Survey on Fungal, Parasites and Epibionts Infestation on The

Archive Iranian of JournalSID of Fisheries Sciences 10(2) 266-275 2011

Survey on fungal, parasites and epibionts infestation on the

Astacus leptodactylus (Eschscholtz, 1823), in Aras Reservoir West

Azarbaijan, Iran

Nekuie Fard A.1, 2*; Motalebi A. A.2; Jalali Jafari B .1; Aghazadeh Meshgi M.1; Azadikhah D.3;Afsharnasab M. 2

Received: February 2010 Accepted: May 2010

Abstract

A total of 394 (255 males,139 females) live freshwater crayfish Astacus leptodactylus from four stations of Aras reservoir in West Azarbaijan Province (North-Western Iran) were studied during the winter until early autumn of 2009 for the presence of parasites, Epibionts and Fungal agents. Parasitological surveys were carried out on gills; exoskeleton and internal organs, mycological examinations on the exoskeleton (the legs, abdominal cuticle and the eggs). 9 epibionts and parasites peritrich protozoans including :Cothurnia sieboldii (68.5%), Zoothamnium spp. (56.6%), Vorticella similis (45.6%), Chilodonella spp. (0.5%), Podophrya fixa (7.8%), Epistylis chrysemidis (53.2%), Pyxicola annulata (66%), Opercularia articulata (19.8%), Tetrahymena pyriformis (0.5%) were recorded. From Metazoan parasites group, Branchiobdella kozarovi (71%) as the first observation was the only parasite recorded from exoskeleton with prevalence (100%) during spring and summer of the study year. Infected gills were heavily damaged with Aeolosoma hemprichi (Annelid) in winter with 90% prevalence. Other Epibiont fouling organisms such as Rotatoria; free living Nematods were observed in this survey. Furthermore, on the mycotic agents identified Penicillium expansum; Aspergillus flavus; Alternaria sp. ; Fusarium sp. and Saprolegnia sp. were isolated in IM media and identified with slides cultured from cuticular melanized lesions and eggs of infected specimens. This is the first investigation on epibionts, parasites and fungal organisms of the endemic crayfish in Aras reservoir, Iran.

Keywords: Astacus leptodactylus, Fungi,Epibiont, Aras reservoir, Iran

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1- Faculty of specialized veterinary sciences, Islamic Azad University, Science and Research Branch, Tehran, Iran. 2- Iranian fisheries Research Organization, P.O.Box:14155-6116, Tehran, Iran. 3-Veterinary Department, Islamic Azad University, Urmieh, Iran. *Corresponding author: [email protected]

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Introduction Aras dam is one of the important water many cases of mass mortality remains sources for irrigation and electric power in undiagnosed (Edgerton et al., 2004). Iran which was constructed on Aras River Except Asgharnia (2008) who described and completed in 1970 with a total area of prevalence and intensity of Branchiobdella about 11000-15000 (ha). Fish composition hexodenta (Annelid: Clitella) in carapace species in Aras Lake included 15 species and gills of cultured Astacus leptodactylus belonging to Cyprinidae 86%, Siloridae in research ponds in the north of Iran. This and Percidae individualy 7% (Azadikhah study completely focused on fungal, et al., 2008). Furthermore, Astacus epibionts and parasites on cultured Astacus leptodactylus is the most economic non- leptodactylus’s natural population in Iran. fish species introduced in Aras reservoir. But among the neighboring countries, the Fresh water crayfish Astacus leptodactylus epibiont and parasitic infections have been with a limited geographical distribution in well studied in Turkey. The parasite fauna the country is the only crayfish existing in of crayfish was investigated by Harioglu Iran. This species is in Anzali lagoon (1999), some studies carried out by Baran where it is translocated to a few rivers and et al. (1987) and Baran and Soylu (1989) reservoirs in the Northwestern part of the on the parasite fauna of cultured and wild country. The amount of the local crayfishes of Turkey may concern similar consumption of crayfish in Iran is very low problems as those of the neighboring (Gorabi, 2003). Therefore, almost all of Northwestern part of Iran where Aras the catches are exported to European basin is situated. Concerning mycotic countries (shilat, 2009). Pathological disease (plague) of crayfish, which is a investigations are essential either obtaining well-known infection to Europe and information about the health status of wild especially in Turkey, research work done populations or guaranteeing the success of by Söderhäll, K., and Cerenius, L. (1999) crayfish culturing by preventing the spread confirmed that Astacus leptodactylus is the of diseases. The two most dangerous most sensitive species among other pathogenic groups are fungal and viral. Of members of Astacus. From 1985, the these, the most virulent toward crayfish is European Union and Turkey are well the fungus, Aphanomyces astaci, and the known countries where the plague has agent of crayfish plague in freshwater been established. The objective of the crayfish (Edgerton et al., 2002a). Some study was to evaluate the health status of authors have reported the association of an native freshwater crayfish population in intranuclear bacilliform virus with the near North-West of Iran. extirpation of Astacus pallipes, complex from the Nant watershed in France Material and methods (Edgerton et al., 2002a; Edgerton, 2003; During the winter until early autumn of Edgerton et al., 2004). A serious mortality 2009 (Jan-October), a total of 394 (255 in crayfish populations has often been males, 139 females) live fresh water attributed to pollution but without any crayfish Astacus leptodactylus, from wild proof. However, the causative agent in populations were collected from Aras

