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A mycosis caused by Lagenidium sp. in laboratory-reared larvae of the Dungeness crab, Cancer magister, and possible chemical treatments
JOURNAL
OF INVERTEBRATE
PATHOLOGY
28,
329-336
(1976)
A Mycosis Caused by Lagenidium sp. in Laboratory-Reared Larvae of the Dungeness Crab, Cancer magister, and Possible Chemical Treatments’ DAVID
A. ARMSTRONG,2 AND RICHARD
DAVID V. BUCHANAN,3 S. CALDWELL
Department of Fisheries and Wildlife, Oregon State University, MarineScience Center, Newport, Oregon 97365 Received
December
19, 1975
A mycosis in larvae of the Dungeness crab, Cancer mugister. was observed about 48 hr after they had molted to second-stage zoeae in the laboratory during a chronic bioassay. The fungus was of the genus Lugenidium and caused about 40% mortality of larvae before the test was terminated. Hyphal development and subsequent sporulation of the fungus was observed over a range of O-32”/,, salinity, with an optimum around 16@/,,. Bioassays of eight fungicides and the herbicide trifluralin, using the larvae and fungus in separate tests, were performed to explore means of chemical treatment. Trifluralin was the most satisfactory of the chemicals screened, with captan, Cuz+, and benomyl also giving good results for certain treatment intervals of up to 96 hr. Malachite green was toxic to larvae at concentrations inhibiting the fungus.
INTRODUCTION Natural and laboratory mycoses of marine invertebrates have been reviewed by Johnson (1970). Of particular importance have been infections caused by species of the genus Lagenidium. Parasitism of the eggs of the blue crab, Callinectes sapidus, by L. callinectes was described by Couch (1942), and elaboration of its distribution and pathogenicity were given by Sandoz et al. (1944) and RogersTalbert (1948). More recently, developmental and anatomical features of L. callinectes were discussed by Bland and Amerson (1973) and by Gotelli (1969) for a Pacific Coast Lagenidium sp. Instances of fortuitous mycoses of laboratory-reared larval crustaceans have been reported by Lightner and Fontaine (1973) in the white shrimp, Penaeus setiferus, and by Nilson et ITechnical Paper No. 4152, Oregon Agricultural Experiment Station, Corvallis, Oregon 9733 1. *Present Address: Ecology Group, Department of Water Science and Engineering, University of California, Davis, California 95616. 3Present Address: Oregon Department of Fish and Wildlife, Research Section, Corvallis, Oregon 97331.
al. (1975) in the lobster, Homarus americanus. An infection by Lagenidium sp. of larval Dungeness crab, Cancer magister, in the spring of 1973 is described in this paper. At the outbreak of the disease, larvae were being exposed to several pesticides for evaluation of chronic toxicity (Caldwell et al., unpublished data). High survival of animals exposed to one herbicide, trihuralin, prompted us to explore chemical control of the fungus, and results are discussed relative to culture procedures. MATERIALS
D 1976 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Crab larvae were hatched in the laboratory from ovigerous females collected off Newport, Oregon. During the chronic toxicity tests, larvae were reared in a continuous-flow system of glass aquaria described by Buchanan et al. (1975). All glass in the rearing system was washed twice a week in a chlorine solution, and at this time larvae were fed newly hatched brine shrimp, Artemia salina, at a density of about six shrimp/ml of test solution.
329 Copyright All rights
AND
330
ARMSTRONG.
Nine
Pesticide
Chemicals Active
Common Metiram’ CuZ+ (Bordeaux Folpet Chlorothalonif Benomyl
name
green
Dichlone Captan sCommon name bAssumed. ‘As copper.
AND
CALDWELL
TABLE I Tested for Toxicity against Cancer and the Fungus Lagenidium sp
mugister
Larvae
ingredient
Chemical
Formulation
name
Trade
name
tested Active ingredient (%)
Polyram mixture)
Trifluralin Malachite
BUCHANAN
for a complex
Copper sulfate tribasic (monohydrate) N-(trichloromethylthio)phthalimide Tetrachloroisophthalonitrile Methyl I-(butylcarbamoyl)-2benzimi dazolecarbamate cy,cu,a-Trifluoro-2,6-dinitro-N,Ndipropyl-p-toluidine Tetramethyl-di-p-aminotriphenylcarbinol chloride 2,3-Dichloro-1,4-naphthoquinone IV-trichloromethylthio-4-cyclohexene-1,2dicarboximide of zineb
and polyethylene
Sea water used in the chronic toxicity tests was pumped from Yaquina Bay, Oregon, passed through a coarse sand filter (to about 100 pm) and stored for up to 3 days in a IO,OOO-galtank. The water was adjusted to 2.5 f O.5o/oo salinity during the first 15 days of the test and afterwards was 15O/,, or higher. From the storage tank, water was pumped into a constant-temperature laboratory, refi ltered through a fine sand filter (to about 20 pm), exposed to uv irradiation, and allowed to reach 13” =t 1°C before entering exposure aquaria. The dissolved oxygen concentration of the water always exceeded 90% of saturation and the pH range was 7.6 to 7.9 during the test. The temperature of sea water in Yaquina Bay was less than 13°C when the infection was noted in our laboratory. The fungus was cultured from dead crab larvae onto kernels of hemp seed, Cannabis sativa. Perpetuation of the fungus was achieved by adding a new kernel to a beaker of 25O/,, sea water containing a heavily infected kernel. By this procedure, the fungus was kept in a state of active sporulation. Hyphae would appear on the new kernel within 18 hr of its introduction, and sporulation occurred in 40 to 60 hr.
thiuram
disulfide
IOOb 54’ 50 75 50
Phaltan Bravo Benlate Treflan
E.C.
