are widely used
for controlling detrimental insects, weeds, and other pests and may
be applied in large quantities and geographic areas, it is
essential that test data be obtained prior to field application.
This data will help predict the fate and survival of MPCAs in the
environment and their effect on non-target organisms that would be
exposed as a result of normal field application. Information such
as this is even more important when considering the application of
genetically engineered microorganisms (GEMs).
The purpose of this cooperative agreement was to develop a
simple, functionally closed aquatic multispecies test system in
which the study of MPCAs could be accomplished in a manner that
models ecosystems and utilizes representative types of non-target
organisms.
Dr. Lightner developed an enclosed multispecies test system
in which several species of aquatic animals and plants were tested
experimentally for adverse non-target effects of wild-type and
genetically engineered MPCAs. Both marine and freshwater non-
target organisms representing diverse phylogenetic taxa and trophic
levels were utilized in tests with four different types of model
MPCAs. The fate, persistence, and infectivity of these MPCAs were
evaluated experimentally using traditional microbiological and
histological methods. Additionally, gene probes were used as a
detection method to track genetically altered MPCAs in the test
system water and non-target organisms.
Our role in this project was to provide guidance concerning
the objectives of the research and to be involved with the detailed
planning and review of the research. Additionally, we provided
collaborative interpretation and evaluation of experimental results
which allowed us to compare, contrast, and validate any effects in
non-target species. Some of the non-target organisms used in
testing as well as one of the model MPCAs were provided by GB/ERL
scientists.
It is important to know the fate and/or persistence of the
intact viable MPCA itself, as well as the fate of its genetic
material. Data obtained from this project directly relates to our
ongoing in-house research program which is primarily concerned with
development of methods to determine the effects of MPCAs on non-
target, aquatic species utilizing the endpoints of infectivity,
toxicity, and pathogenicity. It also provides us information that
will be useful in some of our future research that will be
specifically concerned with determining the fate and survival of
bacterial MPCAs in aquatic test systems and the development of
specific methods for the analysis of test system water, sediment,
and non-target organisms.
ix
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INTRODUCTION
Microbial pest control agents (MPCAs), also known as
biological control agents or "biorationals," are microbial agents
intended for use in controlling detrimental insects, weeds, and
other pests (Couch and Rao, 1983; Couch and Martin, 1984).
Because they may be applied in rather large quantities (Mulligan
et al., 1980) or repeatedly applied in. smaller quantities to
areas outside the normal geographic range of the wild type
pathogen (Davidson et al., 1984), it is important that test data
be obtained, prior to field application, which will help to
predict the fate and persistence of MPCAs in the environment and
their effect on non-target organisms that would be exposed to a
given MPCA as a result of normal field application. Acquisition
of such information becomes even more important when the
application of genetically altered MPCAs is considered
(Environmental Protection Agency, 1982).
Controlled tests are clearly needed in which wild-type and
genetically altered MPCAs are tested in self-contained systems in
the laboratory with organisms representing species which are not
pests (non-target organisms, NTOs), but are likely to become
exposed as a result of the field application of MPCAs. Data from
these controlled laboratory studies (which approach, but cannot
mimic, actual field conditions) may be used as guidelines for
further studies, the goals of which will be used to assess the
safety and/or hazards of proposed future uses of genetically
altered MPCAs.
The purpose of the project reported here was to develop a
functionally closed aquatic multispecies test system in which the
study of MPCAs could be accomplished in a manner that models an
ecosystem (Lundgren, 1985) and utilizes as many different, yet
readily available types of NTOs as possible. Therefore, the two
major objectives of the study were: 1) to develop a relatively
simple, easily replicated, and inexpensive recirculating tank
system in which multiple species of aquatic (estuarine, marine,
or freshwater) animals and plants could be maintained in direct
or indirect contact with each other for extended study periods
with an introduced MPCA? and 2) to conduct studies on the fate
and effects of model MPCAs on the non-target organisms in the test
system.
MATERIALS/ METHODS AND RESULTS
TEST SYSTEMS
Five different enclosed recirculating tank designs were
constructed and tested as potential multispecies test systems for
tests with model MPCAs (wild strain or genetically altered).
1
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Test System No. 1
To provide a standard from which to compare various
configurations of multispecies test tanks and aquaria, a 120 L
glass aquarium was assembled to duplicate as closely as possible
the principal design features of those described by Foss and
Couch (undated, internal EPA report) and by Fournie et al. (1987
and 1988).
Test System No. 2
This experimental test system consisted of a 1,000 L round
fiberglass tank equipped with its own undergravel biological
filter, and six compartments separated by radial partitions
constructed of flat fiberglass panels (Figure 1). Seawater
within the tank could be recirculated through the undergravel
filter through a central sump, or be moved from any compartment
to another via six moveable airlift pumps. A number of the
species in Tables 1 & 2 were maintained successfully in the
system for more than 60 days, in numbers large enough to provide
adequate sample sizes for microbiological and histological
sampling in model MPCA tests. However, the system was found to
have one significant deterrent to its practical use: the animals
contained in it could not readily be inspected or even counted
without literally draining the tank.
Test System No. 3
This test system was designed to incorporate the advantages
of systems No. 1 and No. 2, while eliminating their major
disadvantages. System No. 3 was constructed using a 400 L
commercially available Plexiglass tank. Initially, it was
equipped (Figure 2) with both undergravel and vertical biological
filters consisting of crushed oyster shell. In actual use the
undergravel filter was found to be unnecessary, and in subsequent
tests with this tank, only the vertical biological filters were
utilized. The vertical filters were held in place by fiberglass
window screen mounted on rigid frames made from 6.35 mm thick PVC
flat stock, and these assemblies were held in place in the tank
by pairs of vertical PVC strips glued to the tank walls to form
slots. The vertical filters had two functions: to provide
biological filtration, and to physically separate species in the
tank and thus prevent unplanned predation. Salicornia biaelovii
(an estuarine vascular plant) seedlings planted in these dividers
grew normally.
Airlift pumps and an externally affixed electric pump
provided both aeration and circulation of water through the
vertical biological filter matrices. The airlift pumps were
moveable and provided direct mixing of adjacent sections of the
tank. The system performed well and was easily managed; species
reared within it could be easily observed, counted, and sampled;
some test species actually reproduced in the system during the
60-day trial period.
2
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drain line
blofilter
Recirculation pump
surface
Radial
Corrugated fiberglass
1 cm size gravel and oyster shell
Return water line
Airlift pump
Aeration supply line
400 L Plexiglass
Vertical biofilter
and partition
Water level
1000 L Fiberglass tank
\
Air lift
Air supply lines
open sump
tank
Figure 1. Schematic of the multispecies test system No. 2.
Figure 2. Schematic of the multispecies test system No. 3.
3
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Test System No. 4
Despite the advantages of System No. 3, its relatively high
cost limited its practical use. Therefore, Test System No. 4 was
constructed to combine the low cost and component availability
advantages of System No. 1 (the E.P.A. system) with the
functional advantages of System No. 3. This tank system was
constructed using a standard 120 L (30 gallon) glass aquarium.
Parallel strips of 6.35 mm thick PVC flat stock were cemented to
opposite sides of the aquarium using silicone cement (Dow-
Corning1*) . As in System No. 3, the filter matrix was crushed
oyster shell held in place with fiber glass window screen mounted
on a frame of PVC flat stock. One of the principal advantages of
System No. 4 was its vertical biological filter which tended to
remove introduced particulate material (like the model MPCAs or
phytoplankton introduced as food for filter feeding mollusks)
much more slowly than does a bottom undergravel filter. Another
advantage the vertical filter provided was physical separation of
potential predators from prey species, thereby eliminating the
need for several separate containment structures.
Test System No. 5
This system was identical to System No. 4, except that its
biological filter matrix consisted of a polyester fiber pad
(purchased as bulk furnace air filter material) in place of the
crushed oyster shell (Figures 3 & 4). The advantage of the
polyester filter over crushed oyster shell was that the former
could be easily removed, cleaned, and replaced without draining
the tank. The pH of seawater in tanks with polyester pad filters
was maintained by suspending 1 L plastic beakers with perforated
bottoms and half filled with crushed oyster shell or dolomite
under the outlet of an airlift pump (Figures 4 & 5).
A further slight modification was made to eliminate the
unanticipated problem of the non-target test animals becoming
trapped in the biological filter. A perforated plastic sheet
(made from undergravel aquarium filter kits) was installed on
both sides of the vertical polyester pad filter (Figure 5) * This
prevented the filter pad from sagging and thus prevented the
experimental animals from becoming trapped between the sides of
the tank and the biofilter, while still allowing free flow of
tank water through the biofilter. Because of the functional
simplicity and relatively low costs of the original and modified
System No. 5 tank type, this system was used in all tests with
model MPCAs.
4
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1 cm Oyster s
Aeration supply line^ Airlift pump
120 L
Vertical biofilt
Figure 3. Schematic of the multispecies test
Figure 4. Photograph of the multispecies test
used in the saltwater MPCA Trials :
5
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SALTWATER
AIR LINES
Intertidol holophyte
Sbfaoorriid biqdovir
Shore fly
Ephvdrq so,
SeaAnamona SHELL
Bunodosoma cqrrfbrnkt
Sheepshead minnow
Cyprinodorr vqr'tegatua
FLOSS FILTER
Estuarins gross shrimp
Fqlqemorretes puqio
AIR LINES
Anacharis plan
EJodeq canadensis
FRESHWATER
Tubifex worm
Tubifex tubife-
OYSTER
SHELL
Snail
Gyraulus
tIESH
BASKET
FLOSS FILTER Freshwater
grass shrimp .
