Marine leech Zeylanicobdella arugamensis infestation in cultured orange-spotted grouper, Epinephelus coioides

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1 Ž. Aquaculture ž / Marine leech Zeylanicobdella arugamensis infestation in cultured orange-spotted grouper, Epinephelus coioides Erlinda R. Cruz-Lacierda a,), Joebert D. Toledo a, Josefa D. Tan-Fermin a, Eugene M. Burreson b a Southeast Asian Fisheries DeÕelopment Center, Aquaculture Department, Tigbauan 5021, Iloilo, Philippines b School of Marine Science, College of William and Mary, Virginia Institute of Marine Science, Gloucester Point, VA 23062, USA Accepted 9 November 1999 Abstract Heavy infestation of a marine leech occurred among tank-reared juvenile and adult orangespotted grouper, Epinephelus coioides Hamilton, at SEAFDEC AQD, Philippines with a prevalence of 83% and 17%, respectively. The leeches were attached in large numbers on the fins, lower jaw, under the operculum, eyes, and inside the mouth of the fish. The attachment and feeding sites exhibited frayed fins, hemorrhages and swelling of the host s skin. External and internal features indicate that the leech is Zeylanicobdella arugamensis De Silva ŽHirudinea, Piscicolidae.. The parasite can be effectively controlled using 50 ppm formalin bath treatment for 1h. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Grouper; Marine leech; Zeylanicobdella arugamensis 1. Introduction An extremely heavy infestation of a marine leech occurred among tank-reared juvenile and adult orange-spotted grouper, Epinephelus coioides Hamilton, at SEAFDEC Aquaculture Department in Iloilo, Philippines. The fish were being used for broodstock development studies when the parasite infestation occurred. Subsequent observations of ) Corresponding author. address: eclacier@aqd.seafdec.org.ph Ž E.R. Cruz-Lacierda r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 192 ( ) E.R. Cruz-Lacierda et al.raquaculture the same parasite were also recorded in grouper collected from the wild and from earthen ponds. This paper describes the disease outbreaks and a control measure is also presented. 2. Materials and methods Twenty four Ž 24. two-year-old Ž mean BW, 1.0 kg; mean TL, 41 cm. juvenile and six adult Ž mean BW, 6.0 kg; mean TL, 66.5 cm. grouper were stocked in eight canvas and one concrete tank, respectively. The juveniles were being used for experiments on induction of sex inversion while the adults were being monitored for natural spawning. Prior to the sex inversion experiment, the fish were reared in earthen ponds for two years. The juveniles were then transferred to 10-ton, 4-m diameter canvas tanks for 4 months when the parasite infestation occurred. The adult fish were collected from the wild and reared in a concrete tank Ž 5=5=2 m. for more than 1 year prior to parasite infestation. All fish were fed with fish-by-catch. The tanks containing the juvenile fish were supplied with flow-through sand-filtered water while 50 80% of the rearing water was changed at least three times a week for the adult fish. Water temperature and salinity ranges recorded during the experiments were C and ppt, respectively. Mean water temperature and salinity for six days prior to leech infestation were 288C and 22 ppt, respectively. Continuous heavy rains were also noted a few days before the infestation making the incoming water turbid. Heavy infestation of the parasite was observed during regular sampling for experiments on sex inversion and natural spawning. The affected fish were anesthetized with 250 ppm 2-phenoxy-ethanol for 5 min to immobilize the fish. The parasites were then removed manually by carefully rubbing the affected portion with a wet cloth and then transferred to glass jars containing seawater. Many of the parasites could be removed by this method. However, large numbers remained especially those attached on the corners of the fins and the inner surface of the operculum. The live parasites were observed microscopically and measurements were taken. Some were fixed in 10% buffered formalin and processed for histological examination or preserved in alcohol-formalin-acetic acid Ž AFA. and stored in 70% ethyl alcohol. Serial transverse sections were cut at 5 mm for determination of internal anatomy. A mini-bioassay was conducted to determine control measures against the parasite. The leeches Ž n s 10rtreatment. were exposed to 0 Ž 100% freshwater., 15 and 30 ppt Ž control. seawater and 50, 100 and 200 ppm formalin in glass petri plates. The parasites were observed under the stereo microscope every 30 min for 4 h. 3. Results Twenty Ž 20. out of 24 juvenile fish Ž 83%. and one out of six adult fish Ž 17%. were infested by the leech. The parasites were attached in heavy patches Ž several hundreds. in

