United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
Research and Development
EPA/600/S4-89/027 Sept. 1989
&EPA Project Summary
An Enclosed Aquatic
Multispecies Test System for
Testing Microbial Pest Control
Agents with Non-Target Species
D. V. Lightner, R. B. Thurman, and B. B. Trumper
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 avail-
able from aquaculture supply com-
panies, pet shops, and building
material stores. 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 com-
mercial suppliers.
Four different types of model
MPCAs were tested in the multi-
species system. These included two
different strains of the mosquito
pathogen Bacillus sphaericus, 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 experi-
mentally using traditional microbio-
logical and histological methods.
Also used were assays specific for
the model MPCAs that had been
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.
This Project Summary was devel-
oped by EPA's Environmental Re-
search Laboratory, Gulf Breeze, FL, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Microbial pest control agents (MPCAs),
also known as biological control agents or
"biorationals", are microbial agents in-
tended for use in controlling detrimental
insects, weeds, and other pests. Because
they may be applied in rather large
quantities or repeatedly applied in
smaller quantities to areas outside the
normal geographic range of the wild type
pathogen, it is important that test data be
obtained prior to field application. This
data will help to predict the fate and
persistence of MPCAs in the environment
and their effect on non-target organisms
that would be exposed as a result of
normal field application. Acquisition of
such information becomes even more
important when the application of gen-
etically altered MPCAs is considered.
The purpose of the project reported
here was to develop a simple, function-
ally closed aquatic multispecies test
system in which the study of MPCAs
could be accomplished in a manner that
models an ecosystem and utilizes as
many different, yet readily available types
of NTOs as possible.
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Experimental
Test System
The test system consisted of compo-
nents that were inexpensive and readily
available from a variety of commercial
suppliers including aquaculture supply
companies, pet shops, and building sup-
ply stores. Specifically, the system con-
sisted of a standard 120 L glass aquar-
ium that contained a vertical biological
filter. This vertical biological filter also
served as a barrier, functioning to divide
the tank in two halves and to provide a
physical barrier between populations of
experimental animals so as to prevent
unplanned inter specific predation. The
biological filter matrix consisted of a
polyester fiber pad. An obvious advan-
tage of such vertical highly porous pad-
type biological filters over traditional
undergravel filters is that they do not
remove suspended particulates (such as
an introduced MPCA and algal food
organisms) as rapidly as do undergravel
filters. The pH of water in tanks with
polyester pad filters was maintained by
suspending 1 L plastic beakers with per-
forated bottoms and half filled with
crushed oyster shell or dolomite under
the outlet of an airlift pump (Figures 1
and 2).
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 organ-
isms (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; and that
they represent diverse phyla and that, in
the case of the animal species, represent
different levels in the food web (Tables 1
and 2).
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 (of approximately
1500 L each in volume). These were
located in a sheet metal building and a
plastic covered greenhouse on the
grounds of the University of Arizona's
Environmental Research Laboratory
(ERL). Two systems (one each in the
Saltwater
Oyster Shell
Sea Anemone
Bunodosoma California
Turbin Snail
Turbo fluctuosus
-Air Lines,
Intertidal Halophyte
Salacornia biqelovn
Shore Fly
Ephydra sp.
Mesh Basket
Sheepshead Minnow
Cypnnodon vanegaius
Japanese Oyster
Crassostrea qiqas
Freshwater
Snail
Gyraulus sp.
Oyster Shell
Anacharis Plant
Elodea canadensis
Estuarine Grass Shrimp
Palaemonetes pugio
Tubifex Worm
Tubifex tubifex
Mesh Basket
Sailfin Molly /
Poecilia latipinna
- floss Filter
Freshwater Mussel
Marganlifrea margariiifera
Freshwater Grass Shrimp
Palaemonetes kadiakensis
Figures 1 and 2. Schematics of the multispecies test systems that was used to test mot
MPCAs with marine (Figure 1) and freshwater (Figure 2) non-arf,
species.
greenhouse and metal building) were
used for the mass culture of marine
NTOs, while the remaining two systems
were used to mass culture the freshwater
NTOs.