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reservoir in the region of N-W Iran. The maintained in + 4◦C till 24 hours until samples were collected from four different being examined in the Poldasht laboratory stations (Figure 1): 1. 2. 3. 4. Specimens (Table I). In the laboratory, specimens were captured by means of baited traps. were first subjected with bioexamination Only live crayfish were rapidly transported and then parasitological examinations were individually by foam bags and were carried out.

Figure 1: Location of sampling stations on Aras reservoir; West Azarbaijan; Iran

Parasitological investigation Preparations of wet smears from the examined under a microscope at X100-400 exoskeleton, lesions and gills of each magnification. Metazoan parasites were specimen were as follows: the mucus was separated as the same methods used for scraped separately from different organs external epibionts. Line drawing and on to a micro slide and then it spread the length of the species were measured by mucus carefully with the cover slide. The computer and projected by a video camera. protozoas exposed to a fixative for at least Measurements of the parasites were related 15 minutes and then washed for several to the scale of an objective micrometer, minutes in alcohol containing a drop of projected to the screen in the same way. added iodine solution. Then, both wet and The validity of the methods was checked dry smears were mounted in by measuring the same organs with Canadabalsam often dehydration in microscope micrometers. For the accordance with Fernando et al. (1972). identification of epibionts and parasites the Gills need special attention due to the keys given by Hoffman (1967); Matthes sensitivity of this organ to epibiont, and Guhl (1973); Kudo (1977) and parasites and its important rule in Alderman et al. (1988) were used. osmoregulation. The organ was cut off and Branchiobdellidans, were removed from

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-1 each specimen, fixed in 70% ethanol and et al., 1994) with penicillin G (6 mg l ) were then counted and clarified with -1 and oxolinic acid (10 mg l ) (Alderman lactophenol. The specimen were stained and Polglas, 1986), and incubated at 26°C. with Borax Carmine and mounted with The colonies found growing on agar were Glycerin Jelly or Hoyer’s fluid. All of the examined macro- and microscopically, branchiobdellidans were examined and using bright field illumination and slide measured using the optical microscope. culture method, for identification. Moulds Identification of the specimens was made were identified to genus using on the basis of the jaws, the spermatheca morphological features, i.e. colony and spermathecal duct morphology using appearance, hyphae, sexual and/or asexual identification keys (Moszynsky, 1938; reproduction (Barron, 1968; St–Germain Pop, 1965; Gelder et al., 1994). and Summerbell, 1996; De Hoog and Mycological investigation 2 Guarro, 1996). When the colonies had Small pieces (1-2 mm ) and melanized tubular, variably branched, very poorly patches of the exoskeleton were removed septate, hyphae (looking like oomycetes), from the abdomen and legs of infected they were placed in sterile distilled water specimens among 390 crayfishes, rinsed in with at 18°C and 26°C, to develop sterile distilled water, placed on plates sporangia and/or sexual structures. containing glucose-yeast extract agar (Min Table 1: Number of captured and type of examination crayfishes during winter until early autumn (2009)