3 ( ) E.R. Cruz-Lacierda et al.raquaculture pectoral, ventral, anal, and caudal fins Ž Fig. 1.. Large numbers of the leeches were also found in the skin folds behind the lower jaw, under the operculum, eyes, and inside the mouth. The affected fish showed frayed fins, hemorrhages and swollen areas on the parasite s attachment and feeding sites Ž Fig. 2.. Microscopic examination of the live brownish-black parasites revealed an elongated and cylindrical body narrowing at both ends with oral and caudal suckers Ž Fig. 3a.. Total length ranged from 8 12 mm including the suckers. The oval-shaped oral sucker Ž Fig. 3b. measured = mm and contained a pair of eyes located dorsally at the lower portion of the sucker. The discoid-shaped caudal sucker Ž Fig. 3c. measured mm in diameter and its width was equal or wider than the maximum width of the body. From the microscopic examination of live and preserved specimens and the histological sections, the parasites were confirmed as Zeylanicobdella arugamensis De Silva Ž Hirudinea, Piscicolidae.. The leech was also observed in pond-cultured grouper Ž mean BW, 40 g., collected and acclimatized Ž C, ppt. in 600-l fiberglass tanks for one month for a feeding experiment Ž P. Eusebio, personal communication. with a prevalence of 25% and mean intensity of 4. The leeches were observed in the jaw and corners of the fins. The affected fish were found aggregated near the aeration source and mortalities occurred. The fish also showed heavy infestation of monogeneans Ž Pseudorhabdosynochus sp.. and didymozoid trematodes in the gills. The leech was also observed in grouper fingerlings Ž BW, 3 58 g. collected from the wild for transport experiment ŽE. Coniza, Ž. Fig. 1. Adult E. coioides infested with Z. arugamensis arrows. Bars4.5 cm.

4 194 ( ) E.R. Cruz-Lacierda et al.raquaculture Fig. 2. Caudal fin of adult E. coioides infested with Z. arugamensis. The attachment and feeding sites of the Ž. parasite are hemorrhagic and swollen arrows. Bars0.6 cm.. personal communication with a prevalence of 30% and mean intensity of 2. The leech in both observations was confirmed as Z. arugamensis. Fig. 3. Z. arugamensis: Ž. a entire specimen, dorsal view, bars1 mm; Ž. b oral sucker, dorsal view, bars0.25 mm; Ž. c caudal sucker, dorsal view, bars0.5 mm.

5 ( ) E.R. Cruz-Lacierda et al.raquaculture Results of the bioassay showed that all treated leeches died after 1 h exposure to 0 ppt seawater Ž 100% freshwater. and ppm formalin. Those exposed to 15 and 30 ppt seawater were still active even after 4 h. For treatment, fish with remaining attached parasites were treated with 50 ppm formalin bath for 1 h. The fish were supplied with ample aeration during the treatment period and observed closely. At the end of the treatment period, most of the parasites were found detached from the fish and seen at the bottom of the tank. The 50 ppm formalin, however, did not have an adverse effect on the treated fish. 4. Discussion This paper is the first documented report of marine leech infestation in cultured grouper in the Philippines with grouper as a new host record for Z. arugamensis. Zeylanicobdella arugamensis have been observed to infest a number of brackishwater and marine fishes in Sri Lanka, Malay Peninsula off Singapore, and India ŽDe Silva, 1963; De Silva and Fernando, 1965; Sanjeeva Raj et al., This leech is also common in Indonesia and along the coast of Queensland, Australia ŽBurreson, unpublished data.. Bondad-Reantaso Ž reported Piscicola sp. in grouper from Luzon Island, Philippines. Leong and Wong Ž reported an unidentified leech from grouper cultured in floating cages in Malaysia with a prevalence of 0.4%. The checklist of parasites of fishes of the Philippines did not show any record on the occurrence of Z. arugamensis Ž Arthur and Lumanlan-Mayo, However, the heavy infestation by this leech found in the present study is very unusual Ž Burreson, unpublished data.. The pathology of leech infestations has been reviewed in marine fish by Rohde Ž and in fish cultured in the tropics by Kabata Ž Hemorrhages and swelling of affected areas have been reported in yellow fin bream, Acanthopagrus australis and Atlantic cod, Gadus morhua Ž Khan, 1982; Roubal, In the present study, hemorrhages and swelling on the attachment and feeding sites of the leech have been observed. The importance of leeches as vectors of pathogens and as parasites of fish have been reviewed by Burreson Ž It would be appropriate to look at possible viruses and blood parasites associated with heavy infestation of leech in grouper. Despite the heavy infestation in juvenile fish, no mortality occurred among the affected individuals. However, the adult fish died three days after the parasite infestation. This can be attributed to the extremely heavy leech infestation with the consequent loss of large amount of blood, the probable secondary effects of multiple feeding wounds, and the loss of appetite of the affected fish. Mortality has been recorded in Pacific salmon Ž Oncorhynchus spp.. with extremely heavy infestations by leech ŽKhan, The mortalities in grouper fingerlings caught from the wild and grow-out ponds, however, can be attributed to transport stress and the combined effects of multiple parasite infestations, respectively. The epizootic outbreaks of Z. arugamensis in the present study are probably attributable to environmental factors such as high turbidity level of the incoming water. The incoming rearing water was turbid brought about by continuous heavy rains for several days before the infestation. The leeches in adult and juvenile fish were not