Feeds and Feeding Methods for
NTOs
Marine and freshwater NTOs in the
large rearing tanks and in the MPCA test
tanks were fed live food organisms or
frozen and artificial feeds once each day
that were consistent with their feeding
behavior and known nutritional requi
ments. Thus, filter feeding mollus
were fed cultures of planktonic alg
once per day. Finfish and grass shrii
received chopped frozen squid, arter
nauplii, and a commercial flake fc
daily. NTO species that were not 1
directly included the aquatic plai
(Salicornia and Elodea), the snails,
tubifex worms, and the shore fly larvat
Model MPCAs Tested
Four model MPCAs were tested w
marine and freshwater NTOs in our t
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Table 1. Assessment of Marine Non-Target Species Used in Tests with Model MPCAs.
Marine Species Source1 Lab Culture2
Test Organism
Suitability
Plant
Salicornia bigelovii Gulf of CA
(intertidal halophyte)
Invertebrate Animals
Bunodosoma californica Gulf of CA
(sea anemone)
Turbo fluctuosus Gulf of CA
(turbin snail)
Crassostrea gigas Gulf of CA
(Japanese oyster)
Palaemonetes pugio Florida
(estuar/ne grass shrimp)
Ephydra sp. Gulf of CA
(shore fly)
Vertebrate Animal
Cypnnodon vanegatus Florida
(sheepshead minnow)
RP
RP
CW
PS
RP
RP
RP
Poor
Fair
Excellent
Excellent
Excellent
Fair
Good
'Source. Gulf of CA = collection sites near Puerto Penasco in Sonora, Mexico, on the Northern
Gulf of Mexico.
2Lab 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.
system. Used in these studies were
three bacterial MPCAs (a spore forming
Bacillus, a vegetative form of Bacillus,
and a Pseudomonas), and an insect
baculovirus (Table 3).
Spore-Forming Bacillus
sphaericus
Spores of Bacillus sphaericus (modi-
fied strain 2362) containing the plasmid
pLT103 were used in Trials 1 and 5. This
strain of 8. sphaericus possesses
msecticidal activity against mosquitoes
and it is being developed commercially
for use as a mosquito larvacide. In
addition to the strain's natural resistance
to streptomycin, insertion of the plasmid,
pLT103, that encodes for Neomycm
resistance, provided the bacterium with a
unique genetic marker, which was used
to "track" this organism in the MPCA
tests using conventional microbiological
methods.
Vegetative Cells of Bacillus
sphaericus
Vegetative cells of B. sphaericus
(modified strain 1593, thymme deficient
and harboring the plasmid pLT117) were
used in Trial 2 The plasmid (pLT117), a
ligation product of pTG402 and pUB110,
encodes for Neomycin resistance, as well
as containing the xylE gene. The xylE
gene expresses catechol 2,3-dioxy-
genase which converts catechol from
colorless to a yellow product (2-
hydroxymuconic semialdehyde) within a
few minutes when sprayed onto growing
colonies. Culture and detection methods
for this organism consisted of supple-
menting TBAB agar plates with Neo-
mycin (5 ng/ml) and thymme (50 ug/ml).
After overnight incubation, plates were
sprayed with catechol solution and
observed for yellow colonies
Pseudomonas putida with
Genetic Markers
In Trials 4 and 6 vegetative cells of a
genetically altered strain of Pseudo-
monas putida were used. While this
species has no uses as an MPCA, it was
selected for use here as a model for
other members of the genus that are
being developed as MPCAs. This strain
had been modified from the parent strain
PP0200, by transformation with the
plasmid pEPA74 This plasmid was con-
structed by inserting the UC19 multiple
linker sequence and a piece of plant
DMA (approximately 400bp) into a
pseudomonas plasmid pKT230 which
contains Kanamycin resistance. The
resulting pseudomonad was mutated in
two separate genes on the chromosome
to produce a strain resistant to high
levels of nalidixic acid. By using
Pseudomonas F Agar, (a selective
medium for Pseudomonas spp. and
upon which colonies are fluorescent
yellow), supplemented with 500 ng/ml
nalidixic acid and 150 ug/ml Kanamycin,
we were able to select exclusively for
the genetically altered strain of P.
putida.
A gene probe was used, in addition to
the traditional microbiological methods,
to "track" this model MPCA in the test
system The gene probe was prepared
by insertion of the 400 bp segment of
plant DNA into a Pst1/EcoR1 site on a
pUC18 plasmid and then transformed
into E. coli Ac80. This organism pro-
vided plasmid DNA for labeling, which
was used as a gene probe.
The Nuclear Po/yhedros/s Virus
AcMNPV
The nuclear polyhedrosis baculovirus
(AcMNPV) from the lepidopteran Auto-
grapha californica was used as the
model MPCA in Trials 3 and 7. 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
AcMNPV, was used as a gene probe to
"track" this model MPCA in the test
system.
Containment of MPCAS in the
Test Systems
The building in which tests with wild-
type and genetically altered model
MPCAs were conducted was located at
the extreme eastern end of the ERL
grounds. It was isolated from other
occupied buildings at the facility by at
least 30 meters. The building was con-
structed to provide a limited access
"containment" area. A wall separated the
experimental half of the building, where
MPCAs were tested with NTOs in glass
aquaria, from the entry and NTO mass
culture tanks areas of the building. To
further insure containment of the model
MPCAs, rubber boots (disinfected in a
200 ppm chlorine foot bath at the
entrance door) were required for access
to the experimental side of the building.
In addition, nets, labware, and other tools
used in the test tanks for sampling
purposes were labeled and dedicated to
a particular tank (in order to reduce
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Table 2. Assessment of Freshwater Non-Target Species Used in Tests with Model MPCAs.
Freshwater Species Source Lab Culture1
Test Organism
Suitability
Plant
Elodea canadensis
(Anachans plant)
Invertebrate Animals
Tubifex tubifex
(annelid worm)
Gyraulus sp.
(snail)
Margaritifera margaritifera
(freshwater mussel)
Palaemonetes kadiakensis
(freshwater grass
shrimp)
Vertebrate Animal
Poecilia latipinna
(sail fin molly)
Commercial
Commercial
Arizona pond
Commercial
Commercial
Hawaii
PS
RP
PS
RP/PS
RP
Excellent
Fair
Good
Excellent
Good
Excellent
'Lab culture:
RP = reproducing laboratory colony established.
PS = experimental animals purchased from a commercial supplier and maintained in the
laboratory.
Table 3. Model MPCAs and dose rates applied to multispecies aquatic test
systems in Trials 1 through 7*
Trial Number
Model MPCA
Initial Dose Level
1 (M)
2(M>
3(M)
Bacillus sphaencus
spores
B. sphaencus
vegetative cells
AcMNPV
baculovirus occlusions
106 CFU/ml
2 x 106 CFUlml
106 occlusion bodies/ ml
4 (M) Pseudomonas putida
5 (M) B. sphaericus
spores
6 (FW) Pseudomonas putida
7 (FW) AcMNPV
baculovirus occlusions
106 CFU/ml
1.6 x 10f CFUlml
7.5 x 104 CFUlml
1.8 x 105 occlusion bodies:ml
*M = test run in saltwater with marine NTO species.
FW = test run in freshwater with FW NTOs.
CPU = colony forming units.
cross-contamination) and disinfect
separately in 100 ppm iodine (polyvi
providine iodine; Fritz Egg Disinfecte
Fritz Chem. Co., Dallas, TX). The fl<
was mopped with 100 ppm pvp iod
periodically to further reduce the risk
contamination. A 5000 liter concn
sump received waste water from
MPCA trials. Water contained in 1
sump was continuously chlorinated (to
20 ppm chlorine) prior to disposal.
