Analysis On Salinity Related Genes Expression And Histological Structure Of Apostichopus Japonicus

Apostichopus japonicus, as the highest value breed of mariculture, is widely distributed in the coastal area in north of China, which has important social benefits and economic value. The culture ponds of sea cucumbers located in the intertidal zone, the sea salinity of the aquacultural pond suffer severe changes in the rainstorms in summer, freezen in winter and tidal variation. So it’s very importment to study the physiological, biochemical and molecular regulation mechanism and understand the salinity adaptation process of A.japonicus. The study measured the physiological and biochemical indexes, observed the histologic changes of different organs,researched the key gene expression and located Na⁺/K⁺-ATPase’s gene expression under salinity stress. These results supplied more reliable datas for sea cucumber breeding and reducing the losses in sea cucumber industry.The experiment simulated salinity change in the rainy season, which divided into three stages. The first stage simulated the rains suddenly fell, salinity decreased gradually from normal sea water（32 psu） salinity to 18 psu at the speed of 3 psu every 6h. The second stage, the salinity maintained at 18 psu for 96 h, no water changed. Finally, salinity increased from 18 psu to 30 psu at the same rate, and then maintained at 30 psu for 24 h. The variation trends of osmotic pressure in coelomic fluid, total protein concentration, Na⁺, K⁺ and Cl- were consistent with water salinity variation. The concentration of Na⁺, K⁺ and Cl- in coelomic fluid reaches the minimum at salinity18 psu. The concentration of Ca²⁺ was maintained the higher level than the control during the experiment. The Na⁺-K⁺-ATPase activity of coelomic fluid showed significantly higher than other groups at 18 psu for 3d（P<0.05）. The variation in the activity of Na⁺-K⁺-ATPase in coelomic fluid reflected its key effects on osmotic regulation under salinity stress. There was no significant difference on GPT of sea cucumbers’ coelomic fluid under different salinities.The results illustrated that the histological variations of different organs under different salinities. There was significant difference in tube feet, intestine and longitudinal muscle under salinity stress. The low salinity had obvious damage in the integrity of organizations from the variation of the histology. The damage deteriorated with stress time increasing. Tissue damages were significantly observed in intestines, muscles and tube feet under low salinity environment（18 psu, 23 psu, and 27 psu）. The connective tissue in intestines of Apostichopus japonicus exposed to 18 psu and 23 psu damaged and partly separated from the mucosal epithelium. The significant variations occurred in tube feet, which presented the swelling in connective tissue and a fracture in longitudinal muscles under low salinity（18 psu）. The morphological change of tube feet showed the shrinkage of connective tissue under high salinity（40 psu）. The amount of infusoria in the respiratory trees decreased or even disappeared in salinity treatment groups（18psu and 23 psu）. The results concluded that effect of low salinity stress is more significant than high salinity stress.Based salinity stress transcriptome information, the 10 genes from 403 differentially expressed genes,（involving Calcium-transporting ATPase, H⁺-ATPase, Neuronal acetylcholinereceptor subunit alpha-9, Sodium- and chloride-dependent GABA transporter 2, Aquaporin,sodium/potassium-transporting ATPase subunit alpha（Na⁺/K⁺-ATPase）, Calcium-activated chloride channel regulator 4-like（CLCA4）, NADH, GlyR） were analyzed by real-time quantitative PCR. The results showed that different genes had different expression profile in different tissues. The expression of Na⁺/K⁺-ATPase and Ca²⁺-ATPase showed the same tendency in the intestine and respiratory trees and reached the maximum at 6 h. The expression of Ca²⁺-ATPase showed up-regulated in each tissue under low salinity stress. This phenomenon indicated that Ca²⁺-ATPase played a significant role in the regulation of salinity change. Both Sodium- and chloride-dependent GABA transporter 2 and Glycine receptor subunit alpha-4, as nerve conducted receptor, showed the same tendency under the low salinity stress.The result of in-situ hybridization in Na⁺/K⁺-ATPase showed the positive hybridization sites in each tissue. There is no positive hybridization site in the connective tissue of respiratory trees,which illustrated that respiratory tree wasn’t the primary site of the Na⁺/K⁺ exchange. The positive hybridization points of Na⁺/K⁺-ATPase gene emerged in tentacle tissue under low salinity stress. The result shows some positive hybridization points existed in the joint gaps between connective tissues and the muscle layer, which were similar with the previous studies on tube feet tissue. The two tissues belong to the water vascular system, which plays an important role in salinity adapt process. Above all, all these results revealed the important effects on osmotic regulation of A. japonicus.