Freshwater mussel Pqtqgmo1etes kod!qk"n51-
Marqaritifera margon'tifera If
Sallfin molly
Foeciliq kitipinna
Figure 5. Schematics of the multispecies test system No. 5 as
used in:
5a. the saltwater MPCA Trials 1-5.
5b. the freshwater MPCA Trials 6-7.
6
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Table 1. List of marine species of indicator test organisms used
in tests with model MPCAs, their original source, their
culturability in the laboratory, and thier suitability
as test organisms in tests with model MPCAs.
Marine Species
SourceJ
Lab Culture
Test Organism
Suitability
PLANT
Salicornia biqelovii
(intertidal halophyte)
INVERTEBRATE ANIMALS
Bunodosoma californica
(sea anemone)
Turbo fluctuosus
(turbin snail)
Crassostrea qjqas
(Japanese oyster)
Palaemonetes puaio
(estuarine grass shrimp)
Ephvdra sp.
(shore fly)
VERTEBRATE ANIMAL
Cvprinodon varieaatus
(sheepshead minnow)
Gulf of CA
Gulf of CA
Gulf of CA
Gulf of CA
Florida
Gulf of CA
Florida
RP
RP
CW
PS
RP
RP
Poor
Fair
Excellent
Excellent
Excellent
Fair
RP
Good
1 Source: Gulf of CA collection sites near Puerto Penasco in
Sonora, Mexico, on the Northern Gulf of Mexico.
2 Lab culture: RP = reproducing laboratory colony established.
CW = captive wild colony successfully maintained in lab.
PS = experimental animals purchased from a commercial supplier and
maintained in lab.
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Table 2. List of freshwater species of indicator test organisms
used in tests with model MPCAs, their original source,
their culturability in the laboratory, and thier
suitability as test organisms in tests with model MPCAs.
Test Organism
Freshwater Species Source Lab Culture1 Suitability
PLANTS
Elodea canadensis
(Anacharis plant)
INVERTEBRATE ANIMALS
Tubifex tubifex
(annelid worm)
Gvraulus sp.
(snail)
M. maraaritifera
(freshwater mussel)
Commercial
PS
Excellent
Commercial
Arizona pond
Commercial
£. kadiakensis Commercial
(freshwater grass shrimp)
VERTEBRATE ANIMALS
Poecilia latipinna
(sailfin molly)
Hawaii
PS
RP
PS
RP/PS
RP
Fair
Good
Excellent
Good
Excellent:
1 Lab culture:
RP = reproducing laboratory colony established.
PS = experimental animals purchased from a commercial
supplier and maintained in the laboratory.
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Table 3. List of plant and animal species acquired and tested in
recirculating seawater and freshwater systems as
candidate non-target species in multispecies tests with
model MPCAs.
Species
Category
Habitat
Source
PLANTS
Enteromorpha intestinalis algae
marine
Salicornia biaelovii
Azolla caroliniana
Limnobium sponaia
Elodea canadensis
vascular plant "
water fern fresh
duckweed "
anacharis plant 11
Gulf of CA
II If It
pond, AZ
(I 19
commercial
INVERTEBRATES
Bunodosoma californica
Ceratonereis mirabilis
Sipunculus nudus
Tubifex tubifex
Gvraulus sp.
anemone
annelid worm
It II
tl
It
snail
Maraaritifera marqaritifera mussel
Turbo fluctuosus
Cerithidea mazatlanica
Crassostrea aigas
Palaemonetes pugio
Palaemonetes kadiakensis
Penaeus stylirostris
Mvsidopsis bahia
Clibanarius dicrueti
Uca crenulata
Ephvdra sp.
snail
snail
oyster
shrimp
hermit crab
fiddler crab
shore fly
marine
II
tl
fresh
tt
II
marine
it
it
marine
fresh
marine
I
H
I -
marine
Gulf of CA
commercial
pond, AZ
commercial
Gulf of CA
Florida
commercial
Gulf of CA
Florida
Gulf of CA
it ii ii
Gulf Of CA
VERTEBRATE ANIMALS
Cvprinodon varieqatus
Poecilia latioinna
Gambusia affinis
Notropis lutrensis
minnow
molly
mosquito fish
red shiner
marine
fresh
Florida
Hawaii
commercial
9
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MPCA TESTS AND NON-TARGET TEST ORGANISMS
A variety of marine, estuarine and freshwater vertebrate and
invertebrate animals and plants were acquired for potential use
as non-target test organisms (NTOs) in model MPCA tests. The
selection criteria for potential NTOs were that they should be
common and readily available from cultured laboratory stocks,
commercial suppliers, or from easily accessible wild populations.
An effort was made in selecting plants and animals for use in the .
multispecies test system that represented diverse phyla and that,
in the case of the animal species, represented different levels
in the food web. The marine, estuarine, and freshwater species
acquired and evaluated as candidate NTOs with model MPCAs are
listed in Table 3. Those species found to be suitable for
testing are listed in Tables 1 and 2.
MPCA Tests with Marine NTOs
The estuarine and marine NTOs used in MPCA Trials 1-5 were:
the estuarine vascular plant, Salicornia biaelovii; the snail,
Turbo fluctuosus; larvae of the shore fly, Ephvdra sp.; the sea
anemone, Bundosoma californica: the oyster, Crassostrea aiaas:
the grass shrimp, Palaemonetes puaio; and the sheepshead minnow,
Cvprinodon varieaatus (Table 1). Those species not readily
available from commercial suppliers or from laboratory cultures
were collected from wild populations along the Gulf of Mexico
coast of Florida and from the Northern Gulf of California.
The saltwater plant (S. biqelovii) was supported in
hydroponic culture on a styrofoam raft floated in the test tank
seawater with its roots hanging free in the water. Juvenile sea
anemones from their mass culture tank were allowed to grow on
oyster shells, which were transferred into the test tanks. The
shore fly larvae were confined to a net bag suspended in the test
tank water.
MPCA Tests with Freshwater NTOs
The freshwater NTOs used in tests with model MPCAs included
the freshwater anacharis plant Elodea canadensis, the annelid
worm Tubifex tubifex. the snail Gvraulus sp., the freshwater
mussel Maraaritifera maroaritifera. the grass shrimp Palaemonetes
kadiakensis. and the molly (a finfish) Poecillia latipinna. All
of the freshwater NTO species were acquired from commercial
sources (Table 2), with the exception of the snail (Gvraulus sp.)
and the molly (P. latipinna). The snail was collected from a
fish culture pond in Southern Arizona, and the molly was
originally collected from estuarine drainage ditches on the
Island of Oahu, Hawaii.
Mass Culture of NTO Test Species
Culture and holding facilities for laboratory colonies of the
marine and freshwater NTO species consisted of four totally self
contained, recirculating multiple tank systems located in a
10
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temporary sheet metal building and a plastic covered greenhouse
on the grounds of the University of Arizona's Environmental
Research Laboratory (Figure 6).
Specifically, each culture system consisted of three to four
1,500 L cylindrical fiberglass tanks that were plumbed with
center drains. Each tank's center drain was fitted with a
screened vertical stand pipe to regulate water depth and to
prevent escape of the cultured species. Drain lines from tank
sets were connected to large diameter PVC pipes through which
water flowed by gravity to 4,000 L concrete water storage sumps
(two below ground level septic tanks located just outside the
metal building). Water from the sumps was pumped to 1,000 L
biological filters with 1 hp swimming pool-type pumps, and
returned through supply headers to the culture tanks using 0.5 hp
submersible pumps located in the end of each of the biological
filter boxes opposite the water inlet (Figures 6-8).
The biological filters were fabricated "in-house" using
plywood construction. Filter boxes were 4 ft by 8 ft by 2 ft
deep. All surfaces were sealed with fiberglass resin and epoxy
paint. Biological filter material consisted of polyester fiber
pads (bulk furnace filter material) held in five vertical rows
perpendicular to the axis of the filter box with heavy plastic
net material in rigid PVC plastic frames. These filter pad units
were held in place in the filter box in flanged slots fastened to
the box sides. Receiving the incoming water from the storage
sump was a 120 L polyethylene container (a trash can) filled with
a mixture of crushed oyster shell, dolomite, and removable filter
bags of activated charcoal. The oyster shell and dolomite
functioned to regulate pH and alkalinity, while the charcoal was
used to remove dissolved organic pigments and other dissolved
organic materials from the system.
Feeds and Feeding Methods for NTOs
Marine and freshwater NTOs in mass rearing tanks and in the
MPCA test tanks were fed once per day with live food organisms,
frozen, or artificial feeds that were consistent with their
feeding behavior and known nutritional requirements (Table 4).
Thus, filter-feeding mollusks were fed cultures of planktonic
algae once per day (Walne, 1974), finfish and grass shrimp
received chopped frozen squid, artemia nauplii and a commercial
flake food daily, etc. NTO species that were not fed directly
included the aquatic plants (Salicornia and Elodea), the snails,
the tubifex worms, and the shore fly larvae. Except for
Salicornia. all experimental NTOs appeared to do well (in terms
of feeding activity, survival, general appearance, and
histological condition) in test tanks and in mass culture tanks.
