- Email: [email protected]
Signal crayfish, Pacifastacus leniusculus, as a vector for Psorospermium haeckeli to noble crayfish, Astacus astacus
Aquaculture Aquaculture 148 ( 1996) l-9
Signal crayfish, Pacifastacus leniusculus, as a vector for Psorospermium haeckeli to noble crayfish, Astacus astacus Rolf Gydemo Department of‘ Systems Ecology, Section Gotland, Stockholm University, Ar Fleringe, 620 35 F&b;sund, Sweden
Accepted 6 July 1996
Abstract As part of investigations of the ecological impact and transmission of the crayfish parasite Psorospermium haeckeli, a laboratory experiment was performed where noble crayfish, Astacus astacus, and signal crayfish, Pacifastacus leniusculus, from populations in which P. haeckeli had not been detected, were fed with each other in all combinations. Noble crayfish fed noble crayfish did not acquire P. haeckeli during the experimental period of 10 months, while noble crayfish fed signal crayfish did. Signal crayfish fed noble crayfish showed a lower frequency of definitive positive infestation than signal crayfish fed signal crayfish. The results support the theory that Psorospermium haeckeli has been introduced to the Baltic island of Gotland with signal crayfish, and that signal crayfish, when not infected with the crayfish plague fungus Aphanomyces astaci, is more successful in suppressing the parasite. The investigations also showed that the technique of digesting soft tissues might be an inadequate method of observing P. haeckeli in signal crayfish.
Keywords: Crayfish Pacifustucus
parasite; Psorospermium haeckeli; Noble crayfish;
Astcrcus crstacus; Signal
crayfish;
leniusculus
1. Introduction Psorospermium haeckeli is generally described as a parasite of crayfish, and has been connected to mortalities in freshwater crayfish (Vey, 1978, Edgerton et al., 1995) although the pathogenecity is uncertain. It was first described by Haeckel (18571, but is still not well known (Alderman and Polglase, 1988). The taxonomic position of the organism is not established. The distribution is unknown in detail but it has been 00448486/96/$15.00 Copyright PII SOO44-8486(96)01400-7
0 1996 Elsevier Science B.V. All rights reserved.
2
R. Gydemo/Ayuoculrurr
148 (1996) 1-9
reported from Europe, Asia, North America and Australia, i.e. all continents with native crayfish. Grabda (1934) described several different stages and information has been added by Henttonen et al. (1995) and Rug and Vogt (1994, 19951, but the complete life cycle is not known. The transmission of the parasite is also unknown although infection through ingestion has been suggested (Grabda, 1934, Henttonen, personal communication). During the last 15-20 years, an increase in P. haeckeli has been reported. Whether this is due to a true increase of the parasite, or a result of increased interest in crayfish research, is still being discussed. This increase also coincides with the introduction of the American signal crayfish, Pacifustucus leniusculus, to Europe in the early 1960s and a possible connection has been suggested.
2. Background
to the investigation
The first known introduction of the noble crayfish, Astucus astucus, to the Baltic island of Gotland was in 1842 (Steffenburg, 18721, and later introductions, also from the mainland of Sweden, have been made on at least eight different occasions. Crayfish have been introduced or spread naturally to most waters on the island. The island, with a calcareous bedrock, excellent water quality, favourable climate and relative isolation, was exempted early on from introductions of the American signal crayfish, Pacifustucus leniusculus. This species, introduced to Sweden in 1960 to restore crayfish stock after the severe effects of the crayfish plague since its first appearance in 1907 (Sv-dson, 1965, 1995), has been demonstrated to be a vector for the crayfish plague fungus Aphanomyces astuci to noble crayfish (Unestam, 1973). Gotland, together with a few other areas in Sweden, were set aside as refuges for the native noble crayfish. Surveys and investigations on the noble crayfish on the island in the 1980s did not reveal any indications of the presence of crayfish plague. Only porcelain or white-tail disease, caused by the microsporidian Thelohania contejeani, and the burnt-spot disease, caused by the fungus Ramularia astaci, were found in noble crayfish. In 1985, signal crayfish were discovered on the island, in a sympatric population with noble crayfish. Repeated analyses did not reveal any signs of the crayfish plague or any other disease or parasite. The signal crayfish had been illegally introduced from the province of SmHland on the mainland of Sweden to a pond in a former limestone quarry around 1970, while the noble crayfish were descendants of the first introduction in 1842. In 1990, Psorospermium haeckeli was found in noble crayfish but not in signal crayfish in a crayfish farm which had obtained its stocking material, containing both noble and signal crayfish, from the aforementioned quarry pond and (noble crayfish only) from the stream of the original introduction in 1842. Thus there were two possible donor populations of P. haeckeli. When sampling these, only signal crayfish were left in the quarry pond. Analyses did not show the presence of P. haeckeli in signal crayfish from the quarry pond, nor in noble crayfish from the original stream. Approximately 600 signal crayfish from the farmed sympatric crayfish population were kept in indoor concrete basins in high densities (150 m-*) in chlorinated tap water and under stressful conditions of working hour disturbances of noice and vibrations
R. Gydemo/Aquuculture
148 (1996) 1-9
3
during 5 months. Analyses by the Swedish Crayfish Health Control (SCHC) with use of the digestion technique (digestion of soft tissues in a 1% pepsin-HCl solution followed by centrifugation and examination under an inverted microscope (Cerenius et al., 1991)) of 50 specimen did not reveal any P. haeckeli. From the same batch, five signal crayfish were analysed at our laboratory by making squash preparations of scrapings from underneath the carapace and abdominal muscle, and examining the slides under a conventional microscope. In three of these, we observed P. haeckeli. When the SCHC was informed, they analysed more of the material microscopically, too, and discovered a few P. haeckeli in the signal crayfish. They have since included analyses by microscopy in their routines. The question arose as to whether Psorospermium haeckeli originated from the noble crayfish, of the same origin in both cases, or from the signal crayfish.
3. Material and methods In order to investigate whether the signal crayfish can act as a vector for Psorosperan experiment was designed in which were used noble crayfish from a population that had been analysed on several occasions without any signs of P. haeckeli, and signal crayfish from the pond of the original, illegal, introduction around 1970, which also was considered to be P. haeckeli-free according to previous investigations. The noble crayfish population descended from the first introduced noble crayfish on Gotland. As the experimental site, the same four concrete basins, measuring 1.2m X 1.2 m, as mentioned before, were used. The water depth was approximately 25 cm. The water temperature changed from 17 to 12°C and back to 16°C during the experiment. Prior to the start of the experiment, the basins were emptied of water and cleaned, left dry for 5 months, treated with iodine, burned with a propane flame, washed with 70% alcohol and treated once more with iodine in an attempt to exclude the possibility that any stage of P. haeckeli would remain. Four groups, two with A. astacus and two with P. leniusculus, each consisting of 40 individuals of sizes in the range from 7 to 11.5 cm, were placed under investigation. Additional noble and signal crayfish of the same respective origin were kept in separate tanks to serve as food. The experiment began in August 1991, when the basins were stocked according to Table 1. As food, the crayfish received freshly killed crayfish three times a week according to Table 1. A total of 264 noble crayfish and 232 signal crayfish were used as food, i.e. approximately 1 crayfish was fed to each group on each of the 111 feeding occasions. In addition, all crayfish were also fed potatoes and carrots once a week. The difference between stocked numbers and final numbers results from crayfish that died and in most cases were consumed between two feeding occasions. If remnants were found, these were also analysed. The conditions were as previously described, i.e. stressful. In March 1992, a subsample of 10 crayfish was taken from each group for analysis. The experiment ended in May 1992, when the remaining crayfish in each group were killed by freezing. Analysis was performed under a microscope. Preparations were made of tissue from
mium haeckeli,
4
R. Gydemo / Ayuuculture
148 (1996) I-9
Table I Design of and results from an experiment of 10 month duration on the transmission hueckeli infestation in noble crayfish, Astucus astacus, and signal crayfish, Pacijmucus
of Psomspermium Imiusculus
Group I: A. u.stucu.s/ A. mtucus a
Group II: A. ustucu.s/ P. leniusculus a
Group III: P. leniusculu.s/ A. ustucus a
Group IV: P. leniusculu.s/ P. lrniusculus a
No. at start No. in subsample, March No. infested No. at end, May No. infested No. dead outside sampling occasions
40 10 0 12 0 18
40
40 10 Ob 7 Id 23
40 10 lb 12 3 18
No. analysed No. infested No. “missing” Total no. analysed Definite positive infestation Suspected positive infestation Total positive or suspected positive infestation
4 0 14 26 0 0 0
5
5 0 18 22
12 2 4 34 6 2 8
IO
10 4c 20
14 25 6 5 II
6 7
a Food. b + 2 suspect positive infestation. ’ + 5 suspect positive infestation. d + 4 suspect positive infestation.
underneath the carapace, hepatopancreas and abdominal muscle close to the gut and a scraping from the exoskeleton. In addition, preparations were also made of tissue at wound areas, when present. From each site, at least two preparations were made, yielding a minimum of eight preparations per crayfish. As a complement, the digestion of soft tissue in a pepsin-HCl solution, according to the method described by Cerenius et al. (199 1), was performed on the subsample in March 1992.
4. Results In the March material, Psorospermium haeckeli (Fig. 1) was found in all groups except the first. The result was the same in the May material (Table 1). For all infested crayfish, the frequency of infestation was less than 15 P. haeckeli per field of view at 100 X magnification. In signal crayfish, the number of observed P. haeckeli was less than 10 per field of view, which was less than in noble crayfish. The results were similar in crayfish dead outside the sampling occasions. Using the digestion technique, in addition, on the March subsample did not reveal results that were any different. P. haeckeli was only observed in crayfish of both species after 6 months. Furthermore, unidentified, unicellular bodies were observed. In general, the size of these was similar or somewhat smaller, the cell walls were thinner, the nucleus more dispersed inside the
R. Gydemo/Aqucrculturr
148 (1996) I-9
Fig. 1. Psorospermium haeckeli in carapax connective tissue from a signal crayfish, fed signal crayfish. (Fresh mount; 100X magnification; scale bar, 100 km.)
Pacifastacus
Imiusculus,
cell and the typical sutures on the shell were vague or lacking. Since these did not fit into the typical well-known appearance, they were considered to be uncertain. These latter uncertain observations did not influence the general result. A test (Chi-square) of whether the infestation rate was independent of food (treatment) for all combinations and using only certain observations lead to rejection ( p = 0.05). If each species was tested likewise, the significance was even greater. The mortality was equally high in all groups in the experiment. The mortality increased between March and May, most likely due to the increased water temperature and activity coupled to the relative scarcity of food. Most dead crayfish were partially or fully consumed (cannibalised). In the remnants that were able to be analysed, no higher incidence of P. haeckeli was found. Among the noble crayfish fed noble crayfish, group I, no P. haeckeli was found either in the March subsample or in the May final sample, or in any crayfish found dead at any other time. Among the noble crayfish fed with signal crayfish, group II, P. haeckeli was found in both the subsample and at the end, but in different numbers. In the March subsample, P. haeckeli was found in one crayfish. In the May sample, P. haeckeli was found in four out of ten crayfish and in another five, observations of P. haeckeli-like structures were made. Of the five crayfish dead at other times, one showed the presence of P. haeckeli. If the five uncertain observations are included, the total of infested noble crayfish is 11. Among the signal crayfish fed with noble crayfish, group III, two uncertain observations of P. haeckeli were made in the March subsample. In the final, May, sample, one certain observation was made and four uncertain ones. No P. haeckeli was observed in crayfish dead at other times. Including the six uncertain observations gives seven cases of infested signal crayfish.
6
R. Gydemo/Aquuculrure
148 (1996) I-9
Among the signal crayfish fed with signal crayfish, group IV, P. haeckeli w’as observed in both samples as well as in two out of 12 dead outside the sampling periods, and again more of them in the final sample. The number of infested crayfish was six and including the two uncertain observations, the total number was eight.
