UNITED STATES
ENVIRONMENTAL PROTECTION GED Contribution Number 996
AGENCY February 1997
® IZDA Research and
Development
INTERNAL REPORT:
ESTUARINE INVERTEBRATE TESTING OF THE
ENTOMOPATHOGENIC FUNGUS,
PAECILOMYCES FUMOSOROSEUS APOPKA STRAIN 97
Prepared for
Office of Pesticide Programs
Prepared by
Gulf Ecology Division
National Health and Environmental Effects Research Laboratory
Gulf Breeze, Florida 32561
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GED Contribution Number 996
February, 1997
Estuarine Invertebrate Testing of the Entomopathogenic Fungus,
Paecilomyces fumosoroseus Apopka strain 97
by
Fred J. Genthner1, Heidi K. Kumpf2, and Geraldine M. Cripe1
1U.S. Environmental Protection Agency, and
2National Network of Environmental Management Studies
Gulf Breeze, Florida 32561
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LABORATORY
GULF ECOLOGY DIVISION
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This document is intended for internal Agency use only.
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Abstract
Paecilomyces fumosoroseus conidiospores were evaluated for toxicity and
pathogenicity to Mysidopsis bahia and pink shrimp, Penaeus duorarum. Duplicate, static,
acute 96-h tests were conducted with ^24-h-old M. bahia (N=10) using viable and heat-
killed conidiospores. The low mortalities (<20 %) observed in control and experimental
treatments suggested that P fumosoroseus conidiospores were neither toxic nor
pathogenic to mysids. Post-larval Ouvenile) P. duorarum (N=15) were fed a diet
containing conidiospores and mycelia of P. fumosoroseus. The fungus was recovered
from feces of shrimp that were fed viable fungus, but after 30 days no mortalities were
observed. All shrimp appeared healthy with no visible signs of disease.
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Materials and Methods
Cultivation of Fungus and Recovery of Spores
P fumosoroseus (ATCC 20874) Pa 97 10-11 was obtained from Dr. L. S. Osborne,
University of Florida, Central Florida Research and Education Center, Apopka, FL, and
cultured at 25° C on glucose-yeast extract-basal salts medium (GYBS; Boucias ef a/
1988). Solid GYBS medium was prepared by the addition of 2.0% agar. For mysid testing
purposes, conidiospores were scraped from the surface of 7-day-old sporulating cultures
and suspended in filtered (0.22um) seawater at a salinity of 20%o by gentle aspiration in a
hand-held tissue homogenizer.
For the shrimp feeding experiment, 1 liter wide-mouth Erlenmeyer flasks containing
200 ml of GYBS were inoculated with a loopful of conidiospores and incubated at 25- C for
10-14 day without shaking. Mycelial mats containing conidiospores were harvested by
filtration through Whatman #1 filter paper an j stored at -20° C. For both assays, a
hemocytometer was used to estimate conidiospore densities. Viable counts were
estimated by diluting spores or mycelial mats in sterile distilled water containing 0.03%
Triton X-100®1 (Union Carbide, Indianapolis, IN, USA), spreading the dilutions onto GYBS
agar plates, and counting colonies which appeared after a 5-day incubation.
P fumosoroseus was cultured from shrimp feces by streak plating freshly- collected
'Mention of tradenames or commercial products does not imply endorsement by the I' S
Environmental Protection Agency
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fecal strands on GYBS agar plates containing 0.5 mg/ml nalidixic acid to suppress
bacterial growth. Fungal colonies arising from these plates were identified as P
fumosoroseus by the size, shape, color and arrangement of the spores.
Mysid tests
Mysids, <24-h-old, were obtained from laboratory cultures. Seawater for culture and
testing was pumped from Santa Rosa Sound near Gulf Breeze, Florida, filtered to 20um,
diluted to a salinity of 20%0 with dechlorinated tap water, aerated and maintained at 25° C.
Techniques used for conidiospore exposures were similar to the Toxic Substances
Control Act Guidelines: Final Rules (USEPA 1985). Controls included uninoculated
seawater and a heat-killed (autoclaved 20 min, 15 Ib/in2) conidiospore treatment.
Sterilization of conidiospores was verified by spread plating 0.1 ml of the suspension on
GYBS agar plates and scoring for growth after 5 days of incubation. Exposure vessels
were 500 ml glass culture dishes filled with 200 ml of seawater containing suspensions of
the conidiospores. Five mysids were added to each exposure vessel using two vessels
for each control and eacch conidiospore density. Exposure vessels were covered and
incubated with aeration at 25° C under a photoperiod of 14-h light:10-h dark. Each day
mysids were counted, dead animals were removed and the remaining animals were fed
Artemia (48 h post-hydration, ;>30 /4rtem/a/mysid/day). Salinity, dissolved oxygen (D.O.)
and pH were measured daily. Temperature was recorded continuously.