44.5
lOOb Phygon-XL Orthocide-50W containing
80% zineb
50 50 by weight.
This method was used to test the salinity tolerance of the fungus. Three salinities, 0, 16, and 32O/,,, were tested in triplicate during six experiments. A heavily infected and an uninfected kernel were placed in 200 ml of water and not allowed to touch each other. The times to hyphal growth and subsequent sporulation on the new kernel were recorded, as were the extent and quality of these events. The toxicity of eight fungicides and the herbicide trifluralin to crab larvae and to the fungus were tested in separate 96-hr bioassays (Table 1). A logarithmic series of five concentrations of each chemical was tested in duplicate against unfed, first-stage zoeae. In all bioassays, concentrations were adjusted for the percentage of active ingredient in the specific formulation. Tests were performed in 250-ml beakers with 10 larvae per beaker. Solutions were renewed every 24 hr and were kept at 13°C and 25O/,, salinity. The criterion of toxic effect in determining ECso values (the concentration of pesticide producing a sublethal effect in 50% of the test organisms in a specified time period) was cessation of swimming, and for the LCso values (the concentration of pesticide result-
MYCOSIS
OF
LARVAL
ing in death of 50% of the test organisms in a specified time period) the criterion was cessation of all movements and development of an opaque appearance. Such values were calculated as described by the American Public Health Association et al. (197 1). The exposure beakers and water conditions were the same for the fungal bioassay. Four concentrations of each chemical were tested in duplicate with 200 ml of solution per beaker. To each beaker was added a heavily infected hemp kernel on which spores were actively being released. Twenty-four hours later, an uninfected kernel was added to the beaker and was kept from touching the inoculum; the introduction of the second kernel was considered time zero. The time lag in introduction of the two kernels ensured that
FIG. 1. View of the dorsal sp. Scale = 30 pm.
spine of Cancer
mugister
larva
331
CRABS
infecting spores had formed while in the treatment. Data from the fungal and larval bioassays were compared by means of selectivity ratios [SR = (EC,, larvae)/(concentration toxic to fungus)] for a given time period. The effect of a chemical on the fungus was an all or nothing response, i.e., either hyphae were growing on the new kernel by 48 hr or not. This precluded the calculation of 50% response values. Therefore, the denominator was taken as the lowest concentration that inhibited the transfer of the fungus to an uninfected kernel. RESULTS During the chronic toxicity bioassays (Caldwell et al., unpublished data), larvae in
showing
heavy
endobiotic
proliferation
of Lugenidium
332
ARMSTRONG,
BUCHANAN
control and low concentration pesticide treatments molted from first- to secondstage zoeae on the ninth and tenth days of exposure, and survival exceeded 95% at this time. Mortalities among these groups were
AND
CALDWELL
first recorded on day 11, and survival of control larvae had dropped to 60% by day 18 when the test was terminated. Death was apparently caused by an endobiotic fungus of the genus Lagenidiunz
FIG. 2. Typical appearance of branched Lagenidium. Scale = 10 pm. FIG. 3. Discharge vesicle with hyaline membrane filling with endogenous protoplasm of undifferentiated Scale = 10 Wm. FIG. 4. Discharge vesicle nearly filled. Protoplasmic unit (arrow) just entering vesicle. Scale = 10 pm. FIG. 5. Discharge vesicle containing differentiated, motile spores. Note hyaline membrane and empty tube (arrow). Scale = 10 pm.
spores.
discharge
MYCOSIS
OF
LARVAL
(identification has been made by William Denison, Oregon State University; Howard Whistler, University of Washington; and Charles Bland, East Carolina University; cultures of this fungus are presently being maintained by C. Bland for eventual identification as to species). The mycelium was first localized in the visceral region of the larvae. Within 24 hr, hyphae had spread throughout an animal using all available tissue for nutrition (Fig. 1). We believe this mycosis was the primary cause of death. Squashes of moribund and apparently healthy larvae were viewed under oil immersion microscopy. In no case did we find endobiotic protozoans or bacteria to suggest concurrent infection with the fungus. After death, however, zoeae were quickly invaded by ciliates and bacteria. Intramatrical hyphae of the fungus were sparingly septate, extensively branched, irregular in their growth pattern, and 8-12 pm in cross-sectional diameter (Fig. 2). Hyphae were a pale green color and had a granular
FIG.
