In situ hybridization detection of initial infective stages of Marteilia refringens (Paramyxea) in its host Mytilus galloprovincialis

Marteilia refringens is a protistan parasite of the phylum Paramyxea, which affects economically important bivalve species in Europe, such as the European flat oyster Ostrea edulis, and the mussels, Mytilus edulis and M. galloprovincialis (LópezFlores, de la Herran, Garrido-Ramos, Navas, Ruiz-Rejon & Ruiz-Rejon 2004). Infection with Marteilia refringens is listed as a notifiable disease by the World Organization for Animal Health (OIE) because of the detrimental impact of the parasite on infected mollusc cultures. However, the life cycle of the parasite is still unclear although recent research points towards a presumed zooplanktonic intermediary host (Audemard, Le Roux, Barnaud, Collins, Sautour, Sauriau, De Montaudouin, Coustau, Combes & Berthe 2002; Berthe, Le Roux, Adlard & Figueras 2004; Carrasco, Arzul, Furones, Chollet, Robert, Joly & Berthe 2005; Carrasco, López-Flores, Alcaraz, Furones, Berthe & Arzul in press). Marteilia refringens is usually found in the digestive gland of flat oysters and mussels. Young stages of M. refringens have been described in the epithelia of stomach and primary digestive tubules, whereas more mature stages of the parasite can be found in the epithelium of the digestive gland tubules (Grizel, Comps, Bonami, Cousserans, Duthoit & Pennec 1974; Berthe et al. 2004) where sporulation takes place. However, a few studies have reported the presence of young plasmodial stages of the parasite in gill tissues of O. edulis (Comps 1970; Grizel et al. 1974) and M. galloprovincialis (Robledo & Figueras 1995). This location was considered unusual and not the normal location of the parasite (Comps 1970; Grizel et al. 1974; Robledo & Figueras 1995; Berthe et al. 2004). Development of molecular detection techniques for Marteilia, such as in situ hybridization (ISH) (Le Roux, Audemard, Barnaud & Berthe 1999), may be very useful to improve the understanding of the parasite cycle within its host, and to observe early infections which are not easy to detect by routine histological observation. For example, ISH was used to describe the early development of M. sydneyi in its host Saccostrea glomerata (Kleeman, Adlard & Lester 2002). This report presents a preliminary study using ISH for the detection of all parasitic stages of M. refringens occurring throughout the year, paying special attention to initial infective and early stages of the parasite with the aim of improving the understanding of the life cycle of M. refringens and its transmission. Journal of Fish Diseases 2008, 31, 153–157