General Methods for MPCA
Tests
In all tests with model MPCAs, six si
contained 120 L glass aquaria (three b
and three control) were used. T
biological filters in each aquarium w<
pre-conditioned with a commerc
preparation of nitrifying bacteria (Aqi
Gold, LaMonte Environmental Techn
ogy, Saticoy, CA) or by addition of fil
matrix material from "mature" functic
ing filters. Artificial seawater (Foi
Fathoms, Marine Enterprises, Towst
MD) was used in Trials 1 through 5: c
tap water was used to make up t
artificial seawater. City tap water w
used directly in the freshwater Trials
and 7. Salinity, pH, ammonia, nitri
dissolved oxygen, and alkalinity of t
tank water were monitored and ma
tained (by aeration, partial wat
exchanges, manipulation of feeding ra
use of room space heaters/coolers, et
within acceptable limits.
Each of three replicate test and cont
tanks were stocked with 15 NTOs
each species. This provided a total of
NTOs of each species in test and cont
treatments of each MPCA test. Tt
number was sufficient to provide I
microbiological and histologic
sampling, as well as to allow for sor
loss due to possible natural and/
treatment related mortalities, while s
providing for statistical confidence
data evaluation.
Dosing and Sampling Methods
Three test tanks were inoculated
day 0 of a planned 28 to 30 day stui
with the model MPCAs to dose lew
listed in Table 3. The amount of moc
MPCA added to each test system w
representative of amounts found in t
literature or recommended by the ma
ufacturers of our model (or simil.
MPCAs for use in field applications I
insect control. The water was then mix<
for 5 mm, and water samples were tak
from each tank to determine the mit
concentration of recoverable moc
MPCA. At predetermined time mtervj
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Table 4. Summary of Microbiological Assays for Bacillus sphaencus Spores in Trial 5 Run in Seawater
Controls Exposed
NTOs
Tank Water
NTOs
Tank Water
uay ui
Trial
0
12
15
JO
F
GS
0
O
S
0
p
0
A
0 0
nri
9
0
10
0
11
0 0
F GS O S P A 1
0 00000
nrt
2
0
3
0 0
Abbreviations used:
0 = MPCA not recovered from the test system water or NTOs.
•* = MPCA recovered from test system water or NTOs.
i = sample taken immediately after MPCA introduced.
F = sheepshead minnow (fish)
GS = grass shrimp
S = turbin snail
0 = oyster
P = SaJicornia sp. (plant)
A = sea anemone
nd = not done
throughout the study, water and NTO
samples were collected for micro-
biological and histological analyses.
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 assayed. 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 B. sphaericus). For the B.
sphaericus vegetative cell study, the
plates were sprayed with catechol fol-
lowing 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
for 5-10 min. Oysters, mussels, snails
and fish were scrubbed with a brush
before put into two washes of sterile
distilled water. The remaining organ-
isms were washed two times with sterile.
distilled water. For analysis tissue
samples were aseptically removed and
homogenized in 0.01 M Tris buffer at
pH 7.0. One hundred microliters of each
resulting homogenate were plated and
spread onto duplicate plates of the
appropriate media and incubated. The
remainder of the sample was stored at
-20°C for subsequent gene probe
assays. 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 mem-
brane, baked at 80° C for 2 hr, pre-
hybridized, hybridized, washed and
placed on X-ray film to produce an
autoradiogram.
Histological Samples: Samples for histo-
logical examination were preserved in
Davidson's AFA fixative for 24 to 76 hr,
transferred to 50% ethanol for storage,
and later processed and examined using
routine histological methods. Mayer's
hematoxylin and phloxine/eosin stain was
used for all NTO specimens. In addition,
Brown and Brenn tissue Gram stain 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 (Table 3), plas-
mid DNA was radio-labeled and used 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 resis-
tance gene. To harvest large amounts of
the DNA, the appropriate E. coli was
grown in LB media supplemented with
30-50 ng/ml Ampicillin. Overnight cul-
tures were pelleted, the bacteria washed
and the plasmid was isolated using the
alkaline lysis procedure.