6. Encysted
spore
on exoskeleton
333
CRABS
cytoplasm composed of refractive lipid vacuoles (Fig. 2). Extramatrical hyphae penetrated the exoskeleton after an animal died. These hyphae were non-septate and usually unbranched. Sporogenesis was completed in extramatrical discharge tubes and vesicles (Figs. 3, 4, 5) in a manner similar to descriptions given for Lugenidium by Fuller et al. (1964), Bland and Amerson (1973), and Lightner and Fontaine (1973). Spores were reniform in shape, measured 7 x 5 pm, and were propelled by two lateral flagella. Spores swam vigorously for some minutes after their release from a discharge vesicle. Gradually their movement would slow into a tightening circle. After stopping, both flagella would separate from the spore and were not seen to be retracted as described by Bland and Amerson (1973). A single germ tube grew from the then-encysted spore and could apparently penetrate some portion of the larval exoskeleton (Fig. 6). Lugenidium was able to transfer, grow,
of dead crab larva
with germ
tube just emerging.
Scale
= IO pm
334
ARMSTRONG,
BUCHANAN
and sporulate in 0 to 32O/,, salinity. However, the time required for these processes varied considerably with salinity and was most rapid at 16’/,,. Infection of the hemp kernel and subsequent hyphal growth required about 14, 40, and 55 hr at 16, 32, and O”/oo, respectively. Discharge vesicles containing spores appeared after 32, 50, and 60 hr in these same salinities. The mycelium was always less dense and the hyphae shorter on the seeds in freshwater. Since seeds used for infection were always cultured in water of 25o/oo, the time response for infection in O”/,, may partially represent the stress of no previous acclimation to zero salinity. Several chemicals with high SR values (Table 2) seem promising as chemical treatments for the fungus; the higher the SR quotient, the less toxic is the chemical to larvae relative to the fungus. Benomyl, CU’+, trifluralin, and captan had 48-hr SR values of > 100,4.5, 34, and 12, respectively. By 96 hr, however, the two former chemicals had become quite toxic to the crab larvae (Table 2), and only trifluralin and captan, with SR values of 1.5 and 6.4, could be considered as potential treatments for this time interval. The data for trifluralin from the chronic
Comparison
AND
toxicity test, where larvae and fungus occurred in the same system, gave a 96-hr SR value of 117. DISCUSSION Reports of Lagenidium infections in laboratory-reared crustacean larvae have increased in recent years (Lightner and Fontaine, 1973; Nilson et al., 1975). There is no evidence in these reports, or in our work, that Lagenidium is a natural pathogen to any of the species affected, however, and only two marine arthropods have been reported as natural hosts for fungi of this genus (Johnson, 1970). Lagenidium in this and other larval culture experiments mentioned seems to appear fortuitously as an opportunistic yet virulent pathogen. The Lagenidium sp. infecting our crabs is probably endogenous to bays and estuaries where it resides on various algae (Fuller et al., 1964; Gotelli, 1969). The euryhaline response of the fungus, with optimal growth in less than full-strength sea water (35°/oo), helps to support the contention of estuarine origins. Lagenidium cultured from Puget Sound, Washington, grew best at 15-30°/oo, and Johnson (1970) reported that the east
TABLE 2 of the Toxicity of Nine Pesticide Chemicals to Cancer Larvae and the Fungus Lagenidium sp. Toxic
Pesticide
concentrations
EC,,
Fungus’
2.4 4.5lOO 0.17 0.12 0.05 0.7
96 hr LC50 6.7 >I00 0.1 0.56 >lOO >0.32 0.16 0.08 >I0
‘Data for inhibition of transfer of fungus to uninfected bSelectivity ratio [(SR = (EC,, larvae)/(concentration ‘Data from Caldwell et al. (1976b).
mugisrer
(mg/liter)
LaXle
48 hr
Metriam Cu*+ (Bordeaux mixture) Folpet Chlorothalonil Benomyl Trifluralin (Chronic test)c Malachite green Dichlone Captan
CALDWELL
EC,,
LGo
0.7 < 1.0
5.9 1.50.48 0.04 0.038 8.0
hemp kernel. toxic to fungus)]
SRb
48 hr
96 hr
48 hr
96 hr
12.5 1.0 I.0 1.o 1.0 0.005
12.5 10.0 1.0 1.0 10.0 0.1 0.004 1 1.0 0.1 0.056
0.19 4.5 0.1 0.17 > 100 34.0
0.06
1.0 0.032 0.056 for a given
time interval.
0.12 0.15 12.0
MYCOSIS
OF
LARVAL
coast L. callinectes sporulated in 5-300/,, sea water with an optimum at 200/,,. The larvae of C. magister are pelagic through the megalops stage (Lough, 1975) and usually occur offshore rather than in estuaries. The use of bay water in our experiments with these larvae probably exposed the animals to a pathogen they do not normally encounter. The manner and timing of infection in the crab larvae was not determined, but it may occur during or just after ecdysis via penetration of the still-soft exoskeleton by a germ tube. Encysted spores with emerging germ tubes were found on the exoskeleton of larvae, but we did not actually view penetration into the animals. The incubation period of the fungus on hemp kernels corresponded closely to the 36-48-hr period between ecdysis and first mortalities of crabs observed in the chronic toxicity test. Lightner and Fontaine (1973) exposed protozoeae I larvae of the shrimp Penaeus aztecus to Lagenidium. Sixty hours later, having molted to protozoeae II, some animals were infected by the fungus. Infection of larvae may have been by spores just entering the aquaria from some point without or by release of spores from well-established mycelium within the culture system. Many dead Artemia in our aquaria were infected by Lagenidium, and live Artemia have been experimentally infected by the fungus (L. Ho and D. Lightner, pers. commun.). Because of the presence of Artemia in our culture system from day 1, their frequency of molting, and their high mortality between cleaning days, the brine shrimp may have served as a host and medium for proliferation of the fungus until it became infective to the crabs. The use of herbicides and fungicides, in conjunction with other water treatments such as uv irradiation, microfiltration, or high salinities may curtail decimation of crustacean larvae by fungi such as Lagenidium. However, we stress that our pesticide toxicity data are meant only to be directional in nature. Any large-scale use of these chemicals should be prefaced by screening
CRABS
335
tests used to ascertain the sensitivity of the particular animal species involved at water conditions typically used for their culture. Of the nine chemicals tested, we believe that trifluralin is the most promising for controlling the fungus at concentrations that are not injurious to the larvae. During the initial chronic toxicity test, when the mycosis apin 0.0015 mg of peared, larvae trifluralin/liter were not affected by the fungus (Caldwell et al., unpublished data). The pesticide was toxic to the larvae in 96 hr only at concentrations nearly a 100 times greater (Table 2). The fungicide captan was the only other compound with a 96-hr SR value > 1. Benomyl was very effective over a 48-hr period (SR value > 100) and might be considered for very short treatments. We believe the SR values were obtained from somewhat extreme exposure conditions and may, therefore, be unusually low. The hemp kernel is probably an easier target for infection by Lagenidium than is a crab larva. Lagenidium spores in a particular concentration of a chemical treatment might infect hemp kernels but have difficulty in overcoming the natural defenses of an animal. Also, test concentrations of compounds in these experiments were usually spaced by IO-fold increases. Because 50% responses could not be calculated for the fungus transfer, the SR denominators were probably higher than closer spaced concentrations could have revealed. Chemical treatments for this parasite should be of a prophylactic nature. Once the fungus is endobiotic it is too late to save that portion of larvae affected. Short-term treatments centered around ecdysis in the molting cycle may be best. In our work with the toxicity of pesticides to crab larvae, we have found up to two orders of magnitude difference between acute and chronic levels (Armstrong et al., 1976; Caldwell et al., unpublished data). We believe, therefore, that prophylaxis based on chronic chemical exposures would be less desirable because lower concentrations of fungicides would be required to avoid chronic toxic affects in the
336
ARMSTRONG.
larvae, yet these concentrations less likely to affect the fungus.
BUCHANAN
would be
ACKNOWLEDGMENTS We thank Drs. C. Bland, W. Fisher, R. Krieger, and E. Nilson for reviewing the manuscript. Michael Myers provided excellent technical assistance. This research was supported by Contract No. 68-01-0188 from the United States Environmental Protection Agency and by Oregon State University Sea Grant College Program Grant No. 04-3-158-4. NOAA, U.S. Department of Commerce.
REFERENCES American Public Health Association, American Water Works Association, and Water Pollution Control Federation. 1971. ‘Standard Methods for the Examination of Water and Wastewater,” 13th ed., 874 pp. American Public Health Association, Washington, D.C. ARMSTRONG, D. A., BUCHANAN, D. V., MALLON, M. H., CALDWELL, R. S., AND MILLEMANN, R. E. 1976. Toxicity of the insecticide methoxychlor to the Dungeness crab, Cancer mugister Dana. Mar. Biol. (in press). BLAND, C. E., AND AMERSON, H. V. 1973. Observations on Lagenidium callinectes: Isolation and sporangial development. Mycologiu. 65,3 10-320. BUCHANAN, D. V., MYERS, M. J., ANDCALDWELL, R. S. 1975. Improved flowing water apparatus for the cul-
AND
CALDWELL
ture of brachyuran crab larvae, J. Fish. Rex Board Canud. 32, 1880-1883. COUCH, J. N. 1942. A new fungus on crab eggs.J. Elisha MitcheliSci. Sot., 58, 158-162. FULLER, M. S., FOWLES, B. E., AND MCLAUGHLIN, D. J. 1964. Isolation and pure culture study of marine phycomycetes. Mycologia, 56.745-756. GOETELLI. D. 1969. “Morphology and Nutrition of the Marine Fungus Lagenidium callinectes,” I05 pp. Ph.D. Thesis, University of Washington, Seattle, Washington. JOHNSON, T. W., JR. 1970. Fungi in marine crustaceans. In “A Symposium on Diseases of Fishes and Shellfishes” (S. F. Snieszko, ed.), Amer. Fish. Sot. Spec. t’ubl.. No. 5,405-408. LIGHTNER, D. V., AND FONTAINE, C. T. 1973. A new fungus disease of the white shrimp Penaeus settyerus. J. Invertebr. Pathol.. 22.94-99. LOUGH, R. G. 1975. “Dynamics of Crab Larvae (Anomura, Brachyura) off the Central Oregon Coast, 1969-1971,” 299 pp. Ph.D. Thesis, Oregon State University, Corvallis, Oregon. NILSON,
E.