Results Epibiont investigation The parasite and epibiont organisms which were found on crayfish from all stations, were removed from the examined Astacus while Chilodonella sp. and Tetrahymena leptodactylus are summarized in Table 2. pyriformis showed a lower infestation rate Peritrichous ciliate Cothurnia sieboldii, only in one station (Figure 2). In Epistylis sp., Vorticella similis, laboratory examination externally, the Zoothamnium sp., Pyxicola annulata, most frequently observed organisms were Opercularia articulata and Podophrya fixa Cothurnia sieboldii, on healthy

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exoskeleton. Colonies of these ciliates the deeper portions of the gill chamber, tended to concentrate in protected cuticular they appeared to trap debris and bacteria. folds on the exoskeleton and Besides the other epibiont protozoan cephalothorax (Figure 3), and in the gill including: Stylonychia mytilus, chamber particularly in well-sheltered Paramecium sp., are observed with low areas, such as at the base of gill filaments. incidence one the branchial cavity of When peritrichous ciliates were located in crayfish.

Table 2: Results of parasitological examination – No. of positives (%)

Branchiobdella kozarovi was found and infestation of B.kozarovi isolated in identified on the exoskeleton and walking crayfishes is showed in Table II. The mean leg, antenna; antennules; base of eyes and total length of specimens was 1.35±0.6 in one of the specimens of the gill of mm (n=30, range 0.8-3 mm,) with a mean crayfishes from all of the stations. The width of 0.32±0.09 mm (n=30, range 0.22- most infestation was observed on 3rd 0.6 mm). Maxilipede (Figure 3). The percentage of

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Figure 2: A=Loricate ciliophora Cothurnia sieboldii (100X), B=Vorticella similis (400X), C= Pyxicola annulata (400X)., D= Opercularia articulate (100X), Attached to the gill filaments of Astacus leptodactylus

Figure 3: A=3rd Maxilipede infestation by Branchiobdella kozarovi (100X), B=Peleopods infestation by cocoons of Branchiobdella kozarovi (1000X) The numerous cocoons were attached on free living nematodes including: the thorax segments (exopodit, protopodit, Mononchus sp. ,Prodesmodora sp. and epipodit) of crayfishes (Figure 3). High Bunonema reticulatum are observed on gill infestation in the presence of chambers of examined specimens in all branchiobdellidan and their cocoons were seasons.Fungi were isolated from both the observed in the exoskeletons of samples abdominal cuticle and the legs, and the during spring and summer (100%). results are summarized in Table 3. Metazoan ectosymbionts included: Penicillium expansum; Aspergillus flavus; Aeolosoma hemprichi (Annelida), Alternaria sp. and Saprolegnia sp. were Philodina acuticornis (Rotatoria), isolated from 83 captured out of 394 Mesocyclops strennus of copepods, and specimens after cultured in IM media from

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cuticular melanized lesions and eggs. coenocytic thallus from the melanized Penicillium expansum was the most areas of the exoskeleton and legs on one frequently isolated being found on 16.8% crayfish was isolated. This, when of the abdomens melanized cuticles, transplanted on to IM media, produced 15.7% on legs and 16.1% on eggs of abundant clavate, pyriform or irregular examined specimens. Most of these gemmae, single, or frequently, catenulate. crayfishes were with marked clinical signs. Cylindrical, clavate or irregular straight Seasonal prevalence of mycoting infection zoosporangia were abundant. The strain was as follows: Winter (53%), Spring failed to produce a sexual form at either (14.4%), Summer (3.6%) and Autumn 18°C or 26°C. The characteristics of (10.8%) in the study year. Positive septate secondary cysts were not observed. Based hyphae on the exoskeleton were often on morphological features, the isolates observed in the presence of melanized were assigned to the genus Saprolegnia, areas and in association with detritus and but further identification should be carried bacteria accumulations. A mycete with out to the species level.

Table 3: Results of mycological investigations: no. of positives (percent).

