Functional Characterization of Neuroendocrine Regulation of Branchial Carbonic Anhydrase Induction in the Euryhaline Crab Callinectes sapidus

Carbonic anhydrase (CA) plays an essential role as a provider of counterions for Na/H and Cl/ HCO3 exchange in branchial ionic uptake processes in euryhaline crustaceans. CA activity and gene expression are low in crabs acclimated to full-strength seawater, with trans- fer to low salinity resulting in large-scale inductions of mRNA and subsequent enzyme activity in the posterior ion-regulating gills (e.g., G7). In the green crab Carcinus maenas, CA has been shown to be under inhibitory neu- roendocrine control by a putative hormone in the x-organ- sinus gland complex (XOSG), located in the eyestalk. This study characterizes the neuroendocrine regulation of CA induction in the blue crab Callinectes sapidus, a commonly used experimental organism for crustacean osmoregulation. In crabs acclimated to full-strength seawater, eyestalk liga- tion (ESL) triggered a 1.8- and 100-fold increase in CA activity and mRNA, respectively. Re-injection with eyestalk homogenates abolished increases in CA activity and frac- tionally reduced CA gene expression. ESL also enhanced CA induction by 33% after 96 h in crabs transferred to 15 ppt salinity. Injection of eyestalk homogenates into intact crabs transferred from 35 to 15 ppt diminished by 43% the CA induction stimulated by low salinity. These results point to the presence of a repressor hormone in the eyestalk. Separate injections of medullary tissue (MT) and sinus gland (SG), two components of the eyestalk, reduced salin- ity-stimulated CA activity by 22% and 49%, suggesting that the putative repressor is localized to the SG. Crabs injected with SG extract harvested from crabs acclimated to 5 ppt showed no decrease in CA activity, demonstrating that the hormone is down-regulated at low salinity. Our results show the presence in the XOSG of an inhibitory compound that regulates salinity-stimulated CA induction.