Apparently, the cultural requirements for Salicornia were not met
with the hydroponic culture method employed for culturing this
plant in the MPCA test tanks. The plant did very well in those
culture systems (Test Tank Systems No. 3 and No. 4) where it was
cultured rooted in sand or oyster shell biological filter
substrates, but it did poorly in hydroponic culture.
11
-------
GREENHOUSE
METAL BUILDING
Figure 6. Schematic of the functional arrangement of culture and
MPCA test systems located within the structures at the
University of Arizona's ERL.
12
-------
Figure 7. Photograph of the culture area in the greenhouse.
Figure 8. Photograph of the culture area in the metal building.
The biological filter (far left) supports the round
culture tanks used to rear saltwater NTO species.
13
-------
Table 4. Feeds and feeding methods for non-target test animals
NTO Species^ Food(s)
Feeding
Frequency
Sea anemone (M)
Turbin snail (M)
Japanese oyster (M)
Grass shrimp (M)
Shore fly larvae (M)
Sheepshead minnow (M)
Tubifex worm (FW)
Snail (FW)
Freshwater Mussel
Grass Shrimp (FW)
Sailfin Molly (FW)
artemia nauplii daily
natural epiphytes, detritus
phytoplankton mixture2: daily
Isochrvsis sp.,
Tetraselmis sp., and
Chaetoceros sp.
Basic Food3, chopped squid daily
and artemia nauplii
natural microflora
Basic Food, chopped squid daily
and artemia nauplii
natural microflora
natural epiphytes, detrtius
Chlorella pvrenoidosa daily
Basic Food, chopped squid daily
and artemia nauplii
Basic Food, chopped squid daily
and artemia nauplii
* M = a marine species
FW = a freshwater species
2 Marine phytoplanktonic species were fed in approximately equal
amounts daily. These were grown separately in mass unialgal
culture using standard algae culture methods as found in
manuals for mollusk culture.
3 Fish Basic Food, Worldwide Aquatics, Gardenia, CA
14
-------
MODEL MPCAS TESTED
Four "model" MPCAs were tested with marine and freshwater
NTOs in our test system. Used in these studies were three
bacterial MPCAs (a spore forming Bacillus, a vegetative form of
Bacillus, and a Pseudomonas), and a nuclear polyhedrosis virus
(Tables 5 and 6).
Spore-Forming Bacillus sphaericus
Spores of Bacillus sphaericus (modified strain 2362; provided
by Dr. A. Youston, Virginia Polytechnic Institute and State
University, VA and Dr. William F. Burke, Arizona State
University, Tempe, AZ) containing the plasmid pLT103 (which
encodes for neomycin resistance) were used in Trials 1 and 5.
Many strains of B. sphaericus are known to possess insecticidal
activity against mosquitoes (Brownbridge and Margalit, 1987;
Davidson, 1981; Davidson et al., 1984). This strain of £.
sphaericus is being developed commercially for use as a mosquito
larvacide (Youston, pers. comm.). The bacillus was also naturally
resistant to streptomycin, and media supplemented with neomycin
and streptomycin provided an excellent method to selectively
isolate and culture the microorganism. Tryptose blood agar base
(TBAB, Difco Laboratories, Detroit, MI), supplemented with 5
ug/ml neomycin (neomycin sulfate, Sigma Chem. Co., St. Louis, MO)
and 100 ug/ml streptomycin (streptomycin sulfate, Sigma Chem.
Co., St. Louis, MO), was used to produce the cultures of the
bacillus from which spores were harvested.
Vegetative Cell3 of Bacillus sphaericus
Vegetative cells of B. sphaericus (modified strain 1593,
thymine deficient; provided by Lisa D. Taylor and Dr. William F.
Burke, Arizona State University, Tempe, AZ) harboring the plasmid
pLTH7 were used in Trial 2. The plasmid (pLT117), a ligation
product of pTG402 and pUBHO, encodes for neomycin resistance,
and it contains the xylE gene. The xylE gene (Zukowski et al.,
1983) expresses catechol 2,3-dioxygenase which converts catechol
(0.5M, Sigma Chem. Co., St. Louis, MO) from colorless to a. yellow
product (2-hydroxymuconic semialdehyde) within a few minutes when
sprayed onto growing colonies. Culture methods for this organism
consisted of supplementing TBAB agar plates with Neomycin (at 5
ug/ml and thymine at 50 ug/ml). After overnight incubation,
plates were sprayed with catechol solution and observed for
yellow colonies. Because this Bacillus strain was a poor spore
former, no effort was made to produce spores.
15
-------
Table 5. List of model microbial pest control agents (MPCAs) used
in this study.
MPCA
Plasmid
in MPCA
Methods of
Detection
Plasmid for
Gene Probe
Bacillus pLT103
sphaericus
- spores
- strain 2362
- streptomycin R
Bacillus
sphaericus
- veg. cells
- strain 1593
Pseudomonas
putida
- PP0200
- NX R
AcMNPV
(baculovirus)
- occlusion
bodies
pLT117
- Neo R
- Xyl E
gene
pEPA7 4
- Kana R
- 400 bp
insert of
plant DNA
n/a
Culture on TBAB
plates with:
- neomycin 5ug/ml
- strep lOOug/ml
Culture on TBAB
plates with:
- neomycin 5ug/ml
- thymine 50 ug/ml
Yellow w/catechol
Culture on Pseudo
F plates with:
- Nx 500ug/ml
-Kana 150ug/ml
Gene probe
Gene probe
n/a
n/a
pEPA90
- in E. coli
Ac 80
- 400 bp
plant DNA
- in £. coli JM83
JM83 (a pUC18
plasmid with a
1000 bp insert
from AcMNPV)
Abbreviations used in Table:
Strep = streptomycin
R = resistant
Kana = Kanamycin
Nx = nalidixic acid
TBAB = tryptose blood agar base
LB = Luria-Bertani culture media (Maniatis et al., 1982).
16
-------
Table 6. List of MPCA trials run with marine and freshwater
non-target organisms.
Trial Test
Number System
MPCA Tested
(form used)
Principal
Detection Method(s)
Marine B. sphaericus 2362
(spores)
Unique genetic markers:
(resistance to neomycin
and streptomycin)
Spore formation
Marine B. sphaericus 1593;
(vegetative cells)
Marine AcMNPV baculovirus
(polyhedra)
Marine P. putida PP0200
+ pEPA74 + Nxr
5 Marine fi. sphaericus 2362
(spores)
6 Freshwater £. putida PP0200
+ pEPA74 + Nxr
7 Freshwater AcMNPV baculovirus
(polyhedra)
Unique genetic marker:
(microbiological assay for
catechol 2,3-dioxygenase)
Gene probe to the gene for
polyhedrin
Unique genetic markers:
(resistance to nalidixic
acid & kanamycin)
Gene probe
Unique genetic marker:
(resistance to neomycin
and streptomycin)
Spore formation
Unique genetic markers:
(resistance to nalidixic
acid & Kanamycin)
Gene probe
Gene probe to the gene for
polyhedrin
17
-------
Pseudomonas putida vith Genetic Markers
For Trials 4 and 6 vegetative cells of a genetically altered
strain of Pseudomonas putida were provided by Dr. Fred Genthner,
U.S. E.P.A., Gulf Breeze, FL. While this species of Pseudomonas
has no uses presently as an MPCA, it was selected for use as a
model MPCA for other members of the genus which are being
developed for such use. This strain had been modified from the
parent strain PP0200, by transformation with the plasmid pEPA74.
The plasmid was constructed by inserting the pUC19 multiple
linker sequence and a piece of plant DNA (approximately 400bp)
into a plasmid pKT23 0 which contains Kanamycin resistance. The
sequence of plant DNA was inserted between an EcoRl site and a
Pstl site. This resulting pseudomonad was mutated in two
separate genes on the chromosome to produce a strain resistant to
high levels of nalidixic acid. The first mutation was made in a
permease gene and the second in a DNA gyrase gene. The resulting
strain is called PPO 220 + pEPA74 + Nx1*. In addition, the 400 bp
of plant DNA was also inserted into a Pstl/EcoRl site on a pUC 18
plasmid and then transformed into £. coli Ac80. This organism
provided plasmid DNA for labeling, which was used as a gene
probe. The genetic manipulations of the bacteria were performed
by Dr. Genthner et al. at EPA Gulf Breeze Laboratory.
Aliquots of the bacteria that had been received from Dr.
Genthner were thawed and plated on Pseudomonas F Agar (PsF)
supplemented with 500 ug/ml nalidixic acid and 150 ug/ml
Kanamycin. The high concentration of divalent cations in PsF
enhances the productions of fluorescein, a yellow pigment. By
using PsF agar, which is a differential medium for fluorescent
pseudomonads, plus a high concentration of nalidixic acid and
Kanamycin in the medium, we were able to selectively isolate and
culture the genetically altered strain of P. putida from the MPCA
test system and NTOs.