Seasons Winter Spring Summer Autumn Total No. of crayfish examined 42 19 9 13 83 Penicillium expansum 8(19) 2(10.5) 1(11.1) 2(15.4) 13(15.7)

Aspergillus flavus 5 (11.9) 1(5.2) 1(11.1) 3(23) 10(12) Fusarium sp. 2 (4.7) 0 0 0 2 (2.4)

pleopod) Saprolegnia sp. 1(2.4) 0 0 0 1(1.2) Legs (walking (walking Legs Negative 26(61.9) 16(84.3) 7(77.7) 8(61.5) 57(69.3)

Penicillium expansum 9(21.4) 3(15.7) 1(11.1) 1(7.7) 14(16.8)

Aspergillus flavus 3(7.1) 1(5.3) 0 1(7.7) 5(6)

Alternaria sp. 0 1(5.3) 0 1(7.7) 2(2.4)

Saprolegnia sp. 1(2.4) 0 0 1(7.7) 2(2.4) Abdominal cuticle Abdominal Negative 29(69) 15(78.9 ) 8(88.8) 9(69.2) 12(14.4) Penicillium expansum 3(25) 2(10.5 ) 0 0 5(16.1)

Aspergillus flavus 2(16.6) 2(10.5 ) 0 0 4(12.9) Eggs Saprolegnia sp. 10(83.3) 3(15.8) 0 0 13(41.9) Negative 27(64.2) 12(63.1) 0 0 39(47)

Discussion A variety of protozoa generally move Quagliof et al. (2004) found that the about or attach to the body surface and elevated presence of Epistylis spp. was gills of crayfish (Johnson, 1983). Epibiont associated with low dissolved oxygen infestation of the gills and exoskeleton of concentration and high organic pollution. the crayfish examined in our study were The low incidence of Epistylis spp. in common, but no detrimental effects on examined Astacus pallipes complex could crayfish health were macroscopically be related to the optimal environmental observed. In another study, carried out on conditions and water quality which they red swamp crayfish (Procambarus clarkii), experience. Matthes and Guhel (1973)

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have reported Cothurnia sieboldii also as a were found on the external surfaces of commonly occurring species on European Northern American crayfish species, crayfish. Peritrich infestations of Pacifastacus simulans, Pacifastacus freshwater crayfish have been widely clarkii, Pacastacus zonangulus and reported (Edgerton et al., 2002b). In our Fallicambarus hedgpethi (Lahser, 1975). survey, sessile peritrichs are found on the Saprolegnia spp. is common water mould external surfaces, including the branchial and includes species which are responsible chamber. Different species of peritrich for significant infections involving both ciliates show site specificity, some being living and dead fish. In the strain of found predominantly on the gills, some on Saprolegnia spp. Only the form of asexual the appendages and carapace, others reproduction was isolated. Due to lack of distributed widely over most of the body the formation and observation in the (Table 2). Among the epibiont and characteristic of secondary cysts and the parasitic population found, most attention molecular studies we were couldn’t to should be paid to Branchiobdella kozarovi identify the species. As pointed out by which may intensify the success of Söderhäll et al. (1991), although production in future development plans. In Saprolegnia parasitica causes a severe North America, Europe and East Asia a problem in fish, it does not appear to be an wide range of crayfish species are infected important parasite for crayfish. The by Branchiobdella kozarovi. This report is populations of Astacus leptodactylus the first related to parasite and epibiont sampled of Aras reservoir during the organisms and Branchiobdella kozarovi survey is rather in good favorable infestation on Astacus leptodactylus in environmental conditions and habitat. Iran. On the basics of our finding the Opportunistic pathogens and epibionts mycetes of the genera Penicillium were frequently observed in examined expansum, Alternaria spp., Fusarium spp. specimens in the wild and in captivity. and Aspergillus flavus were found in Therefore, particular care must be given to healthy specimens. They can be crayfish culture to prevent environmental considered naturally occurring stressors causing disease outbreaks. The saprophytes, often associated with poor presence of the native crayfish has been water quality. Chinain and Vey (1988) greatly reduced progressively in number, reported disease caused by Fusarium in the recent years in many reservoirs of solani in the crayfish species Astacus Iran. Thus, this species has risk extinction. leptodactylus and Pacifastacus leniusculus This may be the result of epizootic in Europe. They found that of the two diseases, the introduction of alien species, species of freshwater crayfish that were changes in the habitat brought by studied, Astacus leptodactylus was the excavation; work on river-beds and more susceptible species. Other species of streams and general industrialization fungi which have been reported as which very often occur in areas close the epizoites of freshwater crayfish include course of the rivers. In general the Alternaria spp., Hormodendrum spp., epibionts, peritrichous protozoan and Aspergillus spp., Saprolegnia spp., metazoan parasites of Astacus Uncinula spp. and Hormisum spp. which leptodactylus are common fauna of fresh

