Phylogenetic and morphological characterization of the green alga infesting the horse mussel Modiolus modiolus from Vityaz Bay (Peter the Great Bay, Sea of Japan) I.G. Syasina a , A.D. Kukhlevsky a,c , A.L. Kovaleva b , M.A. Vaschenko a,⇑ a A.V. Zhirmunsky Institute of Marine Biology, Far East Branch of Russian Academy of Sciences, Palchevsky Str. 17, 690059 Vladivostok, Russia b V.I. Il’ichev Pacific Oceanological Institute, Far East Branch of Russian Academy of Sciences, Baltiyskaya Str. 43, 690059 Vladivostok, Russia c Far Eastern Federal University, Sukhanova Str. 8, 690600 Vladivostok, Russia article info Article history: Received 28 April 2012 Accepted 2 August 2012 Available online 10 August 2012 Keywords: Trebouxiophycean algae Symbiotic algae Coccomyxa parasitica Mytilus edulis abstract In this work, the ultrastructural features and taxonomic position of the green microalga infesting the horse mussel Modiolus modiolus from the north-western Pacific (Vityaz Bay, Peter the Great Bay, Sea of Japan) are reported. Mussels were collected monthly from May to September of 2009. In different months, the prevalence of mussels with green tissues was 16.6–62.5% (mean 43%). The most affected organs were the mantle, digestive gland and gonad. Histological analysis revealed severe infiltration of the connective tissue by hemocytes containing the alga cells. Electron microscopy showed that the alga was morphologically similar to the green algae from the genus Coccomyxa (Chlorophyta: Chlorococcales). Two new primers were designed to generate partial small subunit (SSU) rRNA sequences of the green alga from M. modiolus. Phylogenetic analysis based on the comparison of the SSU rRNA sequences of the tre- bouxiophyceans confirmed an affiliation of the green alga with the genus Coccomyxa. The sequence (1296 bases) of the green alga from M. modiolus was most closely related to the sequence CPCC 508 (AM981206) (identity 100%), obtained from an acid-tolerant, free-living chlorophyte microalga Coccomyxa sp. and to the sequences EU127470 (identity 99.3%) and EU127471 (identity 99.7%) of the green alga, presumably the true Coccomyxa parasitica, infecting the blue mussel Mytilus edulis from the Flensburg Fjord (North Atlantic). Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction The phenomenon of the symbiosis of unicellular green algae with organisms of different phyla is well known. In Bivalvia, infes- tation by green microalgae has been described for several species from Atlantic coastal waters: the horse mussel Modiolus modiolus (Wiborg, 1946, as cited in Mortensen et al. (2005), the giant scallop Placopecten magellanicus (Naidu and South, 1970; Naidu, 1971; Ste- venson and South, 1974, 1975), the mussels Mytilus edulis chilensis (Gray et al., 1999), M. edulis (Mortensen et al., 2005; Rodríguez et al., 2008) and M. galloprovincialis (Crespo et al., 2009), and the geoduck Panopea abbreviata (Vázquez et al., 2010). Recently, a microalga-infected horse mussel, M. modiolus, from the north- western Pacific (Peter the Great Bay, Sea of Japan) was first re- ported (Syasina, 2011). Based on the morphological features, most researchers have referred green microalgae associated with the tissues of marine bivalve mollusks to the genus Coccomyxa (Chlorophyta: Chlorococcales). The genus Coccomyxa represents a heterogeneous group of microalgae comprising free-living forms (Verma et al., 2009), photobionts of protozoans, lichens and higher plants (Lohtander et al., 2003; Trémouillaux-Guiller and Huss, 2007; Hoshina and Imamura, 2008), and parasitic species affecting several marine invertebrate taxa (Stevenson and South, 1974). The members of the genus Coccomyxa have a generally similar mor- phology; moreover, as shown in Coccomyxa parasitica isolated from P. magellanicus and grown under different conditions, the alga may exhibit high variability in cell shape and size (Stevenson and South, 1974). Thus, the morphology-based identification of Coccomyxa species is extremely difficult and necessitates the use of molecular phylogenetic analysis to identify these species (Rodríguez et al., 2008). To date, there are only two studies employing small subunit (SSU) rRNA sequence analysis to determine the phylogenetic posi- tion of the green microalgae infesting bivalve species from the North and South Atlantic, M. edulis (Rodríguez et al., 2008) and P. abbreviata (Vázquez et al., 2010). The horse mussel M. modiolus is a boreal species widely distrib- uted throughout the North Atlantic (Brown, 1984; Dinesen and Ockelmann, 2005), the Barents and White Seas (Flyachinskaya and Naumov, 2003; Sokolov and Shtrik, 2004), and in the Pacific 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.08.001 ⇑ Corresponding author. Fax: +7 423 2310900. E-mail address: mvaschenko@mail.ru (M.A. Vaschenko). Journal of Invertebrate Pathology 111 (2012) 175–181 Contents lists available at SciVerse ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip