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Isolation of Saprolegnia parasitica from the crayfish Astacus leptodactylus
121
Aquaculture, 92 (1991) 121-125 Elsevier SciencePublishersB.V.,Amsterdam
Isolation of Saprolegnia parasitica from the crayfish Astacus leptodactylus Kenneth S6derh411a,Michael W. Dickb, Gillian Clarkb, Magnus Fiirst’ and Ovidju Constantinescud aDepartment ofPhysiological Botany, yniversity of Uppsala, Box 540, 75121 Uppsala, Sweden bSchool qf Plant Science, Universityof Reading, 2 Ear1e.vGate, PO Box 239, Reading RG6 2.4 I! UK ‘Freshwater Laboratory, 170 11 Drottningholm, Sweden dDepartment ofSystematic Botany, Universityof Uppsala. Box 541. 751 21 Uppsala. Sweden (Accepted 30 May 1990)
ABSTRACT SGderhlll, K., Dick, M.W., Clark, G., Fiirst, M. and Constantinescu, O., 1991. Isolation of Saproiegnia parasitica from the crayfish Astacgs leptodactylus.Aquaculture, 92: 12 I- 125. On the cuticle of crayfish, melanized areas are sometimes present as a result of wounding responses or parasite attack. From such melanized spots on the cuticle of the freshwater crayfish a parasitic oomycete fungus, Saprolegnia parasitica, has been isolated.
INTRODUCTION
The crayfish population in Turkey has been almost eradicated as a result of infection by the parasitic fungus Aphanomyces astaci (Fiirst and Siiderhlll, 1987; Rahe and Soylu, 1989)) causing the disease crayfish plague. During our investigation of the causative agent for the decline in crayfish catches in Turkish waters, we have examined about 250 individuals of Astacus leptoductylus from a number of different lakes. Aphanomyces astaci is found in the cuticle of the crayfish, where its presence stimulates the production of substantial deposits of melanin, which provides the visible symptoms of the disease. We found that about 40% of the investigated crayfish had such visible symptoms of crayfish plague (Fiirst and SiiderhHll, 1987 ) and the fungus was isolated from several specimens ofA. leptodactylus.A recent report by Rahe and Soylu ( 1989) also described the isolation of Aphunomyces astaci from a Turkish specimen of A. leptodactylus. The melanization reaction is due to the phenoloxidase activity of the host, and the melanin is commonly laid down on the surface of the fungal hyphae by the host. This melanization reaction, occurring in the cuticle of crayfish. 0044-848619
l/$03.50
0 199 1 -
Elsevier Science Publishers
B.V.
122
K. SiiDERHALL ET AL.
represents the first line of defence in crustaceans towards invading parasites (Siiderhall et al., 1988 ). Although melanin and its precursors have been shown to be fungistatic towards several oomycetous fungi (SSderhlll and Ajaxon, 1982), the crayfish plague fungus, Aphanomyces astaci, and several other opportunistic parasites can survive the melanization response. Thus it is possible to isolate several different species of fungi from such melanized areas of crayfish cuticle (Cerenius et al., 1988). In our attempts to isolate the Aphanomyces species, we also isolated another oomycete from the melanized areas of the crayfish cuticle. After examining asexual life stages of this fungus both in the light microscope and in the electron microscope, its morphological characteristics were found to correspond with Saprolegnia parasitica, a wellknown pathogen of fish. MATERIAL AND METHODS
Isolation and culture Isolation and culture of oomycete fungi including A. astaci from melanized areas of the cuticle followed the methods described in Cerenius et al. ( 1988). Briefly, a melanized area or spot was cut out from the cuticle, washed carefully first with ethanol and several times with sterile water and then placed on peptone-glucose medium. A sterile glass ring was placed around the inoculum to force hyphae emerging from the piece of cuticle to grow within the agar. To the inoculum inside the glass ring was also added 0.5 ml of 0.05% potassium tellurite to deter bacterial growth. Zoospore production Attempts were made to induce zoospore production scribed by Hallett and Dick ( 1986).