5. Discussion Relying only on the certain observations, the frequency of infestation is similar, six cases, in group II and group IV. Both groups were fed with P. leniusculus. Group III, P. leniusculus fed with A. astacus had only one certain observation. If all uncertain observations, i.e. suspected positive infestations, were considered as certain, there would be 0 incidences in group I, 11 in group II, 7 in group III and 8 in group IV. The similar outcome in groups III and IV also indicates that A. astacus did not have any effect on increasing the rate of infestation and it seems that the signal crayfish in groups III and IV developed Psorospermium haeckeli infestation by ingesting killed conspecifics, by the stressful conditions, or a combination. The results could furthermore indicate that P. haeckeli has a more rapid development to the well-known stage in A. astacus than in P. leniusculus. The similar mortality in all the groups, without any demonstrative differences in the degree of infestation by P. haeckeli, suggests that the mortality was due to interaction and food shortage rather than the parasite. The experiment demonstrates that the noble crayfish used did not carry P. haeckeli. Since the noble crayfish used as food was from the same population as in group I, the only source for P. haeckeli in group II, must be the signal crayfish. This is further substantiated in group III, and by the outcome in group IV, where the only possible source of P. haeckeli is the signal crayfish themselves. It is, however, unsatisfactory that there are relatively many observations of uncertain P. haeckeli. On the other hand, this serves to illustrate the need for more thorough investigations on the various stages in the life cycle of Psorospermium haeckeli. Rug and Vogt (1994, 1995) present photographs of a number of developmental stages, of which some are quite similar to those observed in this material and denoted as being uncertain. Also, Henttonen et al. (1995) have observed similar stages. This strengthens the likelihood that the observations denoted as being uncertain were actually developmental stages of P. haeckeli. The results furthermore indicate that the parasite can develop without any intermediate host, which is in accordance with observations by Rug and Vogt (19951, and since the only feed besides crayfish was vegetables (potatoes and carrots>, the only source of P. haeckeli must be the crayfish. Thus, signal crayfish fed noble crayfish did not obtain any additional supply of the parasite, as did the signal crayfish fed signal crayfish. Under the given (stressful) conditions, the signal crayfish may have responded by “permitting” the parasite to develop to the easily identifiable stage, which did not happen in the original pond population. Since the signal crayfish on Gotland are not carrying the crayfish plague parasite, their immune defence systems do not have to fight a “two-front battle” and
R. Gydemo/Aquuculturr
148 (1996) I-9
7
can cope more efficiently with Psorospetmium haeckeli. There are indications to support this theory. In Lake Skillotsjbn, Sweden, a sympatric population of noble crayfish and signal crayfish is present. P. haeckeli has only been found in the noble crayfish, which Svkdson et al. (1991) suggested to be due to a higher resistance in P. leniusculus. Fenouil et al. (1995) investigated lysozyme activity, considered as being a part of the humoral defence system, in six crayfish species. The American crayfish species Orconectes limosus, Procambarus clarkii and Pacifastacus leniusculus all showed a higher lysozyme activity than the European Astacidae species, Astacus leptodactylus, Astacus astacus and Austropotamobius pallipes. Among the European species, A. leptodactylus exhibited around 20% higher lysis than A. astucus after 120 min, almost corresponding to the records for P. leniusculus. This is in accordance with the findings of Unestam (1969) in which A. leptoductylus showed a higher resistance to crayfish plague than A. astucus. Rirst and SGderhall (1987) reported that A. leptoductylus in Turkey infected with the crayfish plague fungus Aphanomyces astaci suffered more heavily when also infected with P. haeckeli. P. leniusculus, when their resistance is weakened, will easily die from infestation by crayfish plague (Persson and SGderhall, 1983 Persson and SGderhall, 1985, Persson et al., 1987). Cerenius and Sijderhall (1993) stress that the few observations of P. haeckeli in signal crayfish and the low degree of infestation should not be interpreted in terms of the signal crayfish being more resistant to P. haeckeli since the capacity of the signal crayfish immune defence system is reduced when chronically infected with the crayfish plague. The signal crayfish in this investigation were not infected by the crayfish plague. Thus, the more efficient immune defence system in signal crayfish may be effective in suppressing the development of P. haeckeli in the absence of other parasites, but when exposed to stress, the immune system is weakened (Siiderhall and Cerenius, 1992) and P. haeckeli develops to the easy identifiable stage. The experiment demonstrates that noble crayfish fed with signal crayfish can become infested with Psorospermium haeckeli. Signal crayfish fed with noble crayfish can also develop P. haeckeli but at a lower frequency than signal crayfish fed signal crayfish. Unestam (1973) and Cerenius and Sbderhall (1993) did observe P. haeckeli in a few Swedish signal crayfish populations but in low numbers. Similar findings were made by Dieguez-Uribeondo et al. (1993) in signal crayfish from Spain: the signal crayfish may harbour P. haeckeli but so far are observed only in comparatively low numbers. The implication with regard to signal crayfish is that, since it is generally a carrier of the crayfish plague, P. haeckeli may be a severe threat, as Cerenius et al. (1991) suggested that ‘even if a P. haeckeli infection itself is not lethal to the host, the reduced immune capacity of the animal will make it more sensitive to other pathogens’. This was demonstrated by Sijderh’;dll and Cerenius (19921, when noble crayfish and “normal” (i.e. infected by A. astaci) signal crayfish were artificially infested with P. haeckeli, the noble crayfish survived while the signal crayfish died from acute crayfish plague attack. Under natural conditions, signal crayfish that have the heaviest load of crayfish plague will also be the most vulnerable to other factors stressing the immune defence system. Thus, the crayfish succumb, thereby not being able to be analysed. The conclusion from the present experiment must be that signal crayfish can act as a vector for P. haeckeli and are the likely source of introduction of the parasite to
8
R. Gydemo/Aquaculture
148 (1996) 1-9
Gotland. Further surveys of infested noble crayfish populations have infested populations. Both of them have also been in contact with population used in this study. Furthermore, the routinely used technique of digestion in crayfish not be adequate when analysing signal crayfish, though it seems method for detecting Psorospermium haeckeli in noble crayfish.
