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E-cadherin is an evolutionary conserved protein, whose main role as the principal component of adherens junctions is supporting epithelial cell-cell adhesion. It is an essential molecule for the maintenance of the epithelial barrier function and the analysis of its immunohistochemical expression is a valuable resource in morphopathological, ontogenetic and pathogenesis studies in mammals. As well, there is an increasing understanding of the importance of E-cadherin in the physiology of the immune system A c c e p t e d m a n u s c r i p t 2 and the development of the immune response. Mucosal health is a primary issue in aquaculture research; nevertheless, there is a lack of immunohistochemical studies of cell junction proteins in fish species. In this work, an immunohistochemical technique was optimized in Bouin-and formalin-fixed paraffin-embedded tissues of turbot Scophthalmus maximus, employing a commercial antibody raised against human Ecadherin. The specificity of the antibody in recognizing the molecule in this teleost species was tested by western blot and mass spectrometry-based proteomic analyses. The assays showed a good specificity and indicated that the antibody recognizes the well conserved cytoplasmic domain of the protein. Immunohistochemistry showed the localisation of E-cadherin at cell-cell contact in the epithelia of the different organs, between the hepatocytes and the pancreatic acinar cells, as well as in the reticuloepithelial stroma of the thymus. Also, the immunoreaction was observed in the cells constituting the melano-macrophage centres in the spleen and kidney. No immunostaining was detected, as expected, only in the heart and brain. No significant difference was noticed between the two fixative used for collecting the tissues samples. This is the first description of E-cadherin immunohistochemical expression in several tissues of a teleost. The immunohistochemical technique represents a useful tool to be used in the different areas of fish health research.

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& It has been shown that adenosine protects against the lack of oxygen in hypoxia=anoxia intolerant organisms such as mammals, and in some tolerant vertebrates such as freshwater turtles of the genera Chrysemys and Trachemys, and crucian carp. By contrast, little is known about the possible protective role of adenosine in invertebrates, although invertebrates comprise the major number of hypoxia=anoxia tolerant organisms, for example, mussels. With the aim to explore the adenosynergic system in these organisms, we have developed an HPLC method for assessing adenosine, its putative precursors (ATP, ADP, AMP, and cAMP), some of its degradation metabolites (inosine, hypoxanthine, and xanthine), and IMP. The method consists of an ion-pair reversed-phase chromatography with gradient elution and UV detection with a diode array detector that allows us to evaluate all the compounds of interest in a single chromatogram within twenty-five min. The method is suitable for both hemolymph and extracts from different tissues.

Two different isoforms of the regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), had previously been identified in the sea mussel Mytilus galloprovincialis. R(myt1) and R(myt2) were differentially distributed in the various cell types comprising the mussel mantle. R(myt1) was found the only isoform to be present in the auxiliary cells of female follicles and the cubic epithelium of the middle fold of mantle edge. In contrast, only the R(myt2) isoform was detected in the subepithelial connective tissue, the endothelium of haemolymph vessels, the adipogranular and vesicular cells of reserve connective tissue, and the spermatozoa. Finally, both R(myt1) and R(myt2) coexist in some cell types but they show a different cellular localization: R(myt1) was localized in the cytoplasm whereas R(myt2) was mainly detected in the cell apical edge, cell periphery, cilia and sperm flagella. Interestingly, both R(myt1) and R(myt2) were absent in oocytes within the female gonadal follicles but, in contrast, they were highly expressed in follicle-released oocytes collected after spawning. Taken together the results show that mussel PKA isoforms are differentially distributed in the mantle cell types, which suggests that they are involved in the regulation of distinct cellular functions. On the other hand, the expression of R(myt1) and R(myt2) proteins is associated with the meiosis resumption of oocytes at the prophase I stage, which occurs in parallel to spawning.