by the methods de-
Electron microscopy When zoospore production was successful, suspended motile zoospores and cysts were concentrated into a small volume by collection onto a Millipore filter which was then inverted over a large drop of glass-distilled water. Formvar-coated copper grids were attached by their extreme edges with doublesided sticky tape (Scotch Pressure-Sensitive Tape) to a glass slide. Droplets of the concentrated cyst suspension were pipetted onto the grids, exposed to osmium tetroxide vapour for 30 s and allowed to evaporate to dryness at room temperature. The grids were shadowed with platinum and examined by transmission electron microscopy in an Hitachi H-800. RESULTS
AND DISCUSSION
During our attempts to isolate the crayfish plague fungus from excised cuticles of Astacus from Turkey, other species of fungi could occasionally be
ISOLATION OFS.4PROLEGNI.4 PAR4SITIC.4 FROM .4STI4Cl’S LEPTOD.4CTYLL’S
123
isolated. Persson and Siiderhill ( 1983) and Siiderhall et al. ( 1988 ) have previously noted that other oomycetes may be isolated from cuticle of the signal crayfish, Pacifastacus leniusculus, which functions as a vector for Aphanomyces astaci. From several specimens of Astacus leptodactylus we isolated a typical oomycete fungus which could be assigned to Saprolegnia. This isolate was never seen to produce oogonia on the media tested but numerous gemmae and sporangia were seen. Zoospore release was characteristic of Saprolegnia, with morphologically distinct primary and secondary forms of zoospores. Thus we decided to study this fungus more closely. Since ornamentation on primary and secondary cysts is diagnostic for some species of Saprolegnia (Pickering et al., 1979 ), the cysts were examined by electron microscopy to see if their ornamentations matched with those of any of the species so far reported (Hallett and Dick, 1986). Two kinds of ornamentation were seen. Some cysts had scattered unbarbed hairs, whereas others had bundles of long hairs, each hair terminating in a bifurcating hook (Figs. l3 ). This is consistent with the characteristics of primary and secondary cysts, respectively, of S. parasitica. This species has also been assigned to the S. diclina-S. parasitica complex (see Willoughby, 1978; Pickering et al., 1979; for a more detailed account). Saprolegnia parasitica is a severe pathogen on fish, especially salmonid species (Scott, 1964; Willoughby, 1978 ). Although S. parasitica is a severe parasite on fish, it appears not to be an important parasite on crayfish, but during intensive culture of crayfish, Saprolegnia species may cause some mortality and especially threaten females with eggs. Persson and Siiderhall (1983) have shown that other species of fungi can occasionally be isolated from melanized cuticle of Pacifastacus, either in the presence or absence of A. astaci. The melanization reaction in the cuticle is a defence response of the crayfish to wounding and parasite attacks (for overview see Siiderhill et al., 1988). However, there is a delicate balance between the host immune defence network and the growth of A. astaci in the host cuticle; if this balance is shifted in favour of the pathogen, the fungus can grow out from the melanized areas and escape the host defence reactions in the host cuticle and as a result the crayfish dies from its own, normally benign, A. astaci infection (Persson et al., 1987). Shortly after the death of the crayfish, Aphanomyces zoosporangia are produced in large numbers and further spread of the disease occurs by means of zoospores. If S. parasitica can also manage to grow out from the melanized spots and similarly escape the host defence system, this fungus may be able to produce zoosporangia and zoospores and the crayfish may then serve as a vector for this important fish pathogen. ACKNOWLEDGEMENTS
This work has been financed by the Fishery Board of Sweden and the Swedish Forestry and Agricultural Science Research Council (Contract 085 3/89 V 78:l).