revealed two more the signal crayfish health control may to be an adequate
6. Recommendations 1. When analysing signal crayfish for diseases and parasites, slide preparations of tissues should be used as well as the digestion technique. 2. If possible, the signal crayfish should be kept under stressful conditions for a period of several weeks at least before analysis. 3. It is of utmost importance that the complete life cycle of the parasite is clarified in order to be able to clearly identify the different developmental stages. 4. No further transplanations or introductions of signal crayfish should be made until it is clearly established which signal crayfish populations/stocks are carrying the parasite. 5. Policies for crayfish diseases and introductions of non-native crayfish species should be reconsidered. 6. The severity of the threat of Psorospermium haeckeli to the crayfish species should be determined.
Acknowledgements Thanks are due to Per Ramnelius who skilfully participated in the experiment, Gunnar Pettersson who kindly supplied the experimental facilities, and Pia Keyser who analysed part of the material. Financial support was obtained from the Carl Trygger Foundation, the Swedish Farmers Foundation for Agricultural Research and the Gunvor and Josef Aneers Foundation, which is gratefully acknowledged.
References Alderman, D.J. and Polglase, J.L., 1988. Pathogens, parasites and commensals. In: D.M. Holdich and R.S. Lowery (Eds.), Freshwater Crayfish - biology, management and exploitation. Croom Helm, London and Sydney, pp. 167-212 Cerenius, L. and Siiderhlll, K., 1993. The distribution of Psorospermium haeckli in Sweden; a preliminary survey. Freshwater Crayfish, 9: 280-285. Cerenius, L., Henttonen, P., Lindqvist, 0. and SiiderhBll, K., 1991. The crayfish pathogen Psorospermium haeckeli activates the prophenol-oxidase activating system of freshwater crayfish in vitro. Aquaculture, 99: 225-233 Edgerton. B., Owens, L., Harris, L., Thomas, A. and Wingfield, M., 1995. A health survey of farmed redclaw crayfish, Cheru qudricurinatus, (van Martens), in tropical Austalia. Freshwater Crayfish, 10: 322-338
R. Gydemo/Ayuaculturr
148 (1996) I-9
9
Fenouil, E., Roth, P. and de Hureaux, D., 1995. Immune defense in Astacidae and Cambaridae: presence of lysozyme activity. Freshwater Crayfish, 8: 614-622 Ftirst, M. and Sikierhall, K., 1987. The crayfish Astucus leptodtictylus in Turkey. Diseases and present distribution of the crayfish plague fungus, Aphanomyce.s ustaci. FAO Report, Rome, pp. 26. Grabda, E., 1934. Recherches sur un parasite de l’ecrevisse (Porcrmohius pcrllipes L.) connu sous le nom de Psorospermium haeckeli. Hilgd. Mem. Acad. Pol. Sci. Lett., S.B. Math. u Naturw., Cracow, 6: 123- 142 (in French). Haeckel, E., 1857. Uber die Gewebe des Flusskebses. Arch. Anat. Physiol., 24: 469-568 (in German). Henttonen, P., Hurter, J.V. and Lindqvist, O.V., 1995. Observations on Psorospermium hoeckeli in noble crayfish Astucus astacus CL.) (Decapoda, Astacidae) populations in Central Finland. Freshwater Crayfish, IO: 339-351. Persson, M. and SBderhall, K., 1983. Pacifastacus lrniusculus Dana and its resistance to the parasitic fungus, Aphanomyces ustuci Shikora. Freshwater Crayfish, 5: 292-298. Persson, M. and SGderhiill, K., 1985. Psorospermium haeckeli - parasit pl kraftor. (Psorospennium - a crayfish parasite). PM. Inst. Fys. Botanik, Uppsala Universitet, pp. 5 (in Swedish). Persson, M., Cerenius, L. and Siiderhall, K., 1987. The influence of haemocyte number on the resistance of the freshwater crayfish, Pacij2zstacu.s leniusculus Dana, to the parasitic fungus, Aphanomyces astuci. J. Fish Dis., 10: 471-477. Rug, M. and Vogt, G., 1994. Developmental stages of the crayfish parasite Psorospermium haeckeli in thoracic arteries of Astacus ustucus. J. Invertebrate Pathol., 64: 153- 155. of developing and mature spores of two Rug, M. and Vogt, G., 1995. Histology and histochemistry morphotypes of Psorospermium haeckeli. Freshwater Crayfish, 10: 374-384. A., 1872. Bidrag till kannedomen om flodkiaftans naturalhistoria. (A contribution to the Steffenbug, knowledge on the natural history of the crayfish). Reprinted 1972 in Information f&t Sbtvattenslaboratoriet, Drottningholm (14), p. 23 (in Swedish) Svardson, G., 1965. The American crayfish Pacifirstacusfeniusculus (Dana), introduced into Sweden. Rep. Inst. Freshwater Res., Drottningholm, Vol. 46, p. 90-94 Svlrdson, G., 1995. The early history of signal crayfish introduction into Europe. Freshwater Crayfish, 8: 68-77. Svardson, G., Fiirst, M. and Fjalling, A., 1991. Population resilience of Pacijtistacus leniusculus in Sweden. Finn. Fish. Res., 12: 165-177 Siiderhall, K. and Cerenius, L., 1992. Crustacean immunity. Annu. Rev. Fish Dis., 2: 3-23. Unestam, T., 1969. Resistance to the crayfish plague in some American, Japanese and European crayfishes. Rep. Inst. Freshwater Res., Drottningholm, Vol. 49, p. 202-209. Unestam, T., 1973. Significance of diseases in freshwater crayfish. Freshwater Crayfish, I: 135-150. Vey, A., 1978. Recherches sur une maladie des ecrevisses due au parasite Psom.~permium haeckeli. Freshwater Crayfish, 4: 41 l-41 8 (in French).