H.,
FISHER,
1975. A new mycosis americanus). J. Invertebr.
W.
S.,
AND
SHLESER,
R.
A.
larval lobster (Homarus Pathol. 22, 177-183. ROGERS-TALBERT, R. 1948. The fungus Lugenidium cullinectes Couch (1942) on eggs of the blue crab in Chesapeake Bay. Biol. Bull.. 95,214-228. SANDOZ.
M.
D.,
ROGERS,
of
R.,
1944. Fungus infection of Callinectes sapidus Rathbun.
AND
NEWCOMBE,
C.
L.
eggs of the blue crab Science, 99, 124-125.
OF INVERTEBRATE
PATHOLOGY
28,
329-336
(1976)
A Mycosis Caused by Lagenidium sp. in Laboratory-Reared Larvae of the Dungeness Crab, Cancer magister, and Possible Chemical Treatments’ DAVID
A. ARMSTRONG,2 AND RICHARD
DAVID V. BUCHANAN,3 S. CALDWELL
Department of Fisheries and Wildlife, Oregon State University, MarineScience Center, Newport, Oregon 97365 Received
December
19, 1975
A mycosis in larvae of the Dungeness crab, Cancer mugister. was observed about 48 hr after they had molted to second-stage zoeae in the laboratory during a chronic bioassay. The fungus was of the genus Lugenidium and caused about 40% mortality of larvae before the test was terminated. Hyphal development and subsequent sporulation of the fungus was observed over a range of O-32”/,, salinity, with an optimum around 16@/,,. Bioassays of eight fungicides and the herbicide trifluralin, using the larvae and fungus in separate tests, were performed to explore means of chemical treatment. Trifluralin was the most satisfactory of the chemicals screened, with captan, Cuz+, and benomyl also giving good results for certain treatment intervals of up to 96 hr. Malachite green was toxic to larvae at concentrations inhibiting the fungus.
INTRODUCTION Natural and laboratory mycoses of marine invertebrates have been reviewed by Johnson (1970). Of particular importance have been infections caused by species of the genus Lagenidium. Parasitism of the eggs of the blue crab, Callinectes sapidus, by L. callinectes was described by Couch (1942), and elaboration of its distribution and pathogenicity were given by Sandoz et al. (1944) and RogersTalbert (1948). More recently, developmental and anatomical features of L. callinectes were discussed by Bland and Amerson (1973) and by Gotelli (1969) for a Pacific Coast Lagenidium sp. Instances of fortuitous mycoses of laboratory-reared larval crustaceans have been reported by Lightner and Fontaine (1973) in the white shrimp, Penaeus setiferus, and by Nilson et ITechnical Paper No. 4152, Oregon Agricultural Experiment Station, Corvallis, Oregon 9733 1. *Present Address: Ecology Group, Department of Water Science and Engineering, University of California, Davis, California 95616. 3Present Address: Oregon Department of Fish and Wildlife, Research Section, Corvallis, Oregon 97331.
al. (1975) in the lobster, Homarus americanus. An infection by Lagenidium sp. of larval Dungeness crab, Cancer magister, in the spring of 1973 is described in this paper. At the outbreak of the disease, larvae were being exposed to several pesticides for evaluation of chronic toxicity (Caldwell et al., unpublished data). High survival of animals exposed to one herbicide, trihuralin, prompted us to explore chemical control of the fungus, and results are discussed relative to culture procedures. MATERIALS
D 1976 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Crab larvae were hatched in the laboratory from ovigerous females collected off Newport, Oregon. During the chronic toxicity tests, larvae were reared in a continuous-flow system of glass aquaria described by Buchanan et al. (1975). All glass in the rearing system was washed twice a week in a chlorine solution, and at this time larvae were fed newly hatched brine shrimp, Artemia salina, at a density of about six shrimp/ml of test solution.
329 Copyright All rights
AND
330
ARMSTRONG.
Nine
Pesticide
Chemicals Active
Common Metiram’ CuZ+ (Bordeaux Folpet Chlorothalonif Benomyl
name
green
Dichlone Captan sCommon name bAssumed. ‘As copper.