The Nuclear Polvhedrosi3 Virus AcMNPV
The nuclear polyhedrosis baculovirus (AcMNPV) from the
lepidopteran Autographa californica was used as the model MPCA in
Trials 3 and 7. Dr. Max Summers (Texas A&M University, College
Station Texas) and Dr. Pat Vail (U.S.D.A., Riverside, CA)
provided the strain of AcMNPV used (Summers and Smith, 1987). In
addition, Dr. Summers provided the JM83 strain of E. coli that
harbors a pUC18 plasmid, which contains a pAC HindV insert (1000
bp) of the central region of the polyhedrin gene of AcJJNPV
(Norander et al., 1983).
Experimental quantities of AcIJNPV were produced from
laboratory-reared colonies of the cabbage looper moth
Trichoolusia nl: the original moths were provided by Dr. Tom
Henneberry (U.S.D.A. Western Cotton Research Laboratory, Phoenix,
AZ). Stock colonies of the moth were cultured under ambient
laboratory conditions as follows: Adult cabbage loopers were held
in 4 L glass jars with gauze tops until mating and egg laying had
occurred. Larvae that emerged from the egg deposits were
18
-------
collected and transferred to trays of an autoclaved lima bean
diet (modified from Patana, 1969) supplemented with a mold
inhibitor (0.3% propionic acid), and covered with a plastic film
to prevent dehydration. After completion of the larval stages,
pupae were collected from the trays and transferred to the 4 L
jars to complete a colony culture cycle.
Larvae used to produce AcMNPV virus polyhedra were infected
with the virus by addition of AcMNPV polyhedra to the lima bean
culture media on day 6 (from egg). Day 15 larvae showing gross
signs of heavy infection were collected. AcMNPV polyhedra were
purified by homogenization, filtration, and centrifugation
(Shapiro, 1981). Purified polyhedra were stored at 4 °C or at
-80 °C until used in subsequent MPCA trials.
CONTAINMENT OF MPCAs IN TEE TEST SYSTEMS
Figure 6 shows a schematic of the metal building in which the
studies with model genetically altered MPCAs were conducted. The
building is located at the extreme eastern end of the
Environmental Research Laboratory grounds and is isolated from
other experimental buildings by at least 30 meters (approximately
100 feet). The building was modified to provide a limited access
"containment" area. A 2.2 m (-8 ft) high wall separated the
experimental half of the building, where MPCAs were tested with
NTOs in glass aquaria, from the entry portion of the building
which also housed two of the four recirculating culture tanks
systems used to culture the laboratory colonies of NTOs. Access
to the experimental side of the building required use of rubber
boots disinfected in a 200 ppm chlorine foot bath (prepared fresh
every seven days from household bleach solution).
In each MPCA trial six multispecies aquaria were set up in
the experimental half of the building (three test, three
control). It was found that aerosol cross-contamination of the
model MPCA (by Bacillus sphaericus spores) from exposed to
control tanks took place in Trial 1. Cross-contamination was
eliminated in subsequent trials by separating test and control
tanks by at least 3 m (- 10 ft), by installation of covers on the
top of the tanks to contain aerosols, and by separating exposed
and control tanks from each other by a plastic curtain room
divider.
Nets used to remove NTOs from the tanks for sampling purposes
were labeled and dedicated to a particular tank (to reduce
cross-contamination) and disinfected separately in 100 ppm iodine
(polyvinyl providine iodine; Fritz Egg Disinfectant, Fritz Chem.
Co., Dallas, TX). The floor was mopped with 100 ppm pvp iodine
periodically to further reduce the risk of cross-contamination. A
common 5000 liter concrete sump (a modified septic tank) received
waste water from both halves of the building. Water contained in
the sump was continuously chlorinated (to > 20 ppm chlorine)
using a floating swimming pool chlorinator and chlorine tablets,
prior to periodic disposal when the sump had been filled to
capacity.
19
-------
GENERAL METHODS FOR MPCA TESTS
For all trials with model MPCAs, six of the Test System Type
5 (self-contained 120 L glass tanks: three test and three
control) were used. Each of three replicate test and control
tanks were stocked with 15 NTOs of each animal species. This
provided a total of 45 animal NTOs of each species in test and
control treatments of each MPCA test. This number was sufficient
to provide for microbiological and histological sampling, as well
as to allow for some loss due to possible natural and/or
treatment related mortalities, while still providing for
statistical confidence in data evaluation.
The biological filters in each tank were preinoculated with a
commercial preparation of nitrifying bacteria (Aqua-Gold, LaMonte
Environmental Technology, Saticoy, CA), or by addition of filter
matrix material from "mature" functioning filters. This insured
that the biological filters were functional when the test
organisms (the NTOs and the MPCA) were introduced. Artificial
seawater (Forty Fathoms, Marine Enterprises, Towson, MD) was used
in Trials 1 through 5; city tap water was used to make up the
artificial seawater. City tap water was used directly in the
freshwater Trials 6 and 7. Salinity, pH, ammonia (Kordon Aquatru
Water Test Kit, Hayward, CA), nitrite (TetraTest Nitrite,
TetraWerke, Ulrich Baensch, West Germany), and alkalinity (Model
AL-AP Test Kit, Hatch Co., Loveland, CO) of the tank water were
monitored and maintained (by partial water exchanges,
manipulation of feeding rate, use of room space heaters/coolers,
etc.) within the limits listed (Tables 7 and 8).
Table 7. Ranges of environmental parameters in test and control
tanks during each MPCA trial run in seawater.
Uater Temp. Salinity Ammonia Nitrite Total Alkalinity
Trial (°C) pH (ppt) (mg/L) (rib/I) (mg/L CaCOj)
1
19.5-22.0
7.9-8.0
25-27
0
0.15-0.6
2
23.5-26.0
8.2-8.4
24
0-0.2
0-0.1
205-221
3
21.0-24.0
7.9-8.2
25-28
0.1-0.2
0.1-0.4
187-255
4
22.5-25.0
8.2-8.5
24-27
0-0.8
0.1-2.5
157-255
5
21.0-23.0
7.8-8.7
25-32
0-0.2
0.1-0.25
119-306
BSSSSi
ns:38S8=aaa
N
n
N
u
II
II
u
n
N
It
II
U
N
II
II
II
N
n
N
N
a
:SS3838S838>i:
S8S8383SS388888888881
20
-------
Table 8. Ranges of environmental parameters in test and control
tanks during each MPCA trial run in freshwater.
Trial Water Temp. Disotved Amnonia Nitrite Total Alkalinity Lighting
(°C) pH Oxygen (ms/L) (mg/L) (mg/L) (mg/L CaCOj) (micro E)
6 18.5-23.0 7.2-8.2 5.5-11.0 0 0.1-0.5 136-170 0.45-2.4
7 18.0-24.5 7.6-8.9 5.2-6.8 0-2.i 0-0.5 136-187 1.2-3.0
Hampi-inq Methods
Water-
Sterile pipets were used to collect approximately 5 ml of
water from each control and test tank. The water was placed into
sterile plastic tubes and kept on ice until it was brought to the
lab. For microbiology assays, 0.1 ml was dropped into the middle
of the appropriate media plate, spread with a sterile "hockey
stick" (a bent glass rod) and placed in an incubator (30°C for P.
putida and 37°C for £. sphaericus). For the B. sphaericus
vegetative cell study, the plates were sprayed with catechol
following overnight incubation. For gene probe assays, the water
was stored at -20°C until the gene probe assay was performed.
Tissues
NTOs were placed in plastic bags on ice once removed from the
experimental tanks. At the lab, the organisms were surface
sterilized by soaking them in Fritz's egg disinfectant (a 10%
iodine solution; Fritz Chemical Co., Dallas, TX) for
approximately 5-10 min. Oysters, mussels, snails and fish were
scrubbed with a brush before being washed twice in sterile
distilled water. The remaining organisms were also washed two
times with sterile distilled water.
For the shrimp, fish, oysters, and mussels, the gills,
intestines, and pieces of muscle were aseptically removed and
placed in a sterile blender containing 20 ml of 0.01 M Tris
buffer, pH=7.0. The tissues were then homogenized for 30 seconds.
Plants, worms, shorefly larvae, and anemones were homogenized by
hand using tissue homogenizers. One hundred microliters of each
resulting homogenate were plated on duplicate plates of the
appropriate media, and spread with a sterile hockey stick. The
plates were then incubated. The remainder of the sample was
stored at -20°C for subsequent gene probe assays or for storage.
For the gene probe assay, 0.5 ml of each sample was added to 0.5
ml deionized formamide and incubated for 30 min at 80°C to
liberate nucleic acid. The samples were then applied to a Gene
Screen plus hybridization membrane, baked at 80°C for 2 hr,
prehybridized, hybridized, washed and placed on X-ray film to
produce an autoradiogram ("blot").
21
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Histological Samples
Samples for histological examination were preserved in
Davidson's acetic acid formalin alcohol fixative (Humason, 1972)
for 24 to 76 hr, transferred to 50% ethanol for storage, and
later processed and examined using routine histological methods
(Luna, 1968) . Mayer's hematoxylin and phloxine/eosin stain
(Sheehan and Hrapchak, 1980) was used for all NTO specimens. In
addition, Brown and Brenn tissue Gram stain (Luna, 1968) was used
in those trials in which bacterial model MPCAs were used.
Method for Obtaining Plasmid DNA for Gene Probes
For the studies using the MPCAs AcMNPV and P. putida (Tables
5 and 6), it was necessary to harvest plasmid DNA for labeling as
gene probes. Both plasmids originated from the pUC 18 family of
plasmids and were placed in E. coli strains, and both plasmids
contained an Ampicillin resistance gene. To harvest large
amounts of the DNA, the appropriate £. coli was grown in LB media
supplemented with 30-50 ug/ml Ampicillin. Overnight cultures
were pelleted, the bacteria washed and the plasmid was isolated
using the alkaline lysis procedure (Maniatis et al., 1982).
TRIALS WITH MODEL MPCAs
Background Studies
Background studies were conducted before beginning trials
with model MPCAs in the marine multispecies test tanks. The NTOs
were examined histologically to determine what major parasites
and/or obvious infectious pathogens might be present (and later
encountered in trials with model MPCAs). Tables 9 and 10 list
those parasites, pathogens, symbionts and lesions found to occur
naturally in the marine and freshwater NTOs. In addition to the
histological studies, background microbiological and gene probe
studies were run with NTO tissue homogenates prepared in
phosphate buffer (0.17 M KH2P04, 0.7 M K2HP04, Mallinckrodt,
Inc., Paris, KY); test tank seawater was split: half was seeded
with the model MPCA, while the other half (controls) were not
seeded. Recovery of the model MPCAs from the tissue homogenates
and tank water was attempted to insure that no interfering
substance would cause false positives or negatives.
General Sampling Scheme
Three test tanks were inoculated on day 0 of a planned 28 to
30 day study with the model MPCAs to dose levels listed in Table
11. The water was then mixed for 5 min, and water samples were
taken from each tank to determine initial concentration of
recoverable model MPCA. At predetermined time intervals
throughout the study, water and NTO samples were collected for
microbiological and histological analyses (Table 12).
22
-------
For the trials using B. sphaericus spores, a large quantity
of spores was received from Dr. A. Youston and stored at 4°C.
The spore content of this stock was titered by plate count on
TBAB plates containing 100 ug/ml streptomycin and 5 ug/ml
Neomycin. An appropriate volume of spore suspension to achieve
the dose rate representative of that used in field applications
(-105-6 CFU/ml; Davidson et al., 1984) was then added to the test
tanks (Table 11).
Table 9. Summary of histological observations of lesions, parasites,
and other anomalies present in the marine non-target
organisms (NTOs) used in MPCA Trials 1 through 5, that were
unrelated to treatment effects of the MPCAs tested.
Marine NTO Species
Parasites or Lesions Observed
Sea anemone
Turbin snail
Japanese oyster
Estuarine grass shrimp
Shore fly larvae
Sheepshead minnow
Salicornia
None observed.
Rickettsia in gill epithelial cells;
Probable haplosporidan in gills;
Probable coccidian in gut mucosa;
Ciliate protozoan on gills.
Generalized atrophy of tissues in some
batches used.
Gregarines in midgut and hepatopancreas;
Encysted larval cestodes in various
tissues resulting in large granulomas.
None observed.
Microsporidan cysts in brain;
Thyroid goiters;
Gill hyperplasia, clubbing, capillary
aneurysms and lamellear fusion;
Excessive hemosiderin, lipid and/or
glycogen accumulation in liver.
Yellowing, browning and wilting of
hydroponically grown (estuarine plant)
plants.
23
-------
Table 10. Summary of histological observations of lesions, parasites,
and other anomalies present in the freshwater non-target
organisms (NTOs) used in MPCA Trials 6 and 7, that were
unrelated to treatment effects of the MPCAs tested.
Freshwater NTO Species Parasites or Lesions Observed
Tubifex worm Multinucleate protozoan in hemocoel;
Surface fouling by blue green algae;
Large basophilic cytoplasmic inclusion in
some individuals (viral, chlamydial ?).
Snail None observed.
Freshwater mussel
Grass shrimp
Sailfin molly
Massive storage of grey-blue mineral
granules in loose connective tissues.
Parasitic nematode in gut;
Massive systemic infection by a rickettsia
or chlamydia.
Gill epithelium hyperplasia and lamellar
fusion.
Anacharis plant Surface fouling of thallus by blue green
algae, rotifers, diatoms, bacteria, and
ciliated protozoans.
24
-------
Table 11. Dose levels of model MPCAs used in Trials 1 through 7*.
Model MPCA Initial Dose Level
Trial
Number
1 (M) Bacillus sphaericus
spores
2 (M) £. sphaericus
vegetative cells
3 (M) AcMNPV
baculovirus occlusions
4 (M) Pseudomonas putida
5 (M) B. sphaericus
spores
6 (FW) £. putida
7 (FW) AcMNPV
baculovirus occlusions
106 CFU/ml
2 x 106 CFU/ml
106 occlusion bodies/ml
106 CFU/ml
1.6 x 107 CFU/ml
7.5 X 104 CFU/ml
1.8 x 105 occlusion bodies/ml
* M = test run in saltwater with marine NTO species.
FW = test run in freshwater with FW NTOs.
CFU = colony forming units.
25
-------
Table 12. General sampling scheme used in Trails 1 through 7 with
model MPCAs.*
Non-target Test Organism
Day of Tank
Trial Snail Fish Worm Bivalve Shrimp Plant Water
0
M,H
M,H
M,H
M,H
M,H
M,H
M,C
i
M
nor
done ~~
1
M
H
M
M
M
M
M,C
7
M
M
M
M
M
M
M,C
14
M,H
M,H
M,H
M,H
M,H
M,H
M,C
21
M
M
M
M
M
M
M
30
M,H
M,H
M,H
M,H
M,H
M,H
M, C
* M = samples for microbiological and/or gene probe tests.
H = samples for histological analysis.
C = samples for "chemical" water quality analyses,
i = "initial;" sample taken immediately after MPCA added.
B. sphaericus vegetative cells were inoculated into 1 L of
brain heart infusion broth supplemented with 5 ug/ml Neomycin and
50 ug/ml thymine for Trial 2. The cells were pelleted,
resuspended in Tris buffer, and plated on TBAB agar with 5 ug/ml
Neomycin and 50 ug/ml thymine. The following day, plates were
sprayed with catechol (0.55 g/ml) and colonies counted to
determine the volume needed for addition to the test tanks.
For Trials 3 and 7, the concentration of the stock of AcJINPV
occlusion bodies was estimated using a hemacytometer. The the
appropriate amount to be added to the test tanks was determined.
In Trials 4 and 6, Pseudomonas putida was grown in 1 liter of
LB media supplemented with 500ug/ml nalidixic acid and 150 ug/ml
Kanamycin. Cells were then pelleted, resuspended in Tris buffer
and plated on PsF Agar supplemented with 500 ug/ml nalidixic acid
and 150 ug/ml Kanamycin. The titer was determined by total plate
count, and the appropriate amount added to the test tanks.
Trial l: Bacillus sphaericus 2362 with PLT103; Spores
The model MPCA, B. sphaericus spores, for Trial 1 was
recoverable from the test tank water throughout the 28 day study
(Table 13). Water and NTOs from the control tanks were positive
26
-------
on days 7 and 14. for this MPCA as well. The contamination was
at a low level and was not detected after day 14. For subsequent
studies test tanks were then covered, moved, and physically
separated from control tanks to prevent aerosol contamination of
the model MPCA.
B. sphaericus-like spores and vegetative cells were noted in
the gut contents of some of the test animals examined
histologically (Figures 9-11). Presumably, these Gram positive
large rods and spores were consumed by the NTO. However, despite
their presence in the gut contents of these animals, no lesions,
inflammation, reduced survival, or other signs of infection or
toxicity accompanied their presence (Tables 14 and 15).
Trial 2; Bacillus sphaericua 1593 with PLT117: Vegetative Cells
J3. sphaericus vegetative cells, which expressed the xylE gen<=
for catechol 2,3-dioxygenase, were used as the model MPCA in
Trial 2. The vegetative cells did not persist in the test system
and were recoverable by microbiological assay only immediately
after the test tanks were seeded (Table 16). Similarly, gross
signs, survival, and histological study of control and MPCA
exposed NTOs showed no differences, and no adverse effects
attributable to the MPCA (Tables 14 and 15) .
Trial 3: Baculovirus AcMNPV; Inclusion Bodies
A gene probe to the polyhedrin gene of baculovirus AcMNPV was
used in Trial 3 in an attempt to track the fate and persistence
of this viral MPCA. Preliminary tests and controls run with
saltwater and NTO tissues "spiked" with AcJJNPV inclusion bodies
showed detection of the virus above background levels, but
positive signals were quite weak. In addition, control tissues
from the minnow showed some weak but non-specific binding of the
probe (Figure 12). Despite the weakness of the probe and because
of the absence of an alternative detection method, this trial was
run with the gene probe as the detection method.
The results of this trial indicated that the virus could be
detected in the test tank water using the gene probe on the
initial day of the seeding of the water (Figure 13). On
subsequent days of the study, however, the baculovirus could not
be detected. The control tissues of the NTO's produced negative
results also. For the test tissues, the larval shore fly and
plant tissues produced very faint signals that were only slightly
more intense than the background blot (Figure 12). Therefore,
either the baculovirus did not persist in the test tanks'
seawater or in the tissues of the NTOs beyond day 1 of the trial,
or the probe lacked adequate affinity to the viral DNA to
demonstrate its presence. In either case, gross signs, survival,
and histological study of control and MPCA exposed NTOs showed no
differences and no adverse effects attributable to the MPCA
(Tables 14 and 15).
