Molecular Identification of Parasites Isolated from Mediterranean Mussel (Mytilus Galloprovincialis Lamarck 1819) Specimens

Year 2020, Volume: 15 Issue: 2, 61 - 71, 23.04.2020

Harun Yılmaz , Ercüment Genç , Esra Mine Ünal , Şükrü Yıldırım , Emre Keskin

Abstract

           In this study, Mediterranean mussels (Mytilus galloprovincialis Lamarck, 1819) were collected from the most important mussel distribution/fishing areas in Turkish coastal waters (Çanakkale, Balıkesir, İzmir). The presence of parasite was investigated based on molecular methods. Molecular identification of parasite species was established by designing parasite-specific primers for PCR amplification and resulting nucleotide sequences were analysed. As a result of this study, four parasite species were identified as Mytilicola intestinalis Steuer 1902, Mytilicola orientalis Mori 1935, Urastoma cyprinae Graff 1882, Parvatrema duboisi Dollfus 1923 respectively. All identified species were found in İzmir (Agean Sea) specimens while Urastoma cyprinae and Parvatrema duboisi were found only in Çanakkale (Dardanelle) and Balikesir (Marmara Sea) specimens. Nucleotide composition, pairwise genetic distance and phylogenetic trees of the detected species were given. This research takes place to be the first study on identifying mussel parasites by using molecular techniques. Also, Mytilicola orientalis is the first record from Turkish coastal waters.