AND
CALDWELL
TABLE I Tested for Toxicity against Cancer and the Fungus Lagenidium sp
mugister
Larvae
ingredient
Chemical
Formulation
name
Trade
name
tested Active ingredient (%)
Polyram mixture)
Trifluralin Malachite
BUCHANAN
for a complex
Copper sulfate tribasic (monohydrate) N-(trichloromethylthio)phthalimide Tetrachloroisophthalonitrile Methyl I-(butylcarbamoyl)-2benzimi dazolecarbamate cy,cu,a-Trifluoro-2,6-dinitro-N,Ndipropyl-p-toluidine Tetramethyl-di-p-aminotriphenylcarbinol chloride 2,3-Dichloro-1,4-naphthoquinone IV-trichloromethylthio-4-cyclohexene-1,2dicarboximide of zineb
and polyethylene
Sea water used in the chronic toxicity tests was pumped from Yaquina Bay, Oregon, passed through a coarse sand filter (to about 100 pm) and stored for up to 3 days in a IO,OOO-galtank. The water was adjusted to 2.5 f O.5o/oo salinity during the first 15 days of the test and afterwards was 15O/,, or higher. From the storage tank, water was pumped into a constant-temperature laboratory, refi ltered through a fine sand filter (to about 20 pm), exposed to uv irradiation, and allowed to reach 13” =t 1°C before entering exposure aquaria. The dissolved oxygen concentration of the water always exceeded 90% of saturation and the pH range was 7.6 to 7.9 during the test. The temperature of sea water in Yaquina Bay was less than 13°C when the infection was noted in our laboratory. The fungus was cultured from dead crab larvae onto kernels of hemp seed, Cannabis sativa. Perpetuation of the fungus was achieved by adding a new kernel to a beaker of 25O/,, sea water containing a heavily infected kernel. By this procedure, the fungus was kept in a state of active sporulation. Hyphae would appear on the new kernel within 18 hr of its introduction, and sporulation occurred in 40 to 60 hr.
thiuram
disulfide
IOOb 54’ 50 75 50
Phaltan Bravo Benlate Treflan
E.C.
44.5
lOOb Phygon-XL Orthocide-50W containing
80% zineb
50 50 by weight.
This method was used to test the salinity tolerance of the fungus. Three salinities, 0, 16, and 32O/,,, were tested in triplicate during six experiments. A heavily infected and an uninfected kernel were placed in 200 ml of water and not allowed to touch each other. The times to hyphal growth and subsequent sporulation on the new kernel were recorded, as were the extent and quality of these events. The toxicity of eight fungicides and the herbicide trifluralin to crab larvae and to the fungus were tested in separate 96-hr bioassays (Table 1). A logarithmic series of five concentrations of each chemical was tested in duplicate against unfed, first-stage zoeae. In all bioassays, concentrations were adjusted for the percentage of active ingredient in the specific formulation. Tests were performed in 250-ml beakers with 10 larvae per beaker. Solutions were renewed every 24 hr and were kept at 13°C and 25O/,, salinity. The criterion of toxic effect in determining ECso values (the concentration of pesticide producing a sublethal effect in 50% of the test organisms in a specified time period) was cessation of swimming, and for the LCso values (the concentration of pesticide result-
MYCOSIS
OF
LARVAL
ing in death of 50% of the test organisms in a specified time period) the criterion was cessation of all movements and development of an opaque appearance. Such values were calculated as described by the American Public Health Association et al. (197 1). The exposure beakers and water conditions were the same for the fungal bioassay. Four concentrations of each chemical were tested in duplicate with 200 ml of solution per beaker. To each beaker was added a heavily infected hemp kernel on which spores were actively being released. Twenty-four hours later, an uninfected kernel was added to the beaker and was kept from touching the inoculum; the introduction of the second kernel was considered time zero. The time lag in introduction of the two kernels ensured that
FIG. 1. View of the dorsal sp. Scale = 30 pm.
spine of Cancer
mugister
larva
331
CRABS
infecting spores had formed while in the treatment. Data from the fungal and larval bioassays were compared by means of selectivity ratios [SR = (EC,, larvae)/(concentration toxic to fungus)] for a given time period. The effect of a chemical on the fungus was an all or nothing response, i.e., either hyphae were growing on the new kernel by 48 hr or not. This precluded the calculation of 50% response values. Therefore, the denominator was taken as the lowest concentration that inhibited the transfer of the fungus to an uninfected kernel. RESULTS During the chronic toxicity bioassays (Caldwell et al., unpublished data), larvae in
showing
heavy
endobiotic
proliferation
of Lugenidium
332
ARMSTRONG,
BUCHANAN
control and low concentration pesticide treatments molted from first- to secondstage zoeae on the ninth and tenth days of exposure, and survival exceeded 95% at this time. Mortalities among these groups were
AND
CALDWELL
first recorded on day 11, and survival of control larvae had dropped to 60% by day 18 when the test was terminated. Death was apparently caused by an endobiotic fungus of the genus Lagenidiunz
FIG. 2. Typical appearance of branched Lagenidium. Scale = 10 pm. FIG. 3. Discharge vesicle with hyaline membrane filling with endogenous protoplasm of undifferentiated Scale = 10 Wm. FIG. 4. Discharge vesicle nearly filled. Protoplasmic unit (arrow) just entering vesicle. Scale = 10 pm. FIG. 5. Discharge vesicle containing differentiated, motile spores. Note hyaline membrane and empty tube (arrow). Scale = 10 pm.
spores.
discharge
MYCOSIS
OF
LARVAL
(identification has been made by William Denison, Oregon State University; Howard Whistler, University of Washington; and Charles Bland, East Carolina University; cultures of this fungus are presently being maintained by C. Bland for eventual identification as to species). The mycelium was first localized in the visceral region of the larvae. Within 24 hr, hyphae had spread throughout an animal using all available tissue for nutrition (Fig. 1). We believe this mycosis was the primary cause of death. Squashes of moribund and apparently healthy larvae were viewed under oil immersion microscopy. In no case did we find endobiotic protozoans or bacteria to suggest concurrent infection with the fungus. After death, however, zoeae were quickly invaded by ciliates and bacteria. Intramatrical hyphae of the fungus were sparingly septate, extensively branched, irregular in their growth pattern, and 8-12 pm in cross-sectional diameter (Fig. 2). Hyphae were a pale green color and had a granular
FIG.