124
K. SijDERHiiLL ET P
ISOLATION OF S.4PROLEGNL4 R4RASITIC4 FROM .4ST.4CC’SLEPTOMCTYLLC5’
125
REFERENCES Cerenius, L., Soderhlll, K., Persson, M. and Ajaxon, R., 1988. The crayfish plague fungus, .ilphanomyces astaci - diagnosis, isolation and pathobiology. Freshwater Crayfish, I: I 3 I144. Fiirst, M. and Soderhlll, K., 1987. The crayfish Astacus leptodactvlus in Turkey. Diseases and present distribution of the crayfish plague fungus, .4phanomyces astaci. FAO Report. Rome, 26 PP. Hallett, I.C. and Dick, M.W.. 1986. Fine structure of zoospore cyst ornamentation in the Saprolegniaceae and Pythiaceae. Trans. Br. Mycol. Sot., 86: 457-463. Persson. M. and Soderhill, K., 1983. Pacifastacus leniusculus Dana and its resistance to the parasitic fungus Aphanomyces astaci. Freshwater Crayfish, 5: 292-298. Persson, M., Cerenius, L. and Soderhill, K., 1987. The influence of haemocyte number on the resistance of the freshwater crayfish, Paclfastacus leniusculus Dana, to the parasitic fungus, .lphanomjsces astaci. J. Fish Dis., IO: 47 1-477. Pickering, A.D., Willoughby, L.G. and McGrory, C.B.. 1979. Fine structure of secondary zoospore cyst cases of Saprolegnia isolates from infected fish. Trans. Br. Mycol. Sot.. 72: 427436. Rahe, R. and Soylu, E., 1989. Identification of the pathogenic fungus causing destruction to Turkish crayfish stocks (Astacus leptodactylus). J. Invertebr. Pathol., 54: lo- 15. Scott, W.W., 1964. Fungi associated with fish diseases. In: Developments in Industrial Microbiology, Vol. 5: American Institute of Biological Sciences, Washington, DC, pp. 109- 123. Soderhall. K. and Ajaxon, R., 1982. Effect of quinones and melanin on mycelial growth of Aphanomyces spp. and extracellular protease of Aphanomyces astaci, a parasite on crayfish. J. Invertebr. Pathol., 39: 105-109. Siiderhall. K., Johansson, M.W. and Smith, V.J., 1988. Internal defence mechanisms in crayfish. In: D. Holdich and R. Lowery (Editors), Freshwater Crayfish: Biology and Exploitation. Croom Helm, London, pp. 2 13-235. Willoughby. L.G., 1978. Saprolegnias of salmonid fish in Windermere: a critical analysis. J. Fish Dis., 1: 5 1-67. Figs. l-3. Zoospore cysts of Saprolegnia parasitica isolated from .4stacus. SEM. Fig. 1. Secondary cyst with tufts of hooked hairs. Scale bar 5 ,um. Fig. 2. Primary cyst with short, simple hairs scattered over the surface. Scale bar 1 pm. Fig. 3. Secondary cyst, enlargement of Fig. 1 upper left, to show the bifurcated hooks on the ends of the hairs. Scale bar 2 Dm.
Aquaculture, 92 (1991) 121-125 Elsevier SciencePublishersB.V.,Amsterdam
Isolation of Saprolegnia parasitica from the crayfish Astacus leptodactylus Kenneth S6derh411a,Michael W. Dickb, Gillian Clarkb, Magnus Fiirst’ and Ovidju Constantinescud aDepartment ofPhysiological Botany, yniversity of Uppsala, Box 540, 75121 Uppsala, Sweden bSchool qf Plant Science, Universityof Reading, 2 Ear1e.vGate, PO Box 239, Reading RG6 2.4 I! UK ‘Freshwater Laboratory, 170 11 Drottningholm, Sweden dDepartment ofSystematic Botany, Universityof Uppsala. Box 541. 751 21 Uppsala. Sweden (Accepted 30 May 1990)
ABSTRACT SGderhlll, K., Dick, M.W., Clark, G., Fiirst, M. and Constantinescu, O., 1991. Isolation of Saproiegnia parasitica from the crayfish Astacgs leptodactylus.Aquaculture, 92: 12 I- 125. On the cuticle of crayfish, melanized areas are sometimes present as a result of wounding responses or parasite attack. From such melanized spots on the cuticle of the freshwater crayfish a parasitic oomycete fungus, Saprolegnia parasitica, has been isolated.
INTRODUCTION
The crayfish population in Turkey has been almost eradicated as a result of infection by the parasitic fungus Aphanomyces astaci (Fiirst and Siiderhlll, 1987; Rahe and Soylu, 1989)) causing the disease crayfish plague. During our investigation of the causative agent for the decline in crayfish catches in Turkish waters, we have examined about 250 individuals of Astacus leptoductylus from a number of different lakes. Aphanomyces astaci is found in the cuticle of the crayfish, where its presence stimulates the production of substantial deposits of melanin, which provides the visible symptoms of the disease. We found that about 40% of the investigated crayfish had such visible symptoms of crayfish plague (Fiirst and SiiderhHll, 1987 ) and the fungus was isolated from several specimens ofA. leptodactylus.A recent report by Rahe and Soylu ( 1989) also described the isolation of Aphunomyces astaci from a Turkish specimen of A. leptodactylus. The melanization reaction is due to the phenoloxidase activity of the host, and the melanin is commonly laid down on the surface of the fungal hyphae by the host. This melanization reaction, occurring in the cuticle of crayfish. 0044-848619
l/$03.50
0 199 1 -
Elsevier Science Publishers
B.V.
