Physiological function of free D-alanine as an iso-osmotic effector in crustaceans

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Free D- and L-amino acids were determined on the various tissues of crayfish Porcambarus clarkii, marsh crab Holometopus dehaani, and shore crab Hemigrapsus penicillatus during seawater acclimation from freshwater.Along with the hyper-osmotic stress, several free amino acids increased in the muscle as well as the other tissues of these crustaceans. Of several D-amino acids found in the tissues, only D-alanine increased significantly during seawater acclimation. The ratio of D-alanine to total alanine also increased to about 50% in seawater. These data clearly indicate that D-alanine is a major osmo-effector for maintaining the tissue iso-osmotic pressure in these crustaceans.Woolly-handed crab Eriocheir japonicus, which migrates downstream during spawning in fall, did not show clear increase of free amino acids and D-alanine during seawater acclimation before summer. D-Alanine in the tissues of this crab is much higher in the individuals obtained from summer to fall than those from spring to summer. During seawater acclimation of this species obtained after summer, the increase of D-alanine was high and reached to almost the same level as that in naturally migrated individuals captured at seashore. During seawater acclimation, hemolymph showed the increase of only inorganic ions and no change of free amino acids and betaines. Thus, these data indicate that only inorganic ions are responsible for the osmotic regulation in hemolymph but D-alanine as well as other several amino acids are also responsible up to 45% of total osmolytes for the tissue iso-osmotic regulation of this species during downstream spawning migration.In this study, the physiological function of D-alanine in the tissues of crustaceans was elucidated to be as a major iso-osmotic effector. The level of D-alanine in the muscle of crayfish was also known to be maintained even after starvation in seawater and after administration of D- or L-alanine in the muscle.