6 196 ( ) E.R. Cruz-Lacierda et al.raquaculture evident one week prior to infestation. Thus, an effective and good filtering system for the water intake may prevent the entry of leeches in culture facilities. The average salinity of the rearing water six days before the outbreak was 22 ppt, indicating that Z. arugamensis can tolerate brackishwater conditions. The 50-ppm formalin bath treatment for 1 h proved to be effective in controlling the leech. The use of freshwater to control the parasite may also be effective but has limited applications in strictly marine species, such as E. coioides. These methods will remove leeches from the fish, but if the leeches have deposited cocoons in the culture facilities, then the total elimination of the parasite may be difficult. Cocoons are very resistant to chemical treatment Ž Burreson, Fish that have been treated can quickly become re-infected as leeches hatch from cocoons present in the tank or pond. Fish should be examined carefully for leeches prior to stocking to prevent parasite infestation. Cocoons can also be eliminated by thorough drying of culture facilities. Acknowledgements We appreciate the technical assistance of the Staff of Fish Health and Breeding Sections of SEAFDEC AQD. Catherine T. Tamse acted as our personal courier and Mila Castanos edited an early draft of the manuscript. References Arthur, J.R., Lumanlan-Mayo, S., Checklist of the Parasites of Fishes of the Philippines. FAO Technical Paper. No Rome, 102 pp. Bondad-Reantaso, M.G., Fish health problems and programs in the Philippines. In: Langdon, J.S., Enriquez, G.L., Sukimin, S. Ž Eds.., Proceedings of the Symposium on Tropical Fish Health Management in Aquaculture, May 1991, Bogor, Indonesia. pp , Biotrop Spec. Publ. No. 48. Burreson, E.M., Phylum Annelida: Hirudinea as vectors and disease agents. In: Woo, P.T.K. Ž Ed.., Fish Diseases and Disorders: Vol. 1. Protozoan and Metazoan Infections. CAB International, UK, pp De Silva, P.H.D.H., Zeylanicobdella arugamensis gen. nov. and sp. nov. from Arugam Kalapu, Eastern Province, Ceylon. Spolia Zeylanica 30, De Silva, P.H.D.H., Fernando, C.H., Three marine leeches Ž Piscicolidae, Hirudinea. from the Malay Peninsula. Spolia Zeylanica 30, Kabata, Z., Parasites and Diseases of Fish Cultured in the Tropics. Taylor and Francis, London, 318 pp. Khan, R.A., Biology of the marine piscicolid leech Johanssonia arctica Ž Johansson. from Newfoundland. Proc. Helminthological Soc. Washington 49, Leong, T.S., Wong, S.Y., A comparative study of the parasite fauna of wild and cultured grouper Ž Epinephelus malabaricus Bloch et Schneider. in Malaysia. Aquaculture 68, Rohde, K., Diseases caused by metazoans: helminthes. In: Kinne, O. Ž Ed.., Diseases of Marine Animals 4 Biologische Anstalt, Helgoland, pp Roubal, F.R., Histopathology of leech, Austrobdella bilobata Ingram, infestation on the yellowfin bream, Acanthopagrus australis Ž Gunther., in northern New South Wales. J. Fish Dis. 9, Sanjeeva Raj, P.J., Jayadev Babu, S., Gladstone, M., Anatomical details of two fish leeches from the Pulicat Lake, South India. J. Mar. Biol. Ass. India 19,