Signal crayfish, Pacifastacus leniusculus, as a vector for Psorospermium haeckeli to noble crayfish, Astacus astacus Rolf Gydemo Department of‘ Systems Ecology, Section Gotland, Stockholm University, Ar Fleringe, 620 35 F&b;sund, Sweden
Accepted 6 July 1996
Abstract As part of investigations of the ecological impact and transmission of the crayfish parasite Psorospermium haeckeli, a laboratory experiment was performed where noble crayfish, Astacus astacus, and signal crayfish, Pacifastacus leniusculus, from populations in which P. haeckeli had not been detected, were fed with each other in all combinations. Noble crayfish fed noble crayfish did not acquire P. haeckeli during the experimental period of 10 months, while noble crayfish fed signal crayfish did. Signal crayfish fed noble crayfish showed a lower frequency of definitive positive infestation than signal crayfish fed signal crayfish. The results support the theory that Psorospermium haeckeli has been introduced to the Baltic island of Gotland with signal crayfish, and that signal crayfish, when not infected with the crayfish plague fungus Aphanomyces astaci, is more successful in suppressing the parasite. The investigations also showed that the technique of digesting soft tissues might be an inadequate method of observing P. haeckeli in signal crayfish.
Keywords: Crayfish Pacifustucus
parasite; Psorospermium haeckeli; Noble crayfish;
Astcrcus crstacus; Signal
crayfish;
leniusculus
1. Introduction Psorospermium haeckeli is generally described as a parasite of crayfish, and has been connected to mortalities in freshwater crayfish (Vey, 1978, Edgerton et al., 1995) although the pathogenecity is uncertain. It was first described by Haeckel (18571, but is still not well known (Alderman and Polglase, 1988). The taxonomic position of the organism is not established. The distribution is unknown in detail but it has been 00448486/96/$15.00 Copyright PII SOO44-8486(96)01400-7
0 1996 Elsevier Science B.V. All rights reserved.
2
R. Gydemo/Ayuoculrurr
148 (1996) 1-9
reported from Europe, Asia, North America and Australia, i.e. all continents with native crayfish. Grabda (1934) described several different stages and information has been added by Henttonen et al. (1995) and Rug and Vogt (1994, 19951, but the complete life cycle is not known. The transmission of the parasite is also unknown although infection through ingestion has been suggested (Grabda, 1934, Henttonen, personal communication). During the last 15-20 years, an increase in P. haeckeli has been reported. Whether this is due to a true increase of the parasite, or a result of increased interest in crayfish research, is still being discussed. This increase also coincides with the introduction of the American signal crayfish, Pacifustucus leniusculus, to Europe in the early 1960s and a possible connection has been suggested.
2. Background
to the investigation
The first known introduction of the noble crayfish, Astucus astucus, to the Baltic island of Gotland was in 1842 (Steffenburg, 18721, and later introductions, also from the mainland of Sweden, have been made on at least eight different occasions. Crayfish have been introduced or spread naturally to most waters on the island. The island, with a calcareous bedrock, excellent water quality, favourable climate and relative isolation, was exempted early on from introductions of the American signal crayfish, Pacifustucus leniusculus. This species, introduced to Sweden in 1960 to restore crayfish stock after the severe effects of the crayfish plague since its first appearance in 1907 (Sv-dson, 1965, 1995), has been demonstrated to be a vector for the crayfish plague fungus Aphanomyces astuci to noble crayfish (Unestam, 1973). Gotland, together with a few other areas in Sweden, were set aside as refuges for the native noble crayfish. Surveys and investigations on the noble crayfish on the island in the 1980s did not reveal any indications of the presence of crayfish plague. Only porcelain or white-tail disease, caused by the microsporidian Thelohania contejeani, and the burnt-spot disease, caused by the fungus Ramularia astaci, were found in noble crayfish. In 1985, signal crayfish were discovered on the island, in a sympatric population with noble crayfish. Repeated analyses did not reveal any signs of the crayfish plague or any other disease or parasite. The signal crayfish had been illegally introduced from the province of SmHland on the mainland of Sweden to a pond in a former limestone quarry around 1970, while the noble crayfish were descendants of the first introduction in 1842. In 1990, Psorospermium haeckeli was found in noble crayfish but not in signal crayfish in a crayfish farm which had obtained its stocking material, containing both noble and signal crayfish, from the aforementioned quarry pond and (noble crayfish only) from the stream of the original introduction in 1842. Thus there were two possible donor populations of P. haeckeli. When sampling these, only signal crayfish were left in the quarry pond. Analyses did not show the presence of P. haeckeli in signal crayfish from the quarry pond, nor in noble crayfish from the original stream. Approximately 600 signal crayfish from the farmed sympatric crayfish population were kept in indoor concrete basins in high densities (150 m-*) in chlorinated tap water and under stressful conditions of working hour disturbances of noice and vibrations
R. Gydemo/Aquuculture
148 (1996) 1-9
3
during 5 months. Analyses by the Swedish Crayfish Health Control (SCHC) with use of the digestion technique (digestion of soft tissues in a 1% pepsin-HCl solution followed by centrifugation and examination under an inverted microscope (Cerenius et al., 1991)) of 50 specimen did not reveal any P. haeckeli. From the same batch, five signal crayfish were analysed at our laboratory by making squash preparations of scrapings from underneath the carapace and abdominal muscle, and examining the slides under a conventional microscope. In three of these, we observed P. haeckeli. When the SCHC was informed, they analysed more of the material microscopically, too, and discovered a few P. haeckeli in the signal crayfish. They have since included analyses by microscopy in their routines. The question arose as to whether Psorospermium haeckeli originated from the noble crayfish, of the same origin in both cases, or from the signal crayfish.