Keywords

Mollusca, Mediterranean, Mytilus, Parasite, Molecular Identification

References

• [1] Figueras, A.J., (1989). Mussel culture in Spain and France. World Aquaculture, 20(4):8-17.
• [2] Rayyan, A., Photis, G., and Chintiroglou, C.C., (2004). Metazoan parasite species in cultured mussel Mytilus galloprovincialis in the Thermaikos Gulf (North Aegean Sea, Greece). Diseases of Aquatic Organisms, 58(1): 55-62. http://dx.doi.org/10.3354/dao058055 .
• [3] Francisco, C.J., Hermida, M.A., and Santos, M.J., (2010). Parasites and symbionts from Mytilus galloprovincialis (Lamark, 1819) (Bivalves: Mytilidae) of the Aveiro estuary Portugal. Journal of Parasitology, 96(1):200-205. http://dx.doi.org/10.1645/GE-2064.1.
• [4] Karagiannis, D., Vatsos, I.N., Theodoridis, A., and Angelidis, P., (2013). Effect of culture system on the prevalence of parasites of the Mediterranean mussel Mytilus galloprovincialis (Lamark, 1819). Journal of the Hellenic Veterinary Medical Society, 64(2):113-122. http://dx.doi.org/10.12681/jhvms.15484.
• [5] Özer, A. and Güneydağ, S., (2014). First report of some parasites from Mediterranean mussel, Mytilus galloprovincialis Lamarck, 1819, collected from the Black Sea coast at Sinop. Turkish Journal of Zoology, 38(4):486-490. http://dx.doi.org/10.3906/zoo-1401-2.
• [6] López-Flores, I., De La Herran, R., Garrido-Ramos, M.A., Navas, J.I., Ruiz-Rejon, C., and Ruiz-Rejon, M., (2004). The molecular diagnosis of Marteilia refringens and differentiation between Marteilia strains infecting oysters and mussels based on the rDNA IGS sequence. Parasitology, 129(4):411-419. https://doi.org/10.1017/S0031182004005827.
• [7] González, C.C., Alvarez, R.R., Domínguez, H.R., Bua, M.S., Iglesias, R., Fernández, C.A., and Estévez, J.G., (2005). In vitro reproduction of the turbellarian Urastoma cyprinae isolated from Mytilus galloprovincialis. Marine Biology, 147(3):755-760.
• [8] Robledo, J.A.F., Santarém, M.M., and Figueras, A., (1994). Parasite loads of rafted blue mussels (Mytilus galloprovincialis) in Spain with special reference to the copepod, Mytilicola intestinalis. Aquaculture, 127(4):287-302. https://doi.org/10.1007/s00227-005-1622-9.
• [9] Villalba, A., Mourelle, S.G., Carballal, M.J., and Lopez, C., (1997). Symbionts and diseases of farmed mussels Mytilus galloprovincialis throughout the culture process in the Rias of Galicia (NW Spain). Diseases of Aquatic Organisms, 31(2):127-139.https://doi.org/10.3354/dao031127.
• [10] Murina, G.V. and Solonchenko, A.I., (1991). Commensals of Mytilus galloprovincialis in the Black sea: Urastoma cyprinae (Turbellaria) and Polydora ciliata (Polychaeta). In Turbellarian Biology (pp:385-387). Springer, Dordrecht.
• [11] Harunoglu, E.M., (2005). Parasites of Mediterranean mussel (Mytilus galloprovincialis Lmk., 1819) from Izmir bay (Aegean Sea, Turkey). Ege University, Graduate School of Natural and Applied Science, Department of Fisheries Basic Science, MSc Thesis (in Turkish), 120 p.
• [12] Guneydag, S., (2013). Parasite fauna of Mytilus galloprovincialis living in Sinop area of Black Sea. Sinop University, Graduate School of Natural and Applied Science, Department of Aquaculture, MSc Thesis (in Turkish), 123 p.
• [13] Gresty, K.A., (1992). Ultrastructure of the Midgut of the Copepod Mytilicola intestinalis Steuer, an Endoparasite of the Mussel Mytilus edulis L. Journal of Crustacean Biology, 12(2):169-177.
• [14] Hockley, A.R., (1951). On the biology of Mytilicola intestinalis (Steuer). Journal of the Marine Biological Association of the United Kingdom, 30(2):223-232.
• [15] Cole, H.A., and Savage, R.E., (1951). The effect of the parasitic copepod, Mytilicola intestinalis (Steuer) upon the condition of mussels. Parasitology, 41(3-4):156-161.
• [16] Stock, J.H., (1993). Copepoda (Crustacea) associated with commercial and non-commercial Bivalvia in the East Scheldt, The Netherlands. Bijdragen tot de Dierkunde, 63(1):61-64.
• [17] Korringa, P., (1952). Epidemiological observations on the mussel parasite Mytilicola intestinalis Steuer, carried out in the Netherlands 1952. Ann Biol, 9:219-224.
• [18] Williams, C.S., (1969). The effect of Mytilicola intestinalis on the biochemical composition of mussels. Journal of the Marine Biological Association of the United Kingdom, 49(1):161-173.
