• hysto-chemical identification of metal storing organelles in the previously identified main target tissues (gill, digestive gland and byssus threads)
• identification of the typical storage form of each selected metal (Hg, Fe and Mn)
• elucidation of the main differences in metal storage between vent species from geochemically different sites of the MAR
• description of experimentally induced uptake/release mechanisms following exposure to extreme levels of selected metals under controlled laboratory conditions using the pressure chamber

2.) The role of amebocytes in Ca2+ transport and biomineralization of the shell
For this objective our primary focus will be the putative calcium binding proteins known to be expressed in bivalves´ mantle epithelia. Thus we expect the following results:

• Ca binding protein expression in hemocytes and also their mRNA levels in Northern blot assays using total RNA extracted from hemocytes, mantle or organic matrix
• direct localization of Ca2+ binding protein using mRNA in-situ hybridization experiments. This will indicate us whether the calcium binding protein genes are up-regulate, where and under what conditions.
• using the gene sequence information available for some bivalve Ca2+ binding proteins PCR cloning strategies will also be considered.
• monitoring of the fate of Ca2+ in its calcium carbonate form or associated to calcium binding proteins, using live tissues, and combining a series of biochemical analysis and live imaging using specific fluorescent Ca2+ markers.
• demonstration of direct Ca2+ binding to proteins and determination of its optimal binding activity and stoichiometry: in vitro studies using Ca2+ isotopes (45Ca and 47Ca) and crude hemocyte membrane preparations subjected to SDS-PAGE and to immobilization onto nitrocellulose filters.
• Hemocyte motility will be monitored by live imaging using optical and fluorescence microscopy.
• Ultra-structural analysis of transport Ca2+ and calcium carbonate deposition into the shell will be achieved by electron microscopy. Electron microscopy will also be used to reveal the ultra-definition and cellular morphological features of the mantle outer epithelial cells as well as the direct presence of hemocytes in this dynamic and interactive milieu which is the shell mineralization front.

3) Shell microstructure in the vent mussel and its role in metal detoxification
From a successful realisation of this objective the following results are foreseen:

• description of the general ultrastructure of the shell in the bivalve (Bathymodiolus azoricus) from hydrothermal vents as compared with their shore analogues
• identification of main differences in crystal ultrastructure of shells originated from vents with different geochemical conditions reflecting the effect of physicochemical factors on the shell calcification
• -investigation of crystal formation under lab-induced stress (pressure, heavy metals, pH and temperature)
• analythical quantification of shell burden of selected metals (Hg, Fe, and Mn)
• description of in vivo metal incorporation/release mechanisms in the shell microcrystals (experimental exposure to toxic metals at different pressure and temperature followed be period of recovery in seawater)
• determination of the effect of environmental factors (pressure, pH, metal exposure levels etc) on the rate of metal uptake in the shells of the vent bivalve
• in vivo metal incorporation mechanisms in the shell microcrystals (experimental exposure to toxic metals at different pressure and temperature)