27
-------
Table 13. Summary of microbiological assays for Bacillus
sphaericus (spores) in Trial 1.
CONTROLS
Day of NTOs Tank Water
Trial F GS 0 S 8 9 10
EXPOSED
NTOs Tank Water
GS 0 S 12 3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i
nd -
nd
+
+
+
1
nd -
nd
+
+
+
7
+
+
+
+
+
+
+
+
0
+
+
+
+
+
14/15
0
0
+
+
0
0
0
+
+
+
+
+
+
+
21/22
0
0
0
0
0
0
0
+
+
+
+
+
+
+
28/29
0
0
0
0
0
0
0
+
+
+
+
+
+
¦ +
Abbreviations used:
0 = MPCA not recovered from the test system water or NTOs.
+ = MPCA recovered from test system water or NTOs.
1 = sample taken immediately after MPCA introduced.
F = sheepshead minnow (fish)
GS = grass shrimp
S = turbin snail
O = oyster
nd = not done
28
-------
Table 14. Initial number, observed mortalities, and adjusted
percent survival* of selected NTOs following exposure
to model MPCAs in seawater Trials 1-5 and freshwater
Trials 6-7.
Shrimp Minnow Snail Oyster
Trial
T
C
T
C
T
C
T
C
Trial 1:
Initial No.
45
45
45
45
45
45
45
45
No. sampled
26
27
26
27
28
26
28
26
Mortalities
1
1
4
3
0
1
0
0
Survivors
12
9
10
12
16
17
15
20
% survival
84
80
80
87
98
96
96
102
Trial 2:
Initial No.
45
45
45
45
45
45
45
45
No. sampled
18
18
18
18
18
18
18
18
Mortalities
3
1
2
1
1
4
6
2
Survivors
25
20
32
31
28
29
27
27
% survival
96
84
111
108
102
104
100
100
Trial 3:
Initial No.
45
45
45
45
45
45
45
45
No. sampled
22
21
22
21
22
21
24
20
Mortalities
1
0
2
2
0
o.
1
5
Survivors
9
13
16
14
16
20
16
19
% survival
69
76
84
78
84
91
89
87
Trial 4:
Initial No.
45
45
45
45
45
45
45
45
No. sampled
18
10
18
11
18
10
19
10
Mortalities
0
0
6
2
2
2
2
3
Survivors
20
31
21
26
27
33
21
32
% survival
84
91
86
82.
100
96
89
93
Trial 5:
Initial No*
45
45
45
45
45
45
45
45
No. sampled
22
19
21 -
19
21
19
21
19
Mortalities
2
1
3
10
3
4
1
0
Survivors
16
14
17
8
13
13
24
28
% survival
84
73
84
60
76
71
100
104
Trial 6:
Initial No.
45
45
45
45
45
45
45
45
No. sampled
28
12
28
12
28
12
28
12
Mortalities
1
0
0
0
0
0
0
2
Survivors
2
16
16
28
13
15
16
30
% survival
66
62
98
89
91
60
98
93
Trial 7:
Initial No.
75
75
45
45
45
45
45
45
No. sampled
18
14
18
14
18
14
18
14
Mortalities
19
3
0
4
0
0
0
0
Survivors
12
26
29
24
30
29
31
25
% survival
40
53
104
84
107
96
109
87
* Adjusted % survival = 100 x [(number survivors + number
sampled) / (initial number)].
29
-------
Table 15. Summary of observations in which organisms, presumed to be
the MPCA being tested, were observed in histological
sections of marine non-target test organisms.
MPCA TESTED (in Trials 1-5)*
Bacillus
Marine NTO Species Spores(1) Rods(2) Pseudomonas(4) AcJJNPV(3)
Sea anemone
Turbin snail GC**
Japanese oyster GC
Estuarine grass GC
shrimp
Shore fly larvae GC
Sheepshead minnow GC
Salicornia
(estuarine plant)
* - = not detected in or on any tissue examined.
GC = "gut contents": signifying that organisms were present in the
gut contents that were identical morphologically to the MPCA
tested. Other than in the gut contents, no organisms were
observed in the tissues of NTOs with morphologies similar to
the MPCAs tested.
GC
GC
GC
GC
30
-------
Figure 9. Light photomicrograph of normal sheepshead minnow gut
showing presumed Bacillus sphaericus spores (SP) and
bacilli (B). Brown-Brenn Gram stain. Bar = lOu.
Figure 10. Light photomicrograph of normal snail gut showing
presumed Bacillus sphaericus spores (SP) and bacilli
(B). Brown-Brenn Gram stain. Bar = lOu.
31
-------
TISSUES
M SN L GS O P B
1x
,1x
1x
.1x
t
V,
¦r
f\
s
j.v-5
" 'x:»
ys-
~
#
%
m
J *
9
M
If
3
*
A
CONTROL
TEST
Figure 11. Light photomicrograph of normal shore fly gut showing
presumed Bacillus sphaericus spores (SP) and bacilli
(B). Brown-Brenn Gram stain. Bar = lOOu.
Figure 12. Autoradiograph showing the results of the gene probe
assay for the detection of AcMNPV in seeded (S) and
unseeded (US) non-target organism tissues. Samples
were applied to the membrane undiluted (IX) and
diluted 1:10 (.IX). M=minnow, SN=snail; L=shore fly
larvae, GS=grass shrimp, 0=oyster; P=plant, and
B=buffer (negative control).
32
-------
TRIAL
DAY
TANK WATER
CONTROL
4 5 6
CONTROL TISSUES
7 14 21 28 C
TRIAL DAY
D-1 D-7 D14D-21 D-28
Figure 13. Autoradiograph showing the results of the gene probe
assay for the detection of AcMNPV in non-target
organism tissues and tank water for Trial number 3.
a) Results of test and control tank water and control
non-target organism tissues, b) Results of test
non-target organism tissues. A=anenome, M=minnow,
O=oyster, P=plant, GS=grass shrimp, L= shore fly
larvae, SN=snail, C=control, + = positive control
(unlabeled plasmid DNA), and - = negative control
(buffer).
33
-------
Table 16. Summary of microbiological assays for e
(vegetative cells) in Trial 2.
Day of
Trial
CONTROLS
NTOs Tank Water
F GS 0 S P D 9 10 11
EXP^
NTOs
F GS O S P r
0
0 00000 0 0 0
0 0 0 0 0 <¦
i
nd
nd
1
nd
nd
2
0 00000 0 0 0
0 0 0 0 0 '
5
0 00000 0 0 0
0 0 0 0 0 c
Abbreviations used:
0 = MPCA not recovered from the test system watev
+ = MPCA recovered from test system water or NTC^
1 = sample taken immediately after MPCA introduce
nd = not done
F = sheepshead minnow (fish)
GS = grass shrimp
O = oyster
S = snail
P = Salicornia root (plant)
D = shore fly larvae (dipteran)
Trial 41 Pseudomonas putida PPQ200 + pEPA74
The results from two detection methods used .
persistence of this model MPCA in Trial 4 correl;
17; Figure 14). Both the gene probe method and i
method showed that the MPCA could not be detectr
the study in either the tank water or the NTO ti.
pseudomonad was detected in the oyster tissues o:
and in the anemone tissues on day 2. Control ti
samples were clearly negative. Similarly, gross
and histological study of control and MPCA expos
differences and no adverse effects attributable
(Tables 14 and 15).
34
-------
TRIAL
DAY
S
US
1
2
5
15
TEST TISSUES
A M 0 P GS SN W
#
CO
1
NTROL TANK NO.
2 3 9 10 11
#
CONTROL TISSUES
Figure 14. Autoradiograph showing the results of the gene probe
assay for the detection of Pseudomonas putida in
.'seeded (S) and unseeded (US) non-target organism
tissues and non-target organism tissues from Trial 4.
.Control tissues were only tested on day 5 of the
Jstudy. l,2,3=test tank numbers, 9,10,11= control tank
numbers, A=anenome, M=minnow, O=oyster, P=plant,
GS=grass shrimp, SN=snail, W=tank water, C=control, +
= positive control (unlabeled plasmid DMA), and - =
negative control (buffer).
35
-------
Table 17. Summary of microbiological assays for Pseudomonas
putida in Trial 4.
CONTROLS
Day of NTOs Tank Water
Trial F GS O S A P 9 10 11
EXPOSED
NTOs Tank Water
FGSOSAP 1 2 3
0
1
1
2
5
15
0 0 0 0 0 0
nd -
nd -
nd
nd
nd
0 0
000000 0 0 0
nd + + +
00 + 000 + + +
00 + 0 + 0 + + 0
000000 0 0 0
000000 0 0 0
Abbreviations used:
0 = MPCA not recovered from the test system water or NTOs.
+ = MPCA recovered from test system water or NTOs.
1 = sample taken immediately after MPCA introduced.
F = sheepshead minnow (fish)
GS = grass shrimp
S = turbin snail
0 = oyster
A = sea anemone
P = Salicornia sp. (plant)
nd = not done
Trial 5: Bacillus sphaericus 2362? Spores
This model MPCA was shown to persist throughout Trial 5 in
all water and NTO test tissue samples (Table 18). In the test
tank, concentration of viable spores in the seawater at the end
of the study remained relatively high, at approximately 104
CFU/ml. Unlike the disconcerting findings of Trial 1# in which
this model MPCA was detected in the control tanks, it was not
detected in the control samples assayed.