122
K. SiiDERHALL ET AL.
represents the first line of defence in crustaceans towards invading parasites (Siiderhall et al., 1988 ). Although melanin and its precursors have been shown to be fungistatic towards several oomycetous fungi (SSderhlll and Ajaxon, 1982), the crayfish plague fungus, Aphanomyces astaci, and several other opportunistic parasites can survive the melanization response. Thus it is possible to isolate several different species of fungi from such melanized areas of crayfish cuticle (Cerenius et al., 1988). In our attempts to isolate the Aphanomyces species, we also isolated another oomycete from the melanized areas of the crayfish cuticle. After examining asexual life stages of this fungus both in the light microscope and in the electron microscope, its morphological characteristics were found to correspond with Saprolegnia parasitica, a wellknown pathogen of fish. MATERIAL AND METHODS
Isolation and culture Isolation and culture of oomycete fungi including A. astaci from melanized areas of the cuticle followed the methods described in Cerenius et al. ( 1988). Briefly, a melanized area or spot was cut out from the cuticle, washed carefully first with ethanol and several times with sterile water and then placed on peptone-glucose medium. A sterile glass ring was placed around the inoculum to force hyphae emerging from the piece of cuticle to grow within the agar. To the inoculum inside the glass ring was also added 0.5 ml of 0.05% potassium tellurite to deter bacterial growth. Zoospore production Attempts were made to induce zoospore production scribed by Hallett and Dick ( 1986).
by the methods de-
Electron microscopy When zoospore production was successful, suspended motile zoospores and cysts were concentrated into a small volume by collection onto a Millipore filter which was then inverted over a large drop of glass-distilled water. Formvar-coated copper grids were attached by their extreme edges with doublesided sticky tape (Scotch Pressure-Sensitive Tape) to a glass slide. Droplets of the concentrated cyst suspension were pipetted onto the grids, exposed to osmium tetroxide vapour for 30 s and allowed to evaporate to dryness at room temperature. The grids were shadowed with platinum and examined by transmission electron microscopy in an Hitachi H-800. RESULTS
AND DISCUSSION
During our attempts to isolate the crayfish plague fungus from excised cuticles of Astacus from Turkey, other species of fungi could occasionally be
ISOLATION OFS.4PROLEGNI.4 PAR4SITIC.4 FROM .4STI4Cl’S LEPTOD.4CTYLL’S
123
isolated. Persson and Siiderhill ( 1983) and Siiderhall et al. ( 1988 ) have previously noted that other oomycetes may be isolated from cuticle of the signal crayfish, Pacifastacus leniusculus, which functions as a vector for Aphanomyces astaci. From several specimens of Astacus leptodactylus we isolated a typical oomycete fungus which could be assigned to Saprolegnia. This isolate was never seen to produce oogonia on the media tested but numerous gemmae and sporangia were seen. Zoospore release was characteristic of Saprolegnia, with morphologically distinct primary and secondary forms of zoospores. Thus we decided to study this fungus more closely. Since ornamentation on primary and secondary cysts is diagnostic for some species of Saprolegnia (Pickering et al., 1979 ), the cysts were examined by electron microscopy to see if their ornamentations matched with those of any of the species so far reported (Hallett and Dick, 1986). Two kinds of ornamentation were seen. Some cysts had scattered unbarbed hairs, whereas others had bundles of long hairs, each hair terminating in a bifurcating hook (Figs. l3 ). This is consistent with the characteristics of primary and secondary cysts, respectively, of S. parasitica. This species has also been assigned to the S. diclina-S. parasitica complex (see Willoughby, 1978; Pickering et al., 1979; for a more detailed account). Saprolegnia parasitica is a severe pathogen on fish, especially salmonid species (Scott, 1964; Willoughby, 1978 ). Although S. parasitica is a severe parasite on fish, it appears not to be an important parasite on crayfish, but during intensive culture of crayfish, Saprolegnia species may cause some mortality and especially threaten females with eggs. Persson and Siiderhall (1983) have shown that other species of fungi can occasionally be isolated from melanized cuticle of Pacifastacus, either in the presence or absence of A. astaci. The melanization reaction in the cuticle is a defence response of the crayfish to wounding and parasite attacks (for overview see Siiderhill et al., 1988). However, there is a delicate balance between the host immune defence network and the growth of A. astaci in the host cuticle; if this balance is shifted in favour of the pathogen, the fungus can grow out from the melanized areas and escape the host defence reactions in the host cuticle and as a result the crayfish dies from its own, normally benign, A. astaci infection (Persson et al., 1987). Shortly after the death of the crayfish, Aphanomyces zoosporangia are produced in large numbers and further spread of the disease occurs by means of zoospores. If S. parasitica can also manage to grow out from the melanized spots and similarly escape the host defence system, this fungus may be able to produce zoosporangia and zoospores and the crayfish may then serve as a vector for this important fish pathogen. ACKNOWLEDGEMENTS
This work has been financed by the Fishery Board of Sweden and the Swedish Forestry and Agricultural Science Research Council (Contract 085 3/89 V 78:l).