3. Material and methods In order to investigate whether the signal crayfish can act as a vector for Psorosperan experiment was designed in which were used noble crayfish from a population that had been analysed on several occasions without any signs of P. haeckeli, and signal crayfish from the pond of the original, illegal, introduction around 1970, which also was considered to be P. haeckeli-free according to previous investigations. The noble crayfish population descended from the first introduced noble crayfish on Gotland. As the experimental site, the same four concrete basins, measuring 1.2m X 1.2 m, as mentioned before, were used. The water depth was approximately 25 cm. The water temperature changed from 17 to 12°C and back to 16°C during the experiment. Prior to the start of the experiment, the basins were emptied of water and cleaned, left dry for 5 months, treated with iodine, burned with a propane flame, washed with 70% alcohol and treated once more with iodine in an attempt to exclude the possibility that any stage of P. haeckeli would remain. Four groups, two with A. astacus and two with P. leniusculus, each consisting of 40 individuals of sizes in the range from 7 to 11.5 cm, were placed under investigation. Additional noble and signal crayfish of the same respective origin were kept in separate tanks to serve as food. The experiment began in August 1991, when the basins were stocked according to Table 1. As food, the crayfish received freshly killed crayfish three times a week according to Table 1. A total of 264 noble crayfish and 232 signal crayfish were used as food, i.e. approximately 1 crayfish was fed to each group on each of the 111 feeding occasions. In addition, all crayfish were also fed potatoes and carrots once a week. The difference between stocked numbers and final numbers results from crayfish that died and in most cases were consumed between two feeding occasions. If remnants were found, these were also analysed. The conditions were as previously described, i.e. stressful. In March 1992, a subsample of 10 crayfish was taken from each group for analysis. The experiment ended in May 1992, when the remaining crayfish in each group were killed by freezing. Analysis was performed under a microscope. Preparations were made of tissue from
mium haeckeli,
4
R. Gydemo / Ayuuculture
148 (1996) I-9
Table I Design of and results from an experiment of 10 month duration on the transmission hueckeli infestation in noble crayfish, Astucus astacus, and signal crayfish, Pacijmucus
of Psomspermium Imiusculus
Group I: A. u.stucu.s/ A. mtucus a
Group II: A. ustucu.s/ P. leniusculus a
Group III: P. leniusculu.s/ A. ustucus a
Group IV: P. leniusculu.s/ P. lrniusculus a
No. at start No. in subsample, March No. infested No. at end, May No. infested No. dead outside sampling occasions
40 10 0 12 0 18
40
40 10 Ob 7 Id 23
40 10 lb 12 3 18
No. analysed No. infested No. “missing” Total no. analysed Definite positive infestation Suspected positive infestation Total positive or suspected positive infestation
4 0 14 26 0 0 0
5
5 0 18 22
12 2 4 34 6 2 8
IO
10 4c 20
14 25 6 5 II
6 7
a Food. b + 2 suspect positive infestation. ’ + 5 suspect positive infestation. d + 4 suspect positive infestation.
underneath the carapace, hepatopancreas and abdominal muscle close to the gut and a scraping from the exoskeleton. In addition, preparations were also made of tissue at wound areas, when present. From each site, at least two preparations were made, yielding a minimum of eight preparations per crayfish. As a complement, the digestion of soft tissue in a pepsin-HCl solution, according to the method described by Cerenius et al. (199 1), was performed on the subsample in March 1992.
4. Results In the March material, Psorospermium haeckeli (Fig. 1) was found in all groups except the first. The result was the same in the May material (Table 1). For all infested crayfish, the frequency of infestation was less than 15 P. haeckeli per field of view at 100 X magnification. In signal crayfish, the number of observed P. haeckeli was less than 10 per field of view, which was less than in noble crayfish. The results were similar in crayfish dead outside the sampling occasions. Using the digestion technique, in addition, on the March subsample did not reveal results that were any different. P. haeckeli was only observed in crayfish of both species after 6 months. Furthermore, unidentified, unicellular bodies were observed. In general, the size of these was similar or somewhat smaller, the cell walls were thinner, the nucleus more dispersed inside the
R. Gydemo/Aqucrculturr
148 (1996) I-9
Fig. 1. Psorospermium haeckeli in carapax connective tissue from a signal crayfish, fed signal crayfish. (Fresh mount; 100X magnification; scale bar, 100 km.)
Pacifastacus
Imiusculus,
cell and the typical sutures on the shell were vague or lacking. Since these did not fit into the typical well-known appearance, they were considered to be uncertain. These latter uncertain observations did not influence the general result. A test (Chi-square) of whether the infestation rate was independent of food (treatment) for all combinations and using only certain observations lead to rejection ( p = 0.05). If each species was tested likewise, the significance was even greater. The mortality was equally high in all groups in the experiment. The mortality increased between March and May, most likely due to the increased water temperature and activity coupled to the relative scarcity of food. Most dead crayfish were partially or fully consumed (cannibalised). In the remnants that were able to be analysed, no higher incidence of P. haeckeli was found. Among the noble crayfish fed noble crayfish, group I, no P. haeckeli was found either in the March subsample or in the May final sample, or in any crayfish found dead at any other time. Among the noble crayfish fed with signal crayfish, group II, P. haeckeli was found in both the subsample and at the end, but in different numbers. In the March subsample, P. haeckeli was found in one crayfish. In the May sample, P. haeckeli was found in four out of ten crayfish and in another five, observations of P. haeckeli-like structures were made. Of the five crayfish dead at other times, one showed the presence of P. haeckeli. If the five uncertain observations are included, the total of infested noble crayfish is 11. Among the signal crayfish fed with noble crayfish, group III, two uncertain observations of P. haeckeli were made in the March subsample. In the final, May, sample, one certain observation was made and four uncertain ones. No P. haeckeli was observed in crayfish dead at other times. Including the six uncertain observations gives seven cases of infested signal crayfish.
6
R. Gydemo/Aquuculrure
148 (1996) I-9
Among the signal crayfish fed with signal crayfish, group IV, P. haeckeli w’as observed in both samples as well as in two out of 12 dead outside the sampling periods, and again more of them in the final sample. The number of infested crayfish was six and including the two uncertain observations, the total number was eight.