6. Encysted
spore
on exoskeleton
333
CRABS
cytoplasm composed of refractive lipid vacuoles (Fig. 2). Extramatrical hyphae penetrated the exoskeleton after an animal died. These hyphae were non-septate and usually unbranched. Sporogenesis was completed in extramatrical discharge tubes and vesicles (Figs. 3, 4, 5) in a manner similar to descriptions given for Lugenidium by Fuller et al. (1964), Bland and Amerson (1973), and Lightner and Fontaine (1973). Spores were reniform in shape, measured 7 x 5 pm, and were propelled by two lateral flagella. Spores swam vigorously for some minutes after their release from a discharge vesicle. Gradually their movement would slow into a tightening circle. After stopping, both flagella would separate from the spore and were not seen to be retracted as described by Bland and Amerson (1973). A single germ tube grew from the then-encysted spore and could apparently penetrate some portion of the larval exoskeleton (Fig. 6). Lugenidium was able to transfer, grow,
of dead crab larva
with germ
tube just emerging.
Scale
= IO pm
334
ARMSTRONG,
BUCHANAN
and sporulate in 0 to 32O/,, salinity. However, the time required for these processes varied considerably with salinity and was most rapid at 16’/,,. Infection of the hemp kernel and subsequent hyphal growth required about 14, 40, and 55 hr at 16, 32, and O”/oo, respectively. Discharge vesicles containing spores appeared after 32, 50, and 60 hr in these same salinities. The mycelium was always less dense and the hyphae shorter on the seeds in freshwater. Since seeds used for infection were always cultured in water of 25o/oo, the time response for infection in O”/,, may partially represent the stress of no previous acclimation to zero salinity. Several chemicals with high SR values (Table 2) seem promising as chemical treatments for the fungus; the higher the SR quotient, the less toxic is the chemical to larvae relative to the fungus. Benomyl, CU’+, trifluralin, and captan had 48-hr SR values of > 100,4.5, 34, and 12, respectively. By 96 hr, however, the two former chemicals had become quite toxic to the crab larvae (Table 2), and only trifluralin and captan, with SR values of 1.5 and 6.4, could be considered as potential treatments for this time interval. The data for trifluralin from the chronic
Comparison
AND
toxicity test, where larvae and fungus occurred in the same system, gave a 96-hr SR value of 117. DISCUSSION Reports of Lagenidium infections in laboratory-reared crustacean larvae have increased in recent years (Lightner and Fontaine, 1973; Nilson et al., 1975). There is no evidence in these reports, or in our work, that Lagenidium is a natural pathogen to any of the species affected, however, and only two marine arthropods have been reported as natural hosts for fungi of this genus (Johnson, 1970). Lagenidium in this and other larval culture experiments mentioned seems to appear fortuitously as an opportunistic yet virulent pathogen. The Lagenidium sp. infecting our crabs is probably endogenous to bays and estuaries where it resides on various algae (Fuller et al., 1964; Gotelli, 1969). The euryhaline response of the fungus, with optimal growth in less than full-strength sea water (35°/oo), helps to support the contention of estuarine origins. Lagenidium cultured from Puget Sound, Washington, grew best at 15-30°/oo, and Johnson (1970) reported that the east
TABLE 2 of the Toxicity of Nine Pesticide Chemicals to Cancer Larvae and the Fungus Lagenidium sp. Toxic
Pesticide
concentrations
EC,,
Fungus’
2.4 4.5
96 hr LC50 6.7 >I00 0.1 0.56 >lOO >0.32 0.16 0.08 >I0
‘Data for inhibition of transfer of fungus to uninfected bSelectivity ratio [(SR = (EC,, larvae)/(concentration ‘Data from Caldwell et al. (1976b).
mugisrer
(mg/liter)
LaXle
48 hr
Metriam Cu*+ (Bordeaux mixture) Folpet Chlorothalonil Benomyl Trifluralin (Chronic test)c Malachite green Dichlone Captan
CALDWELL
EC,,
LGo
0.7 < 1.0
5.9 1.5
hemp kernel. toxic to fungus)]
SRb
48 hr
96 hr
48 hr
96 hr
12.5 1.0 I.0 1.o 1.0 0.005
12.5 10.0 1.0 1.0 10.0 0.1 0.004 1 1.0 0.1 0.056
0.19 4.5 0.1 0.17 > 100 34.0
0.06
1.0 0.032 0.056 for a given
time interval.
