Holbech, Henrik2; Kinnberg, Karin Lund2; Bjerregaard, Poul2; Morthorst, Jane Ebsen2; Pedersen, Knud Ladegaard2
1 Department of Biology, Faculty of Science, SDU2 Department of Biology, Faculty of Science, SDU
Invertebrates and especially molluscs have received increasing attention in relation to endocrine disrupting chemicals (EDs) during the last few years and the development of OECD test guidelines to assess the effect of EDs with molluscs are in progress. One of the main problems with the development of standardized tests in molluscs is that no specific biomarkers or endpoints for endocrine effects have been validated. Some attempts have been made to transfer biomarkers developed for vertebrates – e.g. from fish to molluscs to investigate ED effects. One example is the vertebrate yolk protein vitellogenin that is known to be oestrogen dependent in fish. The yolk proteins in molluscs have been proposed to have the same oestrogenic dependence and used as biomarker for oestrogenic EDs. The present work investigates the possible usability of the main yolk protein in three species of molluscs to function as biomarker for oestrogenic exposure. We have developed specific antibodies against the main yolk protein from the three species (Unio pictorum, Unio tumidus and Lymnaea stagnalis) and with specific ELISAs (enzyme linked immunesorbent assay) and immune-histology we investigate the distribution and concentration of the proteins in molluscs of different sex and life-stage. The main yolk protein was purified from gonads containing eggs of the freshwater bivalves U. pictorum and U. tumidus and from eggs dissected from egg clutches of the pulmonate gastropod L. stagnalis. The ELISAs were used to quantify the concentration of the yolk protein in juveniles and adult male and female U. pictorum and U. tumidus and in the hermaphroditic L. stagnalis. With use of immune-histology, the distribution of the proteins in the tissue of the three species was investigated. The ELISAs revealed that the normal sex specific concentration distribution seen in fish, where vitellogenin is normally seen in very low concentrations in male and juveniles was not seen in the molluscs. The concentration of the protein did not differ among the sexes and was a factor of approximately 10000 higher in male U. tumidus and U. Pictorum than in male fish. Based on The results the authors do not support the use of mollusc yolk protein as biomarker for oestrogenic exposure because it seems to have more than one function in the investigated species. We have shown that the content and distribution of the proteins are not sex specific.