As was the case in Trial 1, B. sohaericus-like spores and
vegetative cells were noted in the gut contents of some test
animals examined histologically. Presumably, these Gram positive
large rods and spores were consumed by some of the NTOs.
However, despite their presence in the gut contents of these
animals, no lesions, inflammation, reduced survival, or other
signs of infection or toxicity accompanied their presence (Tables
14 and 15).
36
-------
Table 18. Summary of microbiological assays for Bacillus
sphaericus spores in Trial 5.
CONTROLS
Day of NTOs Tank Water
Trial F GS 0 S P A 9 10 11
EXPOSED
NTOs Tank Water
FGSOSPA 1 2 3
0
1
1
5
12
15
30
0 00000 0 0 0
nd
0 00000 0 0 0
nd 0 0 0
0 0000 nd 0 0 0
0 00000 0 0 0
0 0000 nd 0 0 0
000000 0 0 0
nd
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + nd + + +
+ + + + + + + + +
Abbreviations used:
0 = MPCA not recovered from the test system water or NTOs.
+ = MPCA recovered from test system water or NTOs.
1 = sample taken immediately after MPCA introduced.
F = sheepshead minnow (fish)
GS = grass shrimp
S = turbin snail
O = oyster
P = Salicornia sp. (plant)
A = sea anemone
nd = not done
Note: Concentration of MPCA spores at day 30 was 104 CFU/ml, down
from the initial dose of 1.6 x 10^ CFU/ml.
Trial 6: Pseudomonas putida PPQ200 + PEPA74
Microbiological results from Trial 6 showed that the MPCA
could be isolated from test system water and from the tissues of
certain NTOs for the first several days of the trial. For the
remainder of the 29-day trial, detection of the MPCA was sporadic
and in low numbers in exposed NTOs' tissues (Table 19). The data
are also represented graphically in Figures 15 and 16.
Interestingly, the results show that the tubifex worms,
freshwater snails, and the mollies harbored the MPCA, in just
detectable amounts, probably for the duration of the trial. In
contrast, the pseudomonad appeared to be cleared from the test
tanks' water by day 7. In view of these findings, one might
37
-------
speculate that this pseudomand had colonized certain of the NTOs,
becoming part of their microflora. All control organisms and
water samples were negative for the presence of £. putida
throughout the trial (Figure 16).
The results of the gene probe study did not correlate well
with the microbiological assay results (Figure 17). Gene probe
results showed cross reactivity with something found in the
tissues and water of the test system. Tissue and water samples
from control and test tanks demonstrated positive results at day
0, even prior to the addition of the MPCA to the test tanks. In
Figure 17 the "+" sign on the blots indicates a positive control
consisting of unlabeled plasmid DNA. This shows that the DNA for
the gene probe had been labeled properly and hybridization did
occur. This means that the other positive signals shown in
Figure 17 may represent the presence of other microorganisms in
the test system and NTOs with homologous nucleic acid sequences.
As in Trial 4, in which this MPCA was tested with marine
NTOs, gross signs, survival, and histological study of control
and MPCA exposed NTOs showed no differences and no adverse
effects attributable to the MPCA (Tables 14 and 20).
Trial 7: Baculovirus AcMNPV; Occlusion Bodies
The baculovirus model MPCA in Trial 7 may have persisted in
some of the test samples. The most intense gene probe signal was
seen at day 30 in the test tank water (Figure 18). However,
non-specific binding of the probe was clearly indicated in this
study. This was evident especially at the cut borders of the
blot, and in day 0 signals that provided false positives when
they should have been negative (Figure 18). Therefore, as was
concluded in Trial 3 run with this MPCA in seawater, the results
of Trial 7 indicated that either the baculovirus did not persist
in the test tanks' water or in the tissues of the NTOs beyond day
1 of the trial, or that the probe lacked adequate affinity to the
viral DNA to demonstrate its presence. While AcMNPV occlusion
bodies were observed in the ingested gut contents of snails in
Trial 7, gross signs, survival, and histological study of control
and MPCA exposed NTOs showed no differences and no adverse
effects attributable to the MPCA (Tables 14 and 20).
38
-------
Table 19. Summary of microbiological assays for Pseuodmonas
putida in Trial 6.
MPCA EXPOSED*
Day of NTOs Tank Water
Trial WSFMGSP4 5 6
0
0
0
0
0
0
0
0
0
0
1
nd
lXlO4
9xl03
lxlO4
1
200
103
104
80
103
10
lxio3
4xl03
6xl03
2
0
700
0
4
240
103
290
50
0
0
0
100
0
7
0
60
70
10
0
0
0
0
0
12
0
140
60
0
0
0
0
0
0
15
nd
0
0
0
0
0
nd
nd
nd
20
20
20
0
0
0
0
0
0
0
29
nd
0
60
0
0
0
0
0
0
* Units shown are MPCA CFU/ml.
Control samples assayed from days 0# 11, and 29 of the study
were uniformly negative for the MPCA.
Abbreviations used:
0 = MPCA not recovered from the test system water or NTOs.
1 = sample taken immediately after MPCA introduced.
W = tubifex worm
S = freshwater snail
F = sailfin molly (fish)
M = freshwater mussel
GS = freshwater grass shrimp
P = anacharis plant
nd = not done
39
-------
Test Tank Organism - Mussels Test Tank Organism - Fish
Test Tank Organism - Plants Test Tank Organism - Shrimp
Test Tank Organism - Snails Test Tank Organism - Worms
Figure 15. Graphs representing the microbiological results for
the detection of Pseudomonas putida in non-target
organism tissues from test tanks from Trial 6.
Results from non-target control organisms are not
represented since the results showed no £. -putida
present in control samples.
40
-------
Water - Test Tank #4 Water - Test Tank #5
Figure 16. Graphs representing the microbiological results for
the detection of Pseudomonas putida in water samples
from the test and control tanks used in Trial 6.
41
-------
D-i
D-1
D-2
D-4
D-7
D-12
D-15
TEST TISSUES
W S F M Sh
TANK NO.
4 5 6
D-20
D-29
f
A
9
.ifl
1
1
J
-5.
r
r
4
.
1 ;
TANK NO.
9 10 11
' ¦ | I
ft
Figure 17. Autoradiographs showing the results of the gene probe
assay for the detection of Pseudomonas putida in
non-target tissues and tank water for Trial 6.
W=tubifex worm, S=snail, F=fish, M=mussel, Sh=shrimp,
P=plant, nd=not done, + = positive control (unlabeled
plasmid DNA. Control samples were taken only on day
0, 11, and 29 of the study.
TRIAL
DAY
DO
D-11
D-29
CONTROL TISSUES
W S F M Sh P
42
-------
D-7
D-14
D-21
D-30
TEST TISSUES
»- W S F M Sh P
TANK NO.
4 5 6
+
CONTROL TISSUES TANK NO.
Tr?JwL w s F M Sh P 9 10 11
fetors
₯5*4
f '
'*¦*
m
f -_t
mm
+
Figure 18. Autoradiography showing the results of the gene probe
assay for the detection of AcMNPV in non-target
organism tissues and tank water for Trial number 7.
W=tubifex worm, S=snail, F=fish, M=mussel, Sh=shrimp,
P=plant, + = positive control (unlabeled plasmid DNA).
Control samples were taken only on day 0, 14, and 30
of the study.
4 3
-------
Table 20. Summary of observations in which organisms, presumed to be
the MPCA being tested, were observed in histological
sections of freshwater non-target test organisms.
MPCA TESTED (in Trials 6 and 7)*
Freshwater NTO Species Pseudomonas (6) Baculovirus (7)
Tubifex worm GC**
Snail GC GC
Freshwater mussel GC
Grass shrimp
Sailfin molly
Anacharis plant
- = not detected in or on any tissue examined.
** GC = "gut contents": signifying that organisms were present in the
gut contents that were identical morphologically to the MPCA
tested. Other than in the gut contents, no organisms were
observed in the tissues of NTOs with morphologies similar to
the MPCAs tested.
44
-------
DISCUSSION
TEST SYSTEMS
An enclosed test system was developed in which multiple
species of aquatic animals and plants can be tested for adverse
non-target effects following experimental exposure to wild-type
and genetically altered microbial pest control agents. Beginning
with a test tank configuration similar in size and design to that
described by Fournie et al., 1987; 1988), a variety of other
possible tank sizes and configurations were constructed and
tested as test systems for evaluation of MPCAs on non-target
aquatic species. In all we developed and tested four additional
tank configurations, which differed in construction materials,
size, water circulation methods, and in configuration and type of
biological filters used.
Each test system that we constructed had its own advantages
and disadvantages. Tank systems No. 2 and 3, for example, were
large tanks (1,000 L and 400 L, respectively) in which a very
large number of species and individuals of NTOs could be tested
with a model MPCA, while unplanned interspecific predation was
controlled. However, the high cost of obtaining the relatively
large amount of model MPCA required to perform a test in three
replicate tanks of 400 to 1,000 L each prohibited their use in
our studies. Therefore, we combined what we saw as the best
features of our Test Systems 2 and 3 with those of the EPA system
(Fournie et al. 1988) in designing Test Systems 4 and 5. Because
of its simplicity in design, construction, and use, we utilized
Test System 5 as our model multispecies test system in Trials 1-7
performed with model MPCAs that are representative of those being
developed for possible use in the United States.