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Shrimp feeding experiment
Post-larval pink shrimp were obtained from spawned, eye-ablated adult Penaeus
duorarum according to the methods of Gripe (1996). Four 64-liter glass aquaria were
used for P. fumosoroseus feeding tests, two for the heat-killed control and two for the
viable fungus. Each aquarium contained 15 shrimp. To prevent cannibalism, each shrimp
was confined to a small cage constructed from a 10 cm diameter glass Petri dish with a
nylon screen collar (1 mm I.D. mesh) attached with silicone sealant. Flow-through
seawater (as described in the previous section) was provided to each aquarium at 1.8
liters/hour. Photoperiod was 12-h light: 12-h dark and dissolved oxygen was monitored
weekly using a YSI Model 57 oxygen meter. Shrimp were allowed 4 days to acclimate to
test aquaria before experimental diet was provided. During the acclimation period each
shrimp was fed 5 pellets per day of Shrimp Production 45/10® (Rangen, Inc., Buhl, Idaho).
The test was initiated by placing shrimp on experimental and control diets. Diets were
prepared by adding viable or heat-killed mycelial mats (0.41 g) and Tetramin® flake fish
food (10 g) to 500 ml of 2% agar that had been sterilized in an autoclave (20 min, 15 Ib/in2
) and cooled to 39° C. This mixture was solidified in sterile plastic Petri plates and stored
at 4 ° C. On alternate days each shrimp was fed a cube (0.25 cm3) of the appropriate test
diet. Before feeding, fecal strands and uneaten food were removed from the bottom of
each cage. In addition, three cages were rotated from the front to the rear of the
aquarium to eliminate position effects during exposure.
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Results and Discussion
Mysid tests
Water quality parameters were very similar for all tests. Test temperatures for all 96-h
tests were 24 ± 2° C. Dilution water salinity was 22 ± 2 %0 for all tests. The D.O. ranged
from 4.79 to 8.24 mg/l and the pH, from 7.7 to 8.6. Both of these physical parameters
were within the acceptable limits for this test.
Mysid Assays
Analysis of mortality data (Table 1) indicated that P. fumosoroseus conidiospores were
neither toxic nor pathogenic to mysids. Seawater control mortality was 5%. The highest
mortality obtained was 20% for a conidiospore density of 1 X 105 per ml. This value was
judged not to be significantly different from control mortality. Dead mysids showed no
visible signs of fungal infection.
Shrimp feeding experiment
All shrimp appeared healthy with no visible signs of disease for the duration of the
study. Each cube of the experimental diet contained viable P. fumosoroseus at a density
of approximately 1 X 106 colony forming units. The agar formulation remained intact in
the water. Shrimp held the cubes with their pleopods and fed vigorously. The dissolved
oxygen in the aquaria ranged from 6.5 to 7.6 mg/l. At 3 weeks, P. fumosoroseus was
cultured from fecal strands obtained from shrimp fed the experimental diet, but not from
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fecal strands obtained from shrimp fed the heat-killed control diet. After 30 days, no
mortalities were observed, and the experiment was terminated.
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References
Boucias, D. G., Pendland, J. C., and Latge, J. P. 1988. Nonspecific factors involved in
attachment of entomopathogenic deuteromycetes in host insect cuticle. Appl. Environ.
Microbiol. 54,795-1805.
Gripe, G. M. 1996. Spawning and larval survival of the pink shrimp, Penaeus duorarum, in
a small culture facility. J. Appl. Aquacult. (ERL, GB 755)
U.S. Environmental Protection Agency (1985) Toxic Substances Control Act Guidelines.
Final Rules. Mysid Shrimp Toxicity Test. U.S. Fed Reg 50:39367-39369
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Table 1. Mortality of Mysidopsis bahia (N=10) exposed to Conidiospores of Paecilomyces
fumosoroseus
Mortality (%)a following treatment with:
Count (conidiospores/ml)
Direct
1 X 104
2X104
1 X105
2X105
1 X106
2X106
Culturable
9X103
NDb
9X104
ND
9X105
ND
Viable
conidiospores
0
0
20
0
0
10
Heat-killed
conidiospores
ND
ND
ND
ND
0
10
aSeawater control mortality was 5%
DND = not determined
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