5. Discussion Relying only on the certain observations, the frequency of infestation is similar, six cases, in group II and group IV. Both groups were fed with P. leniusculus. Group III, P. leniusculus fed with A. astacus had only one certain observation. If all uncertain observations, i.e. suspected positive infestations, were considered as certain, there would be 0 incidences in group I, 11 in group II, 7 in group III and 8 in group IV. The similar outcome in groups III and IV also indicates that A. astacus did not have any effect on increasing the rate of infestation and it seems that the signal crayfish in groups III and IV developed Psorospermium haeckeli infestation by ingesting killed conspecifics, by the stressful conditions, or a combination. The results could furthermore indicate that P. haeckeli has a more rapid development to the well-known stage in A. astacus than in P. leniusculus. The similar mortality in all the groups, without any demonstrative differences in the degree of infestation by P. haeckeli, suggests that the mortality was due to interaction and food shortage rather than the parasite. The experiment demonstrates that the noble crayfish used did not carry P. haeckeli. Since the noble crayfish used as food was from the same population as in group I, the only source for P. haeckeli in group II, must be the signal crayfish. This is further substantiated in group III, and by the outcome in group IV, where the only possible source of P. haeckeli is the signal crayfish themselves. It is, however, unsatisfactory that there are relatively many observations of uncertain P. haeckeli. On the other hand, this serves to illustrate the need for more thorough investigations on the various stages in the life cycle of Psorospermium haeckeli. Rug and Vogt (1994, 1995) present photographs of a number of developmental stages, of which some are quite similar to those observed in this material and denoted as being uncertain. Also, Henttonen et al. (1995) have observed similar stages. This strengthens the likelihood that the observations denoted as being uncertain were actually developmental stages of P. haeckeli. The results furthermore indicate that the parasite can develop without any intermediate host, which is in accordance with observations by Rug and Vogt (19951, and since the only feed besides crayfish was vegetables (potatoes and carrots>, the only source of P. haeckeli must be the crayfish. Thus, signal crayfish fed noble crayfish did not obtain any additional supply of the parasite, as did the signal crayfish fed signal crayfish. Under the given (stressful) conditions, the signal crayfish may have responded by “permitting” the parasite to develop to the easily identifiable stage, which did not happen in the original pond population. Since the signal crayfish on Gotland are not carrying the crayfish plague parasite, their immune defence systems do not have to fight a “two-front battle” and
R. Gydemo/Aquuculturr
148 (1996) I-9
7
can cope more efficiently with Psorospetmium haeckeli. There are indications to support this theory. In Lake Skillotsjbn, Sweden, a sympatric population of noble crayfish and signal crayfish is present. P. haeckeli has only been found in the noble crayfish, which Svkdson et al. (1991) suggested to be due to a higher resistance in P. leniusculus. Fenouil et al. (1995) investigated lysozyme activity, considered as being a part of the humoral defence system, in six crayfish species. The American crayfish species Orconectes limosus, Procambarus clarkii and Pacifastacus leniusculus all showed a higher lysozyme activity than the European Astacidae species, Astacus leptodactylus, Astacus astacus and Austropotamobius pallipes. Among the European species, A. leptodactylus exhibited around 20% higher lysis than A. astucus after 120 min, almost corresponding to the records for P. leniusculus. This is in accordance with the findings of Unestam (1969) in which A. leptoductylus showed a higher resistance to crayfish plague than A. astucus. Rirst and SGderhall (1987) reported that A. leptoductylus in Turkey infected with the crayfish plague fungus Aphanomyces astaci suffered more heavily when also infected with P. haeckeli. P. leniusculus, when their resistance is weakened, will easily die from infestation by crayfish plague (Persson and SGderhall, 1983 Persson and SGderhall, 1985, Persson et al., 1987). Cerenius and Sijderhall (1993) stress that the few observations of P. haeckeli in signal crayfish and the low degree of infestation should not be interpreted in terms of the signal crayfish being more resistant to P. haeckeli since the capacity of the signal crayfish immune defence system is reduced when chronically infected with the crayfish plague. The signal crayfish in this investigation were not infected by the crayfish plague. Thus, the more efficient immune defence system in signal crayfish may be effective in suppressing the development of P. haeckeli in the absence of other parasites, but when exposed to stress, the immune system is weakened (Siiderhall and Cerenius, 1992) and P. haeckeli develops to the easy identifiable stage. The experiment demonstrates that noble crayfish fed with signal crayfish can become infested with Psorospermium haeckeli. Signal crayfish fed with noble crayfish can also develop P. haeckeli but at a lower frequency than signal crayfish fed signal crayfish. Unestam (1973) and Cerenius and Sbderhall (1993) did observe P. haeckeli in a few Swedish signal crayfish populations but in low numbers. Similar findings were made by Dieguez-Uribeondo et al. (1993) in signal crayfish from Spain: the signal crayfish may harbour P. haeckeli but so far are observed only in comparatively low numbers. The implication with regard to signal crayfish is that, since it is generally a carrier of the crayfish plague, P. haeckeli may be a severe threat, as Cerenius et al. (1991) suggested that ‘even if a P. haeckeli infection itself is not lethal to the host, the reduced immune capacity of the animal will make it more sensitive to other pathogens’. This was demonstrated by Sijderh’;dll and Cerenius (19921, when noble crayfish and “normal” (i.e. infected by A. astaci) signal crayfish were artificially infested with P. haeckeli, the noble crayfish survived while the signal crayfish died from acute crayfish plague attack. Under natural conditions, signal crayfish that have the heaviest load of crayfish plague will also be the most vulnerable to other factors stressing the immune defence system. Thus, the crayfish succumb, thereby not being able to be analysed. The conclusion from the present experiment must be that signal crayfish can act as a vector for P. haeckeli and are the likely source of introduction of the parasite to
8
R. Gydemo/Aquaculture
148 (1996) 1-9
Gotland. Further surveys of infested noble crayfish populations have infested populations. Both of them have also been in contact with population used in this study. Furthermore, the routinely used technique of digestion in crayfish not be adequate when analysing signal crayfish, though it seems method for detecting Psorospermium haeckeli in noble crayfish.
revealed two more the signal crayfish health control may to be an adequate
6. Recommendations 1. When analysing signal crayfish for diseases and parasites, slide preparations of tissues should be used as well as the digestion technique. 2. If possible, the signal crayfish should be kept under stressful conditions for a period of several weeks at least before analysis. 3. It is of utmost importance that the complete life cycle of the parasite is clarified in order to be able to clearly identify the different developmental stages. 4. No further transplanations or introductions of signal crayfish should be made until it is clearly established which signal crayfish populations/stocks are carrying the parasite. 5. Policies for crayfish diseases and introductions of non-native crayfish species should be reconsidered. 6. The severity of the threat of Psorospermium haeckeli to the crayfish species should be determined.