124
K. SijDERHiiLL ET P
ISOLATION OF S.4PROLEGNL4 R4RASITIC4 FROM .4ST.4CC’SLEPTOMCTYLLC5’
125
REFERENCES Cerenius, L., Soderhlll, K., Persson, M. and Ajaxon, R., 1988. The crayfish plague fungus, .ilphanomyces astaci - diagnosis, isolation and pathobiology. Freshwater Crayfish, I: I 3 I144. Fiirst, M. and Soderhlll, K., 1987. The crayfish Astacus leptodactvlus in Turkey. Diseases and present distribution of the crayfish plague fungus, .4phanomyces astaci. FAO Report. Rome, 26 PP. Hallett, I.C. and Dick, M.W.. 1986. Fine structure of zoospore cyst ornamentation in the Saprolegniaceae and Pythiaceae. Trans. Br. Mycol. Sot., 86: 457-463. Persson. M. and Soderhill, K., 1983. Pacifastacus leniusculus Dana and its resistance to the parasitic fungus Aphanomyces astaci. Freshwater Crayfish, 5: 292-298. Persson, M., Cerenius, L. and Soderhill, K., 1987. The influence of haemocyte number on the resistance of the freshwater crayfish, Paclfastacus leniusculus Dana, to the parasitic fungus, .lphanomjsces astaci. J. Fish Dis., IO: 47 1-477. Pickering, A.D., Willoughby, L.G. and McGrory, C.B.. 1979. Fine structure of secondary zoospore cyst cases of Saprolegnia isolates from infected fish. Trans. Br. Mycol. Sot.. 72: 427436. Rahe, R. and Soylu, E., 1989. Identification of the pathogenic fungus causing destruction to Turkish crayfish stocks (Astacus leptodactylus). J. Invertebr. Pathol., 54: lo- 15. Scott, W.W., 1964. Fungi associated with fish diseases. In: Developments in Industrial Microbiology, Vol. 5: American Institute of Biological Sciences, Washington, DC, pp. 109- 123. Soderhall. K. and Ajaxon, R., 1982. Effect of quinones and melanin on mycelial growth of Aphanomyces spp. and extracellular protease of Aphanomyces astaci, a parasite on crayfish. J. Invertebr. Pathol., 39: 105-109. Siiderhall. K., Johansson, M.W. and Smith, V.J., 1988. Internal defence mechanisms in crayfish. In: D. Holdich and R. Lowery (Editors), Freshwater Crayfish: Biology and Exploitation. Croom Helm, London, pp. 2 13-235. Willoughby. L.G., 1978. Saprolegnias of salmonid fish in Windermere: a critical analysis. J. Fish Dis., 1: 5 1-67. Figs. l-3. Zoospore cysts of Saprolegnia parasitica isolated from .4stacus. SEM. Fig. 1. Secondary cyst with tufts of hooked hairs. Scale bar 5 ,um. Fig. 2. Primary cyst with short, simple hairs scattered over the surface. Scale bar 1 pm. Fig. 3. Secondary cyst, enlargement of Fig. 1 upper left, to show the bifurcated hooks on the ends of the hairs. Scale bar 2 Dm.