0.12 0.15 12.0
MYCOSIS
OF
LARVAL
coast L. callinectes sporulated in 5-300/,, sea water with an optimum at 200/,,. The larvae of C. magister are pelagic through the megalops stage (Lough, 1975) and usually occur offshore rather than in estuaries. The use of bay water in our experiments with these larvae probably exposed the animals to a pathogen they do not normally encounter. The manner and timing of infection in the crab larvae was not determined, but it may occur during or just after ecdysis via penetration of the still-soft exoskeleton by a germ tube. Encysted spores with emerging germ tubes were found on the exoskeleton of larvae, but we did not actually view penetration into the animals. The incubation period of the fungus on hemp kernels corresponded closely to the 36-48-hr period between ecdysis and first mortalities of crabs observed in the chronic toxicity test. Lightner and Fontaine (1973) exposed protozoeae I larvae of the shrimp Penaeus aztecus to Lagenidium. Sixty hours later, having molted to protozoeae II, some animals were infected by the fungus. Infection of larvae may have been by spores just entering the aquaria from some point without or by release of spores from well-established mycelium within the culture system. Many dead Artemia in our aquaria were infected by Lagenidium, and live Artemia have been experimentally infected by the fungus (L. Ho and D. Lightner, pers. commun.). Because of the presence of Artemia in our culture system from day 1, their frequency of molting, and their high mortality between cleaning days, the brine shrimp may have served as a host and medium for proliferation of the fungus until it became infective to the crabs. The use of herbicides and fungicides, in conjunction with other water treatments such as uv irradiation, microfiltration, or high salinities may curtail decimation of crustacean larvae by fungi such as Lagenidium. However, we stress that our pesticide toxicity data are meant only to be directional in nature. Any large-scale use of these chemicals should be prefaced by screening
CRABS
335
tests used to ascertain the sensitivity of the particular animal species involved at water conditions typically used for their culture. Of the nine chemicals tested, we believe that trifluralin is the most promising for controlling the fungus at concentrations that are not injurious to the larvae. During the initial chronic toxicity test, when the mycosis apin 0.0015 mg of peared, larvae trifluralin/liter were not affected by the fungus (Caldwell et al., unpublished data). The pesticide was toxic to the larvae in 96 hr only at concentrations nearly a 100 times greater (Table 2). The fungicide captan was the only other compound with a 96-hr SR value > 1. Benomyl was very effective over a 48-hr period (SR value > 100) and might be considered for very short treatments. We believe the SR values were obtained from somewhat extreme exposure conditions and may, therefore, be unusually low. The hemp kernel is probably an easier target for infection by Lagenidium than is a crab larva. Lagenidium spores in a particular concentration of a chemical treatment might infect hemp kernels but have difficulty in overcoming the natural defenses of an animal. Also, test concentrations of compounds in these experiments were usually spaced by IO-fold increases. Because 50% responses could not be calculated for the fungus transfer, the SR denominators were probably higher than closer spaced concentrations could have revealed. Chemical treatments for this parasite should be of a prophylactic nature. Once the fungus is endobiotic it is too late to save that portion of larvae affected. Short-term treatments centered around ecdysis in the molting cycle may be best. In our work with the toxicity of pesticides to crab larvae, we have found up to two orders of magnitude difference between acute and chronic levels (Armstrong et al., 1976; Caldwell et al., unpublished data). We believe, therefore, that prophylaxis based on chronic chemical exposures would be less desirable because lower concentrations of fungicides would be required to avoid chronic toxic affects in the
336
ARMSTRONG.
larvae, yet these concentrations less likely to affect the fungus.
BUCHANAN
would be
ACKNOWLEDGMENTS We thank Drs. C. Bland, W. Fisher, R. Krieger, and E. Nilson for reviewing the manuscript. Michael Myers provided excellent technical assistance. This research was supported by Contract No. 68-01-0188 from the United States Environmental Protection Agency and by Oregon State University Sea Grant College Program Grant No. 04-3-158-4. NOAA, U.S. Department of Commerce.
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AND
CALDWELL
ture of brachyuran crab larvae, J. Fish. Rex Board Canud. 32, 1880-1883. COUCH, J. N. 1942. A new fungus on crab eggs.J. Elisha MitcheliSci. Sot., 58, 158-162. FULLER, M. S., FOWLES, B. E., AND MCLAUGHLIN, D. J. 1964. Isolation and pure culture study of marine phycomycetes. Mycologia, 56.745-756. GOETELLI. D. 1969. “Morphology and Nutrition of the Marine Fungus Lagenidium callinectes,” I05 pp. Ph.D. Thesis, University of Washington, Seattle, Washington. JOHNSON, T. W., JR. 1970. Fungi in marine crustaceans. In “A Symposium on Diseases of Fishes and Shellfishes” (S. F. Snieszko, ed.), Amer. Fish. Sot. Spec. t’ubl.. No. 5,405-408. LIGHTNER, D. V., AND FONTAINE, C. T. 1973. A new fungus disease of the white shrimp Penaeus settyerus. J. Invertebr. Pathol.. 22.94-99. LOUGH, R. G. 1975. “Dynamics of Crab Larvae (Anomura, Brachyura) off the Central Oregon Coast, 1969-1971,” 299 pp. Ph.D. Thesis, Oregon State University, Corvallis, Oregon. NILSON,
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