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water crayfish. Most of them attach to the of Iran. . 6th internatl symposium on exoskeleton and gills of crayfish, and feed Monogenea Cape Town,Sout Africa. primarily on bacterial cells associated with Barron, G. L., 1968. The Genera of Hyphomycetes from soil. The eurotropic reservoir which generally Williams & Wilkins Company, increase in summer and reduce in winter. Baltimora, USA. Therefore, we can come to a conclusion Cereniuse, C. and Sölderhall, K., 2004. that parallel to increasing of eutrophication The prophenoloxidase- activating of Aras reservoir, prevalence and intensity system in invertebrates. of epicommensals like Epistylis spp. on Immunological Reviews, 198, 116- crayfish population will be significantly 126. Chinain, M. and Vey A., 1988. increased and may have an adverse effect Experimental study of Fusarium on health status which may lead to disease solani: infections in Astacus outbreak and mortality. leptodactylus and Pacifastacus leniusculus (Crustacea, Decapoda) Acknowledgements Diseases of Aquatic Organisms, 5, The study was supported by Iranian 215-223. fisheries Research Institute. The authors De hoog, G. S. and Guarrio, J., 1996. wish to acknowledge Prof. S. Gelder, Dr. Atlas of Clinical Fungi. Centralbureau Yahya zadeh , Mr. Shiri and Mr. voor Schimmelcultures/Universitat Rovira I Virgili, Baarrn and Delft, The Shirvalilo for helping to do this study. Netherlands. Edgerton, B. F., 2003. Further studies References reveal that A. pallipes bacilliform Alderman, D. J. and Polglase, J. L., virus (ApBV) is common in 1986. Aphanomyces astaci. Isolation populations of native crayfish in and culture. Journal of Fish Disease, south-eastern France. Bulletin of the 9, 367-379. European Association of Fish Alderman, D. J. and Polglase, J. L., pathologists, 23, 7-12. 1988. Pathogens, parasites and Edgerton, B. F., Henttonen, P., Jussila, commensals. In:Holdrich, D.M., J., Mannonen, A., Paasonen, P. , Lowery, R.S. (Eds.), Freshwater Taugbíl, T., Edsman, L. and Souty- Crayfish: Biology, Management and Grosset, C., 2004. Understanding the Exploitation. Croom Helm, Sydney, cause of disease in European pp. 167–212. freshwater crayfish. Conservation Asgharnia, M., 2005. A Survey to Biology, 18, 1466-1474. parasitic infection in freshwater Edgerton, B. F.and Orwens, L., 1999. crayfish “Astacus leptodactylus” in Histopathological surveys of the the rearing environment of Sefidrood th redclaw freshwater crayfish, Cherax fishery Research station, Astaneh , 6 quadricarinatus, in Australia. congress of Marin science and Aquaculture, 180, 23–40. technology. Tehran-Iran. Edgerton, B. F., Watt H., Becheras J. M. Azadikhah, D., Nekuiefard, A., Mirzayi, and Bonami J. R., 2002a. An F., Nasrabadi, S. A. and Jalali, J. B., intranuclear bacilliform virus 2008. On the less known associated with near extirpation of Ancyrocephalidae (Bychowsky, 1937) Austropotamobius pallipes (Monogenea, Polyonchoinea) species Lereboullet from the Nant watershed in freshwater and Caspian Sea fishes in Ardèche, France. Journal of Fish Disease, 25, 523–531.

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