LOVETT, D.L.*; RICART, T.M.; TANNER, C.A.; TOWLE, D.W.; The College of New Jersey, Ewing; Mt. Desert Island Biol. Lab., ME: Chronic exposure of the blue crab, Callinectes sapidus, to low salinity stimulates expression of Na⁺,K⁺-ATPase α-subunit mRNA and protein in gills

When estuarine crabs are transferred from high to low salinity, the specific activity of Na⁺,K⁺-ATPase (ATPase) in homogenates of posterior gills increases. In the blue crab, Callinectes sapidus, there is a slight increase in ATPase activity within 24 h following transfer, but substantial increase is not observed until 4 d after transfer. Maximal ATPase activity is reached by 8 d following transfer, and then activity decreases slightly by 18 d (when the crab has acclimated fully). Using Western blot analysis, we have shown previously that the amount of ATPase α-subunit protein in gill homogenates changes during acclimation in a pattern that is reflective of that observed for change in ATPase activity. This trend was observed using either total protein or actin to normalize results. To assess whether the increase in protein expression was due to an increase in gene expression, we recently have quantified ATPase α-subunit mRNA from posterior gill homogenates using real-time PCR. When the concentration of ATPase mRNA is normalized using the concentration of actin mRNA in each sample, the same pattern as that observed for acclimation changes in both ATPase activity and α-subunit protein expression is obtained. Although levels of actin mRNA increase two-fold at 24 h and then decrease, the pattern of ATPase mRNA levels remains similar even when values are not normalized. These results suggest that changes in concentrations of ATPase mRNA in the gill may effect the change in ATPase activity that is observed during acclimation of crabs to low salinity. (Supported by MDIBL NIA Fellowship to DLL, NSF grant DBI-0100394 to DWT, and NSF REU Site at MDIBL DBI-0139190).