• [19] His, E., (1977). Observations préliminaires sur la présence de Mytilicola orientalis Mori (1935) chez Crassostrea gigas Thunberg dans le basin d’Arcachon. Bull Soc Géol amis Mus Havre, 64:7-8.
• [20] Holmes, J.M.C., and Minchin, D., (1995). Two exotic copepods imported into Ireland with the Pacific oyster Crassostrea gigas (Thunberg). The Irish Naturalists' Journal, 25(1):17-20.
• [21] Minchin, D., (1996). Management of the introduction and transfer of marine molluscs. Aquatic Conservation: Marine and Freshwater Ecosystems, 6(4):229-244.
• [22] Grizel, H., (1985). Mytilicola orientalis MORI, parasitism parasitose a Mytilicola orientalis. MORI. Fiches d’identification des maladies et parasites des poissons, crustaces et mollusques. Conseil international pour l’exploration de la mer, Copenhagen, 1-4.
• [23] Torchin, M. E., and Kuris, A.M., (2005). Introduced marine parasites. In: Marine parasitology, Rohde, K. (Ed.), Csiro Publishing. 358-366.
• [24] Mialhe, E., Bachere, E., Le Bec, C., and Grizel, H., (1985). Isolement et purification de Marteilia (Protozoa: Ascetospora) parasites de bivalves marins. Comptes rendus de l'Académie des sciences. Série 3, Sciences de la vie, 301(4):137-142.
• [25] Mialhe, E., Bachere, E., Boulo, V., and Cadoret, J.P., (1995). Future of biotechnology-based control of disease in marine. Molecular marine biology and biotechnology, 4(4):275-283.
• [26] Sogin, M.L., and Gunderson, J.H., (1987). Structural diversity of eukaryotic small subunit ribosomal RNAs a: Evolutionary implications. Annals of the New York Academy of Sciences, 503(1):125-139.
• [27] Figueras, A.J., (1988). Aber disease of edible oysters caused by Marteilia refringens. Amer. Fish. Soc. Spec. Pub., 18:38-46.
• [28] Gosling, E.G., (1992). The Mussel Mytilus: Ecology, Physiology. Genetics and Culture. Amsterdam: Elsevier.
• [29] Berthe, F.C.J., Burreson, E.M., and Hine, M., (1999). Use of molecular tools for mollusc disease diagnosis. Bulletin of the European Association of Fish Pathologists, 19(6):277.
• [30] Le Roux, F., Audemard, C., Barnaud, A., and Berthe, F., (1999). DNA probes as potential tools for the detection of Marteilia refringens. Marine Biotechnology, 1(6):588-597.
• [31] Pernas, M., Novoa, B., Berthe, F., Tafalla, C., and Figueras, A., (2001). Molecular methods for the diagnosis of Marteilia refringens. Bulletin of the European Association of Fish Pathologists. 21:200–208.
• [32] Fuentes, J., López, J.L., Mosquera, E., Vázquez, J., Villalba, A., and Álvarez, G., (2002). Growth, mortality, pathological conditions and protein expression of Mytilus edulis and M. galloprovincialis crosses cultured in the Rı́a de Arousa (NW of Spain). Aquaculture, 213(1-4):233-251. https://doi.org/10.1016/S0044-8486(02)00046-7.
• [33] Kalkan, E., Kurtuluş, A., Maraci, Ö., and Bilgin, R., (2011). Is the Bosphorus Strait a barrier to gene flow for the Mediterranean mussel, Mytilus galloprovincialis (Lamarck, 1819)?. Marine Biology Research, 7(7):690-700. https://doi.org/10.1080/17451000.2011.558098.
• [34] McManus, D.P., and Bowles, J., (1996). Molecular genetic approaches to parasite identification: their value in diagnostic parasitology and systematics. International Journal for Parasitology, 26(7):687-704.
• [35] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S., (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution, 28(10):2731-2739. https://doi.org/10.1093/molbev/msr121.
• [36] Sharp, P.M., and Li, W.H., (1986). An evolutionary perspective on synonymous codon usage in unicellular organisms. Journal of molecular evolution, 24:28-38.
• [37] Felsenstein, J., (1993). Phylogeny Inference Package (PHYLIP). Version 3.5. University of Washington, Seattle, United States of America.
• [38] Saitou, N. and Nei, M., (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 4:406-425.
• [39] Bandelt, H.J., Forster, P., and Röhl, A., (1999). Median-joining networks for inferring intraspecific phylogenies. Molecular biology and evolution, 16(1):37-48.
• [40] Boxshall, G., (2015a). Mytilicola intestinalis Steuer, 1902. In: T.C Walter, G. Boxshall, World of Copepods database. http://www.marinespecies.org/aphia.php?p=taxdetailsandid=128900 on 2015-12-07.
• [41] Boxshall, G., (2015b). Mytilicola orientalis. In: T.C. Walter, G. Boxshall, World of Copepods database. http://marinespecies.org/aphia.php?p=taxdetailsandid=128901 on 2015-12-07.
• [42] Tyler, S., (2015). Urastoma cyprinae (Graff, 1882). http://www.marinespecies.org/aphia.php?p=taxdetailsandid=142950 on 2015-12-07.
• [43] Gibson, D., (2015) Parvatrema duboisi (Dollfus, 1923). http://www.marinespecies.org/aphia.php?p=taxdetailsandid=109235 on 2015-12-07.
• [44] Bolster, G.C., (1954). The biology and dispersal of Mytilicola intestinalis Steuer: a copepod parasite of mussels. Her Maj. Stat. Office.
• [45] Korringa, P., (1968). On the ecology and distribution of the parasitic copepod Mytilicola intestinalis Steuer. Bijdragen tot de Dierkunde. 38:47-57.
• [46] Camacho, A.P., Villalba, A., Beiras, R., and Labarta, U., (1997). Absorption efficiency and condition of cultured mussels (Mytilus edulis galloprovincialis Linnaeus) of Galicia (NW Spain) infected by parasites Marteilia refringens Grizel et al. and Mytilicola intestinalis Steuer. Oceanographic Literature Review. 11: 1351.
• [47] Canestri, T.G., Baccarani, E.M., Giannetto, S., Giuffrida, A., and Paesanti, F., (1998) Prevalence of Mytilicola intestinalis (Copepoda: Mytilicolidae) and Urastoma cyprinae (Turbellaria: Hypotrichinidae) in marketable mussels Mytilus galloprovincialis in Italy. Diseases of aquatic organisms. 32:145-149.
• [48] Rohde, K., Noury‐Sraïri, N., Watson, N., Justine, J.L., and Euzet, L., (1990). Ultrastructure of the flame bulbs of Urastoma cyprinae (Platyhelminthes, ‘Prolecithophora’, Urastomidae). Acta zoologica. 71:211-216.
• [49] Cáceres-Martínez, J., Vásquez-Yeomans, R., and Sluys, R., (1996). Urastoma cyprinae in natural and cultured mussel (Mytilus galloprovincialis LMK) populations in México. Bulletin of the European Association of Fish Pathologists. 16:200-202.
• [50] Comps, M. and Tige, G., (1999). Procaryotic infections in the mussel Mytilus galloprovincialis and its parasite the turbellarian Urastoma cyprinae, France. Diseases of Aquatic organisms. 38: 211-217.
• [51] Crespo-Gonzalez, C., Alvarez, R.R., Domínguez, H.R., Búa, M.S., Iglesias, R., Fernández, C.A., and Estévez, J.G., (2005). In vitro reproduction of the turbellarian Urastoma cyprinae isolated from Mytilus galloprovincialis. Marine Biology. 147:755-760.
• [52] Crespo-González, C., Rodríguez-Domínguez, H., Segade, P., Iglesias, R., Arias, C., and García-Estévez, J.M., (2010). Seasonal Dynamics and Microhabitat Distribution of Urastoma cyprinae in Mytilus galloprovincialis: Implications for Its Life Cycle. Journal of Shellfish Research. 29:187-192. https://doi.org/10.2983/035.029.0113.
• [53] Holodkovskaya, E.B., (1989). Fauna parazitov i kommensalov midii Mytilus galloprovincialis (Lamark, 1819). V severpo-zapadnoi chasti Chernogo mopya (sistematika, ekologia, prakticheskoe znachenie). Moscow State University, PhD Thesis (in Russian).
• [54] Gaevskaya, A.V. and Machevsky, V.K., (1995). Impact of man-made coastal structures on formation and function of parasite systems. In: ECOSET‘95 Sixth International Conference on Aquatic Habitat Enhancement; 29 Oct.-02 Nov. 1995; Tokyo. 531-536.
• [55] Holodkovskaya, E.B., (2002) Biological diversity of parasites of Mytilus galloprovincialis in the Black Sea. In: Minicheva GG, editor. The Black Sea Ecological Problems. Odessa, Ukraine: SCSEIO. 265-269.
• [56] Holodkovskaya, E.B., (2003). Study on symbiont fauna of commercial Black Sea bivalve molluscs. Ecol Morya. 63:66–72.
• [57] Popov, M.A., Kovrigina, N.P., Machevsky, V.K., Lozovsky, V.L., and Kozintsev, A.F., (2010). Influence of the anthropogenic factor on hydrochemical parameters, mussel Mytilus galloprovincialis LAM., and its endosymbionts in a Balaklava Bay. Ternopol нац. Nat пед. Ped ун-ту. University Сер. AvgБiол. 44:205-208.
• [58] Zrncic, S., Le Roux, F., Oraic, D., Sostaric, B., and Berthe, F., (2001). First record of Marteilia sp.in mussels Mytilus galloprovincialis in Croatia. Diseases of aquatic organisms. 44: 143-148. https://doi.org/10.3354/dao044143 .
• [59] Balseiro, P., Montes, A., Ceschia, G., Gestal, C., Novoa, B., and Figueras A., (2007). Molecular epizootiology of the European Marteilia spp., infecting mussels (Mytilus galloprovincialis and Mytilus edulis) and oysters (Ostrea edulis): an update. Bulletin of the European Association of Fish Pathologists. 27:148-156.