NON-TARGET SPECIES
A number of marine, estuarine, and freshwater animal and
plant species were collected and evaluated for possible use as
non-target species in multispecies test systems with wild-type
and genetically altered MPCAs. Some proved to be excellent
experimental species in terms of their availability, ease of
laboratory culture, and representation of important phylogenetic
groups in aquatic ecosystems. Others, while important
ecologically, were not used as test animals and plants in our
test system (Tables 1-3).
While no adverse effects were noted in some NTOs as a result
of exposure to model MPCAs (i.e. in terms of survival, gross
appearance, and histology of control and exposed specimens), data
from the sea anemone, saltwater plant, and shore fly larvae were
difficult to interpret due to problems with their use in the
enclosed aquaria. The sea anemones moved between sampling times,
making them difficult to find, and, therefore, they were not
sampled during each scheduled sampling period.
45
-------
Nematocyst filaments of the sea anemone stained Gram positive
and fragments of these in histological sections were so similar
in size to Bacillus vegetative rods as to be difficult to
distinguish from the Gram positive bacilli used as the model
MPCA, The saltwater plant we used in Trials 1-5 lost vitality
during the trials, browned, and wilted. Salicornia is an
estuarine plant and may require better lighting conditions or
higher nutrient levels than were possible in the aquaria water in
which the plants were grown hydroponically. The shore fly larvae
were difficult to study as they pupated and adults emerged
usually well before the end of a 28 day trial.
Of the freshwater species listed in Table 2, only the tubifex
worms presented problems in their use as NTOs. Tubifex worms did
not survive until the end of either of the freshwater
experiments. It appeared that algae (mostly a filamentous blue
green, probably Schizothrix calcicola and certain diatom species)
colonized the exposed surfaces of the worms and overwhelmed them.
By the last day of Trial 6, the netted bag containing the worms
was coated with algae. In addition, the worms lived as a matted
ball in their net container, making it difficult to pull apart a
sample without damaging many of the worms. They were also more
difficult to surface sterilize with iodine and rinse prior to
microbiological assays. Because they were so fragile, they had
to be processed separately from the other organisms. For Trial 7
we started with a larger mass of worms, but again, by the end of
the study the worms were overwhelmed with algae. In nature
tubifex worms dwell embedded in bottom sediments, which protect
them from light and surface fouling organisms. If tubifex worms
could be protected from predation in a multispecies test system,
while being provided with a substrate in which to burrow, they
may make an excellent NTO species.
KPCAs AND DETECTION METHODS
The model MPCAs utilized in these studies provided a range of
fates and persistences in the enclosed multi-species test system.
In the two trials (1 and 5) in which B. sphaericus spores were
used as the model MPCA, the organism persisted in saltwater
throughout the 28 day duration of the two studies. This was not
unanticipated because B. sphaericus spores are known to remain
viable in soil for considerable periods of time (Hertlein et al.,
1979) and to remain visibly unaffected during passage through the
gut of mosquitoes {Davidson, 1979; 1981).
The detection method used to track B. sphaericus in Trials 1
and 5 was simple and easy to use. The organism's presence or
absence could be readily detected and accurately enumerated.
Histological studies of NTOs in Trials 1 and 5 showed the
presence of large numbers of Gram positive bacilli and spores in
the gut contents of some of the NTOs from the exposed tanks.
This observation suggests that the model MPCA may have cycled
through the food chain. However, although this model MPCA did
persist for at least 30 days in the test system while losing
three logs activity, it did not cause observable pathological
46
-------
anomalies in the NTOs used in this study (Tables 13, 14, 15 and
18) .
In marked contrast to the findings when bacillus spores were
used as the model MPCA, the vegetative cells of the strain of J3.
sphaericus used in Trial 2 became undetectable in the seawater
system within the first 24 hr (Table 16). Histological study of
the NTOs in this trial also suggested that the NTOs consumed the
MPCA, but that its presence caused no pathological anomalies
(Table 15). Davidson (1979; 1981) noted that B. sphaericus
vegetative cells are digested very rapidly after entering the
anterior midgut of mosquito larvae. Broken cell walls were
detected within 30 min of ingestion, and defecation removed
nearly all the bacteria from the mosquito larva's gut within 1
hr. Spores may remain visibly unaffected by digestion. Thus,
the Gram positive bacilli noted in the gut contents of some of
the NTOs in our studies may be cells which were in the gut less
than 30 min before we sampled and preserved the specimens in
Davidson's fixative.
Brownbridge and Margalit (1987) noted that many factors
contribute to the disappearance and inactivation of such
bacterial strains in the environment. Our inability in Trial 2
to recover viable g. sphaericus vegetative cells after 24 hr from
our test system suggests that the bacterial cells were destroyed
by environmental effects and possibly by the NTOs. This route of
MPCA clearence from the test tanks is a possibility because one
large oyster may filter nearly 400 L of seawater in 24 hr (Bailey
and Biggs, 1968). As each 120 L tank contained 15 oysters at the
start of each trial, the entire volume of tank water may have
passed through the oysters as many as 50 times in the first 24
hr. If only a fraction of the viable B. sphaericus cells were
inactivated during each passage through the gut of an oyster, it
is possible that the entire dose of MPCA could be reduced to zero
in a single day.
As was the case with the detection method used for £.
sphaericus in Trials 1 and 5, the detection method for this
strain of the MPCA was also simple. Both of these Bacillus
detection methods consisted of using standard microbiological
culturing techniques to detect viable organisms with unique
genetic markers.
The effects of the B. sphaericus toxin are not well
understood at the molecular and cellular levels. Kellen et al.
(1965) noted that signs of toxicity in susceptible larvae when
fed less insecticidal strains of fi. sphaericus may not occur for
three days, but then posterior midgut cells demonstrated
vacuolation and sloughing. Other toxin-mediated bacterial and
fungal infections of insects may produce similar changes in
midgut cells (Ebersold et al., 1977; Zacharuk, 1971). Digestion
of j£. sphaericus cells may release the toxin, possibly causing
neurotoxicity (Davidson, 1981). Rapid swelling of the midgut of
larval mosquitos resulting from an influx of fluid into the gut
lumen is the first visible symptom of intoxication in susceptible
47
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larvae. The gut contents within the peritrophic membrane display
zigzag folds as swelling progresses until the midgut wall is
displaced against the outer body wall, eliminating most of the
hemocoelic space. Ten hours after feeding |J. sohaericus to
susceptible mosquito larvae, cytolysosomes in the midgut become
larger and more numerous. Midgut cells swell and separate from
each other at the bases. Loss of microvilli from anterior midgut
cells and cytolysis and sloughing of posterior midgut cells
occurs, with larvae becoming moribund or dead by 24 hr (Davidson,
1979). The classic signs and lesions of £. sohaericus toxicity,
described by Davidson (1979; 1981), were not noted during
histological examination of paraffin sections of exposed NTOs in
this study.
Pseudomonas putida used as a model MPCA in Trials 4 and 6
showed variable results. In the saltwater test system, it did
not survive more than 5 days (Table 17), but in the freshwater
system it survived and was detectable in some samples assayed for
the 29-day study (Table 19). This is not surprising since
Pseudomonas spp. are naturally found in freshwater and can even
persist in distilled water (Doudoroff and Palleroni, 1974) . The
microbiological culturing method was excellent for tracking this
organism in the test system and in the tissues of the NTOs. The
combination of the two antibiotic resistance genes, in addition
to the biochemical properties inherent in this Pseudomonas sp.
(i.e., turning Ps F Agar yellow under its colonies), simplified
isolation, identification, and enumeration of this genetically
engineered microorganism.
The gene probe assay for tracking this organism (Pseudomonas)
in saltwater (Trial 4) also worked very well. The positive
results were indicated by strong signals and there was not any
background nor any non-specific binding of the probe. However,
in the freshwater system (Trial 6), this assay was not
sufficient. Something appeared to cause non-specific binding or
cross reaction. It is easy to imagine that in the freshwater
system that one or more other Pseudomonas sp. existed which
caused interference with the assay. If a gene probe method for
this organism is to be used in the future in a freshwater system,
investigations will be necessary with the gene probe and
determine the extent of the interference.
The results of Trials 3 and 7, in which the baculovirus
AcMNPV was used, are difficult to assess. In these trials we
employed a gene probe in an attempt to detect and track the fate
and persistence of the viral DNA of this model MPCA. However,
this method was not as simple and easy as the microbiological
culturing methods used with the bacterial model MPCAs. Hence, in
our hands, it was not possible to ascertain whether or not the
MPCA persisted in either the seawater or freshwater test systems,
since the initial background studies indicated that the gene
probe did not produce strong signals (and therefore not a strong
affinity) to target viral DNA (of virus in the occlusion bodies).
The sequence itself contained in the insert may not have a strong
enough affinity for its target sequence. If this MPCA is used in
48
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future studies, it will be necessary to investigate more fully a
gene probe to this MPCA.
Further studies should include attempts to analyze water and
tissue homogenates for specific antigen or nucleic acid from the
model MPCAs using either monoclonal antibodies or gene probes.
It is of interest to study the fate of persistence of not only
the intact viable model MPCA itself, but its genetic material.
Use of the polymerase chain reaction to amplify the genetic
material (Steffan and Atlas, 1988) may enhance sensitivity of
detection when using gene probes.
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