Acknowledgements Thanks are due to Per Ramnelius who skilfully participated in the experiment, Gunnar Pettersson who kindly supplied the experimental facilities, and Pia Keyser who analysed part of the material. Financial support was obtained from the Carl Trygger Foundation, the Swedish Farmers Foundation for Agricultural Research and the Gunvor and Josef Aneers Foundation, which is gratefully acknowledged.
References Alderman, D.J. and Polglase, J.L., 1988. Pathogens, parasites and commensals. In: D.M. Holdich and R.S. Lowery (Eds.), Freshwater Crayfish - biology, management and exploitation. Croom Helm, London and Sydney, pp. 167-212 Cerenius, L. and Siiderhlll, K., 1993. The distribution of Psorospermium haeckli in Sweden; a preliminary survey. Freshwater Crayfish, 9: 280-285. Cerenius, L., Henttonen, P., Lindqvist, 0. and SiiderhBll, K., 1991. The crayfish pathogen Psorospermium haeckeli activates the prophenol-oxidase activating system of freshwater crayfish in vitro. Aquaculture, 99: 225-233 Edgerton. B., Owens, L., Harris, L., Thomas, A. and Wingfield, M., 1995. A health survey of farmed redclaw crayfish, Cheru qudricurinatus, (van Martens), in tropical Austalia. Freshwater Crayfish, 10: 322-338
R. Gydemo/Ayuaculturr
148 (1996) I-9
9
Fenouil, E., Roth, P. and de Hureaux, D., 1995. Immune defense in Astacidae and Cambaridae: presence of lysozyme activity. Freshwater Crayfish, 8: 614-622 Ftirst, M. and Sikierhall, K., 1987. The crayfish Astucus leptodtictylus in Turkey. Diseases and present distribution of the crayfish plague fungus, Aphanomyce.s ustaci. FAO Report, Rome, pp. 26. Grabda, E., 1934. Recherches sur un parasite de l’ecrevisse (Porcrmohius pcrllipes L.) connu sous le nom de Psorospermium haeckeli. Hilgd. Mem. Acad. Pol. Sci. Lett., S.B. Math. u Naturw., Cracow, 6: 123- 142 (in French). Haeckel, E., 1857. Uber die Gewebe des Flusskebses. Arch. Anat. Physiol., 24: 469-568 (in German). Henttonen, P., Hurter, J.V. and Lindqvist, O.V., 1995. Observations on Psorospermium hoeckeli in noble crayfish Astucus astacus CL.) (Decapoda, Astacidae) populations in Central Finland. Freshwater Crayfish, IO: 339-351. Persson, M. and SBderhall, K., 1983. Pacifastacus lrniusculus Dana and its resistance to the parasitic fungus, Aphanomyces ustuci Shikora. Freshwater Crayfish, 5: 292-298. Persson, M. and SGderhiill, K., 1985. Psorospermium haeckeli - parasit pl kraftor. (Psorospennium - a crayfish parasite). PM. Inst. Fys. Botanik, Uppsala Universitet, pp. 5 (in Swedish). Persson, M., Cerenius, L. and Siiderhall, K., 1987. The influence of haemocyte number on the resistance of the freshwater crayfish, Pacij2zstacu.s leniusculus Dana, to the parasitic fungus, Aphanomyces astuci. J. Fish Dis., 10: 471-477. Rug, M. and Vogt, G., 1994. Developmental stages of the crayfish parasite Psorospermium haeckeli in thoracic arteries of Astacus ustucus. J. Invertebrate Pathol., 64: 153- 155. of developing and mature spores of two Rug, M. and Vogt, G., 1995. Histology and histochemistry morphotypes of Psorospermium haeckeli. Freshwater Crayfish, 10: 374-384. A., 1872. Bidrag till kannedomen om flodkiaftans naturalhistoria. (A contribution to the Steffenbug, knowledge on the natural history of the crayfish). Reprinted 1972 in Information f&t Sbtvattenslaboratoriet, Drottningholm (14), p. 23 (in Swedish) Svardson, G., 1965. The American crayfish Pacifirstacusfeniusculus (Dana), introduced into Sweden. Rep. Inst. Freshwater Res., Drottningholm, Vol. 46, p. 90-94 Svlrdson, G., 1995. The early history of signal crayfish introduction into Europe. Freshwater Crayfish, 8: 68-77. Svardson, G., Fiirst, M. and Fjalling, A., 1991. Population resilience of Pacijtistacus leniusculus in Sweden. Finn. Fish. Res., 12: 165-177 Siiderhall, K. and Cerenius, L., 1992. Crustacean immunity. Annu. Rev. Fish Dis., 2: 3-23. Unestam, T., 1969. Resistance to the crayfish plague in some American, Japanese and European crayfishes. Rep. Inst. Freshwater Res., Drottningholm, Vol. 49, p. 202-209. Unestam, T., 1973. Significance of diseases in freshwater crayfish. Freshwater Crayfish, I: 135-150. Vey, A., 1978. Recherches sur une maladie des ecrevisses due au parasite Psom.~permium haeckeli. Freshwater Crayfish, 4: 41 l-41 8 (in French).