Effects of low salinity on osmoregulation, 

Na+/K+ -ATPase activity and related gene 

expression of juvenile Epinephelus fuscoguttatus

Abstract: Brownmarbled grouper Epinephelus fuscoguttatus is a popularly important species in tropical mariculture due to its delicious meat, fast growth and strong disease resistance, and has been widely cultivated in Fujian, Guangdong, Hainan and other places. Salinity is an important environmental factor that affects the growth, development and reproduction of fish. Euryhaline teleosts can maintain the stability of internal environment homeostasis through effective osmotic regulation to adapt to the changes of salinity in their living environment. Epinephelus fuscoguttatus has a wide range of salinity tolerance and has been reported that can live in waters with salinity ranging from 11 ‰ to 41 ‰, but little is known about its salinity adaptation mechanism. In order to study the effect of low salinity on osmoregulation of juvenile Epinephelus fuscoguttatus, juveniles with an average weight of (56.8 ± 3.5) g temporarily reared in natural seawater ( 32 ‰ ), were transferred into the water with salinity of 6 ‰, 12 ‰, 24 ‰ and 32 ‰ (control group) for 10 d. Serum osmolality, the concentration of Na+, Cl-, K+ in serum, muscle water content, the Na+/K+ATPase activity and the expressions of NKA α1, NKA β1b, NKA α2a, NKA α3 in gill were determined, and changes of gill microstructure and oxygen consumption rate were observed at the end of the experiment. Results showed that with the decrease of salinity, serum osmolality of juvenile Epinephelus fuscoguttatus decreased, and the serum osmolality of salinity 6 ‰ group was significantly lower than that of the other groups (P<0.05), while salinity 12 ‰, 24 ‰, and 32 ‰ groups had no significant differences (P>0.05). The concentrations of Na+ and K+ in serum decreased with the decrease of salinity, and the concentrations of Na+ and K+ in salinity 6 ‰ group were significantly lower than that in the control group (P<0.05), while salinity 12 ‰, 24 ‰, and 32 ‰ groups had no significant differences (P>0.05). There was no significant difference in the concentration of Cl- in serum among the groups (P>0.05). Muscle water content increased with the decrease of salinity, but there was no significant difference among the groups (P>0.05). With regression analysis, there was a linear correlation between serum osmolality and ambient water osmolality: Y=0.0781X+317.14 (R2 = 0.82, P<0.05), and the intersection of the regression line and the isosmotic pressure line was the isosmotic point calculated as 343.73 mOsm·kg-1, and the corresponding salinity was 12.59 ‰. The activity of Na+/K+ATPase in gills first decreased and then increased in a “U” shape with the decreasing salinity. The lowest activity of Na+/K+ATPase appeared in salinity 12 ‰ group, which was significantly lower than that of other groups (P<0.05), and there was no significant difference between salinity 6 ‰, salinity 24 ‰ or salinity 32 ‰ (P>0.05). With salinity decreasing, the expression of NKA α1 in gill had no significant difference among the groups (P>0.05). The expression of NKA β1b in gill first decreased and then increased, and the expression in salinity 12 ‰ group was the lowest, which was significantly lower than that of other groups (P< 0.05). The expression of NKA α2a in gill first increased and then decreased with the decreasing salinity, the expression at salinity 12 ‰ was the highest and significantly higher than that at salinity 6 ‰, 24 ‰, 32 ‰ (P< 0.05), and there was no significant difference in salinity 6 ‰, 24 ‰, 32 ‰ (P>0.05); the expression of NKA α3 in gill decreased with the decreasing salinity, the expression at salinity 6 ‰ was the lowest and significantly lower than that at salinity 12 ‰, 24 ‰, 32 ‰ (P< 0.05), and the expression of salinity 32 ‰ was the highest and significantly higher than that at salinity 6 ‰, 12 ‰, 24 ‰ (P<0.05). Gill chloride cells of juvenile Epinephelus fuscoguttatus were mainly distributed in gill filaments and the base of lamellae of all salinity groups. With the decreasing salinity, the number of chloride cells in gill filaments first decreased and then increased; the least appeared at salinity 12 ‰, which was significantly lower than that at salinity 6 ‰ and 32 ‰ (P<0.05). The number of chloride cells in gill lamella was the highest at salinity 32 ‰, significantly higher than that at salinity 6 ‰ and 12 ‰ (P<0.05). There was no significant difference in the diameter of chloride cells in gill filaments and lamella among experiment groups (P>0.05). The oxygen consumption rate increased as salinity decreased, and the lowest rate was at salinity 32 ‰, which was significantly lower than that at salinity 6 ‰ and 24 ‰ (P<0.05), but not significantly different from that at salinity 12 ‰ (P＞0.05). There was no significant difference in oxygen consumption rate among salinity 6 ‰, salinity 12 ‰ or salinity 24 ‰ (P>0.05). The findings indicated that juvenile Epinephelus fuscoguttatus had a strong osmoregulatory ability and could effectively adapt to salinity changes in the range of 32 ‰ within 10 d.  However, in salinity 6 ‰ group, feeding almost stopped and activity decreased, indicating that juvenile Epinephelus fuscoguttatus had been subjected to stress at salinity 6 ‰. The changes of Na+/K+ATPase activity in gills were affected not only by the expression of NKA genes, but also by the number of chloride cells in gill. The energy consumption on osmoregulation may occupy a relatively small part of total metabolism in Epinephelus fuscoguttatus.  It is suitable for its growth in a natural habitat within a salinity of 32‰. In summary, it is helpful to understand the theoretical mechanism of osmotic regulation and salinity control of Epinephelus fuscoguttatus aquaculture.