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Darwinian selection approach is widely adopted from single coding gene comparisons to whole genome investigations, to trace the evolutionary forces affecting shape and function of several molecular traits. Positive selection has been demonstrated to drive an enhanced evolution and promote rapid divergence in several genes as in immune defense, host pathogen recognition and reproductive proteins. On the contrary, purifying selection opposes to gene mutations preserving the functional constraints and the molecular architecture, as in housekeeping genes or in trans-membrane protein domains. With the goal of analyzing the natural selection forces which have driven the molecular adaptation of respiratory proteins we have comparatively evaluated 17 groups of non-vertebrate oxygen transporters. The results show a wide dominance of purifying selection in almost all protein families, suggesting that the structural and functional constraints required for the reversible oxygen binding are strictly conserved during evolution. The single exception is constituted by crustacean hemocyanin for which the analyses reveal several sites under diversifying selection. The structural and functional heterogeneity of crustacean hemocyanin is considered to be the result of a complex evolutionary process originated from the aggregation of simpler molecules. The hemocyanins existing today have been created by the gene fusion of ancestral proteins, which constitute the actual three domains and which share, in the new molecule, their ancestral biological functions. Besides to the oxygen binding, achieved by the highly conserved active site, arthropod hemocyanins contribute to other physiological processes, like immune response to pathogens, phenoloxidase activity, wound repair and molting. Furthermore, the analysis of selecting forces, acting on all available genes of the crustacean hemocyanins reveal a complex pattern of accelerated evolution events occurred after gene duplication followed by t

The successful colonization of new environments is often achieved through adaptations or key innovations of existing physiological or biochemical mechanisms. The oxygen supply in marine invertebrates represent a complex and deeply integrated system which plays a fundamental role in animal adaptive plasticity. In particular, species which inhabit highly stochastic environments as shallow water or intertidal bands, have to cope with extremely different regimes of oxygen availability and effectively maintain a stable aerobic metabolism. Within this framework, we have focused on comparative physiology of Portunid Crabs hemocyanin, to evaluate the role molecular heterogeneity and functional plasticity of oxygen transport regulation. The analyses concerned the Mediterranean green crab Carcinus aestuarii, that inhabits shallow brackish waters with soft bottoms and mussel beds and two obligate aquatic species, the grey swimming crab Liocarcinus vernalis, that populates sandy bottoms from lower shore to a depth of -40 m, and the swimming crab L. depurator, distributed below the thermocline on fine sandy and muddy bottom. To assess the adaptive regulation of oxygen transport among these species, we have considered the effects that physiological and environmental modulators (lactate, pH and temperature) play on hemocyanin functioning. The biochemical parameters undergo to drastic changes during hypoxic events and show significant variations when animals move from aerobic to anaerobic metabolism. At molecular level we have verified that both lactate and pH play a central role in modulating the hemocyanin oxygen-binding properties and we have singled out the effect of each parameter on the oxygen affinity in resting and hypoxic conditions. From this study, a clear separation between subtidal and intertidal species emerges, evidencing the physiological plasticity that tolerant species exhibit when subjected to stressful conditions and underlying the importance that the oxygen ava