Quantitative real-time polymerase chain reaction (qPCR) is probably the most used method for gene expression quantification because of its high sensitivity and specificity. Nevertheless, this technology can undergo experimental errors and variations. Normalization of the results using a reference gene is therefore necessary to minimize these variations. As the study of immune genes in bivalve mollusks has increased in the last years, the establishment of adequate and stable reference genes for bivalves is strongly required. We analyzed the behavior of four putative reference genes: ribosomal RNA 18S, actin, elongation factor 1 − α and α-tubulin. The suitability of these four genes as internal control for qPCR was evaluated in mussel (Mytilus galloprovincialis) and clam (Ruditapes philippinarum) hemocytes after bacterial challenge. Four independent approaches (BestKeeper, GeNorm, NormFinder and DeltaCt ) were used to assess the suitable genes for stable expression. For these particular circumstances, the most stable gene in hemocytes was elongation factor 1 − α for mussels and α-tubulin for clams.

An histology and pathological survey on carpet-shell clam cultured in the south of Galicia (NW Spain) (Ruditapes decussatus) was carried out for a two years period (2000-2001). A temporal distribution of the potentially pathogenic agents were determined by histological analysis. In addition, stocks of molluscs reported to suffer mortalities during the studied period were also analyzed and the pathogenic burden determined. Pathogens do not follow a temporal pattern over studied period and no significant differences were detected nor on the presence nor in prevalence of these pathogens among all the analysed stocks. Therefore, no direct relationship can be established between infection by any pathogen and mortality episodes.

Bivalve molluscs are filter feeders and as a consequence they may bioaccumulate in their tissues viruses that infect humans and higher vertebrates. However, there have also been described mortalities of bivalve molluscs associated with viruses belonging to different families. Mass mortalities of adult Portuguese oysters, Crassostrea angulata, among French livestocks (between 1967 and 1973) were associated with irido-like virus infections. Herpesviruses were reported in the eastern oyster, Pacific oyster and European flat oyster and lately in scallops and clams. Disseminated neoplasia, a proliferative cell disorder of the circulatory system of bivalves, although of uncertain aetiology, has been suggested to be caused by retroviral infections. Other viruses described in bivalves are interpreted as members of the Papovaviridae, Togaviridae, Reoviridae, Birnaviridae and Picornaviridae. However, the lack of bivalve cell lines renders difficult virus isolation from molluscs although some viruses can be isolated in fish cell lines.