Results and Discussion
Test Systems
An enclosed test system was devel-
oped in which multiple species of aquatic
animals and plants were tested for
adverse non-target effects following
experimental exposure to wild-type or
genetically altered MPCAs. The test
system was used to test several model
MPCAs representative of those being
developed for possible registration and
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 model MPCAs. Some
species selected as NTOs proved to be
excellent experimental species in terms
of their availability, ease of laboratory
culture, and representation of important
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7esf Tank Organism - Mussels
Test Tank Organism - Fish
CFU/ml
CFU/ml
CFU/ml
(x 1000)
80
3* I • I T~T~* t •
Day 0 Day 1\ Day 4 Day 12\ Day 20
0*—, r—| , 1 > • • <
Day 0 | Day 1 \ Day 4 \Day »2| Day 20
Day i Day 2 Day 7 Day 15 Day 29
Sample Day
Test Tank Organism - Plants
Day i Day 2 Day 7 Day 15 Day 29
Sample Day
Test Tank Organism • Shrimp
4.00
CFU/ml
1000)
Day 0 | Day r | Day 4 \Day J2|Oay 20
Day i Day 2 Day 7 Day 15 Day 29
Sample Day
Test Tank Organism - Snails
1.70-
—i 1 » f • i • O
Day 0| Day 1 \ Day 4\ Day l2\Day 20
Day i Day 2 Day 7 Day 15 Day 29
Sample Day
Test Tank Organism - Worms
240
CFU/ml
0.10
0.00
Day 0
Day i Day 2 Day 7 Day 15 Day 29
Sample Day
Day 01 Day f | Day 4 |Day J2| Day 20
Day / Day 2 Day 7 Day 15 Day 29
Sample Day
Figure 3. Graphs representing microbiological results for detection of Pseudomonas putida in non-target
organisms from test tanks in Trial 6. No P. putida was detected in control samples.
phylogenetic groups in aquatic ecosys-
tems (Tables 1 and 2).
While no adverse effects were noted in
any NTO 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, were
difficult to find, and, therefore, were not
sampled during each scheduled sampling
period. In addition, nematocysts filaments
of the sea anemone stained Gram
positive and fragments of these in
histological sections were so similar in
size to Bacillus sphaericus vegetative
rods as to be difficult to distinguish. The
saltwater plant Salicornia that was used
in Trials 1-5 frequently browned and
wilted. 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 liste
Table 2, only the tubifex worms
sented problems in their use as N
by not surviving well. Surface foulir
the worms by a filamentous blue g
probably Schizothrix calcicola and
tain diatom species, was considere
be the cause of the poor survival of t
worms. In nature tubifex worms <
embedded in bottom sediments (not
vided in the test system described r
which protect them from light and su
fouling organisms. If tubifex worms <
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be protected from predation in a multi-
species test system, while being pro-
vided with a substrate in which to burrow,
they might otherwise make an excellent
NTO species.
MPCAs Tests 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 through-
out the 28 day duration of the two
studies. This was anticipated because B.
sphaericus spores are known to remain
viable in soil for considerable periods of
time and to remain visibly unaffected
during passage through the gut of
mosquitoes.
Spore-Forming Bacillus
sphaericus
The detection method used to track B.
sphaericus in Trials 1 and 5 was simple
and easy to use. The presence of the
MPCA was readily determined, and it was
accurately enumerated. NTO histological
studies in these Trials showed the
presence of abundant Gram positive
bacilli 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 path-
ological anomalies in the NTOs (Table 4).
Vegetative Cells of Bacillus
sphaericus
In marked contrast to the findings when
bacillus spores were used as the model
MPCA, the vegetative cells of the strain
of B. sphaericus used in Trial 2 became
undetectable in the seawater system
within 24 hr. Histological study of the
NTOs in this trial also suggested that the
NTOs consumed the MPCA, but that its
presence caused no pathological anom-
alies.
Our inability in Trial 2 to recover viable
8. sphaericus vegetative cells after 24 hr
from our test system may suggest that
the bacterial cells were destroyed by
environmental effects and possibly by the
NTOs. This latter route of MPCA clear-
ance from test tanks is a possibility
because one large oyster may filter
nearly 400 L of seawater in 24 hr. As
each 120 L aquarium 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 8. sphaericus cells were inacti-
vated 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.
Pseudomonas putida with
Genetic Markers
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, but in the
freshwater system it did survive and it
was detectable in some samples for the
duration of the 29 day study (Figure 3).
Interestingly, the results showed that the
tubifex worms, freshwater snails, and the
mollies harbored the MPCA, although m
just detectable amounts, for the duration
of the Trial. In contrast, the MPCA was
not detectable in the test tanks' water by
day 4. These findings suggest that P.
putida had colonized certain of the NTOs,
becoming part of their microflora. In both
marine and freshwater trials with this
MPCA, gross signs, survival, and histo-
logical study of control and MPCA
exposed NTOs showed no differences
and no adverse effects attributable to the
MPCA.
The microbiological culturmg method
was excellent for tracking P. putida in the
test system and in the tissues of the
NTOs. The combination of the two anti-
biotic resistance genes, in addition to the
biochemical properties inherent m this
Pseudomonas sp. (i e , turning Pseudo F
agar yellow under its colonies), simplified
isolation, identification, and enumeration
of this genetically engineered micro-
organism. The gene probe assay for
tracking this organism (Pseudomonas) in
saltwater (Trial 4) also worked very well
However, in the freshwater system (Trial
6), the assay was not sufficiently specific,
as there was some non-specific binding
or cross reaction with the probe. Pos-
sibly, this was due to the presence in the
freshwater system of one or more other
Pseudomonas sp. If a gene probe
method for this organism is to be used in
the future in a freshwater system, it will
be necessary to do more investigations
with the gene probe and determine the
extent of the interference.
Nuclear Polyhedrosis Virus
AcMNPV
In Trials 3 and 7 a gene probe to the
polyhedrin gene of the baculovirus
AcMNPV was employed to detect and
track the fate and persistence of the viral
DNA of this model MPCA. The results of
these Trials 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, but not on subse-
quent days of the study in test system
water or associated with the NTOs. be
detected in the test tank water using the
gene probe on the initial day of the
seeding of the water, but not on subse-
quent days of the study in test system
water or associated with the NTOs.
Therefore, either the baculovirus did
not persist in the test tanks' seawater or
m the tissues of the NTOs beyond day 1
of the two trials, or the probe lacked
adequate affinity to the viral DNA to
demonstrate its presence. While
AcMNPV occlusion bodies were ob-
served m the gut contents of snails
sampled 24 hr after exposure 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.
Conclusions and
Recommendations
Further studies with model MPCAs in
multispecies aquatic test systems should
include analysis of test system water and
NTO tissue homogenates for specific
antigens or nucleic acid from the model
MPCAs using either monoclonal anti-
bodies or gene probes. It is important to
know the fate or persistence of not only
the intact viable model MPCA itself, but
the fate of its genetic material as well.
Furthermore, the use of the recently
developed polymerase chain reaction to
amplify the genetic material may en-
hance sensitivity of MPCA detection
when using gene probes. Because the
microorganisms present in the gut con-
tents of several NTO species in the
present study may have been the model
MPCAs used, future studies that posi-
tively identify these organisms "in situ"
using specific antibodies or gene probes
are recommended.
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D. V. Lightner, R. B. Thurman, and B. B. Trumper are with the University of Arizona
Tucson, AZ 85706.
John A. Couch and John W. Fournie are the EPA Project Officers (see below).
The complete report, entitled "An Enclosed Aquatic Multispecies Test System for
Testing Microbial Pest Control Agents with Non-Target Species," (Order No. PB
89-231 526/AS; Cost: $15.95, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officers can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, FL 32561
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S4-89/027
0o00s°I5.!l
CHICAGO
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