Preliminary Study Of Portunus Trituberculatus Na~+, K~+-ATPase In Salinity Regulation Mechanism

Swimming crab, which has the great economic significance, is one of the mostimportant marine aquaculture species in China. In the embryonic development and thewhole larval stage, swimming crab is so sensitive to the change of environmentalsalinity that the slightest mistake may cause the delays in metamorphosis. High salinitycan cause the difficulty of zoea truning into megalopa and the low megalopametamorphosis rate. Therefore, for artificial breeding processes of swimming crab,salinity is a very important factor. Figuring out the mechanism of swimming crabadapted to environmental salinity has great significance in both theory and artificialbreeding.This project studies the changes of Na~+,K~+-ATPase mRNA expression and Na~+,K~+-ATPase enzyme activities as well as the morphology and immunohistochemicalcharacteristics under different salinity stress. The purpose of this study is to explore therole of swimming crab Na~+,K~+-ATPase in salinity regulation mechanism by gene level,protein level and organization level.After compared with control (25) salinity environment, the semi-quantitative RT-PCR results of Na~+,K~+-ATPase mRNA expression show that the increase in varyingdegrees of Na~+,K~+-ATPase mRNA expression level come from in both high and lowsalinity stress in the6thgill, and the changes of expression level in muscle and intestineare not obvious. A conjecture ban be extrapolated from the results that when theswimming crab subjected to salinity stress, Na~+,K~+-ATPase mRNA can react to thesituation quickly in order to adapt to the changes of the external environment.The most basic function of Na~+,K~+-ATPase is catalyzing ATP hydrolysis, and thenreleasing inorganic phosphate (Pi). The enzyme activity is proportional to the amount ofPi released when Mg2+and ATP are sufficient. Hence enzyme activity can be calculatedthrough measuring the amount of Pi released. The results of Na~+,K~+-ATPase activity inswimming crab measured under salinity stress show that the activities of gill Na~+,K~+-ATPase decreased in the primary period, increased gradually, decreased again andtended to be stable finally under both high salinity and low salinity degree of stress. Inaddition, the Na~+,K~+-ATPase activities of the three posterior pairs of gills wereremarkably higher than those of the five anterior pairs of gills. The three posterior pairs of gills were the main sites of ion transportation and osmotic regulation in P.trituberculatus.Immunohistochemical technique is used on gill Na~+,K~+-ATPase positioning。Theexpression of Na~+,K~+-ATPase in gill increase significantly under high salinity stress,while this phenomenon is not showed significantly under low salinity stress. The resultsindicated that Na~+,K~+-ATPase is more sensitive to high salinity stress than low salinitystress.25salinity samples after H.E. staining is regarded as a control to analyze the resultsof gill tissue samples under salinity stress for1day. The results show that the amount ofchloride cells compared to the control gets a high increase under high salinity stress, andchloride cells are mainly concentrated in the base of gill filaments under low salinitystress.After analyzing the results comprehensively, this study reveals that the Na~+,K~+-ATPase mRNA expression level get a high increase in both high and low salinity stress,and the expression of Na~+,K~+-ATPase increase significantly in high salinity stresspreliminary stage but not in low salinity stress preliminary stage. In addition, Na~+,K~+-ATPase is more sensitive to high salinity stress than low salinity stress, and the osmoticregulation of Na~+,K~+-ATPase in swimming crab might be divided into3phases: passivestress phase, positive regulation phase and adaptive phase.