| To study salinity effects on survival, concentrations of ions and hormones in serum of Epinephelus coioides juveniles, and the changes in gill mitochondrion-rich cells(MRCs) of Epinephelus coioides juveniles, and immunofluorescence staining of Na~+/K~+-ATPase(NKA), Na~+/K~+/2Cl- contrasporter(NKCC) and Na~+/H~+ exchanger-3(NHE3), the methods of physiology, scanning electronmicroscopic(SEM) observations and whole-mount immunocytochemistry were used. The main results were showed as follows: 1. Survival, concentrations of ions and hormones in serum of Epinephelus coioides juveniles under different salinitiesE. coioides juveniles were directly transferred to water with salinities at 0, 5, 10 and 20 from water with salinity at 30(the control group). Na~+, K~+, Cl- concentrations, growth hormone(GH), cortisol(COR) and prolactin(PRL) in serum and the deaths of E. coioides juveniles were investigated at 2 h, 6 h, 12 h, 48 h, 72 h, 7 d and 15 d after transferring. The results showed that death rate of E. coioides juveniles in water with salinity 0 were gradually increasing, and E. coioides juveniles were all dead at 72 h, while in other groups were living well. Na~+ concentration in serum significantly increased with the rise of water salinity(P<0.05). Na~+ concentration in water with salinity 0 and the control group remained stable, but the other groups were just the opposite, they demonstrated a similar tendency of decreasing first to its lowest value at 6 h, increasing thereafter. K~+ concentration in serum in water with salinity 0 increased and then decreased, and being significantly higher than that at salinities 5, 10, 20, and the control group(P<0.05), while K~+ concentration in water with salinities of 5, 10, 20 and the control group showed a downward trend and then increased,and there were no significant differences in them(P>0.05). Cl- concentration in serum increased with the rise of water salinity, and showed a downward trend in the water with salinity 0, and a fluctuation in water with salinities 5 and 20, while there were no significant difference in water with salinity 10 and the control group(P>0.05). GH level increased with the rise of water salinity, and there were no significantly changes in GH in the water with salinity of 20 and the control group(P>0.05), but significantly higher than that at salinities 0, 5 and 10. PRL displayed a significant downward with the rise of salinity(P<0.05), and in salinity stress groups displayed a similar trend of decreasing first, increasing to its peak at 12 h thereafter, and then decreasing, being stable at last. COR also showed a significant downward with the rise of salinity, and displayed the same trend in water with salinities of 0, 5 and 10 as PRL, but significantly higher than that in salinity 20 and the control group(P<0.05). In conclusion, E. coioides juveniles can adapt to the water with salinity 5, but cannot survive for long in fresh water. And according to above results, the fresh water aquiculture of E. coioides is feasible. 2. Studies on structure of gill mitochondrion-rich cells(MRCs) under different salinitiesGill mitochondrion-rich cells(MRCs) play a crucial role in osmoregulation of euryhaline teleosts. So in order to discuss the osmoregulatory function of gill MRCs, the changes in structure of gill MRCs under acute fresh water stress and low salinities stress were observed. Results showed that the short and long diameter of gill MRCs in fresh water were significantly shorter than that in control group(P<0.05), but the diameter of gill lamellaes widened in fresh water(P<0.05) under acute fresh water stress. And under low salinities stress, the volumes and quantities of gill MRCs increased, and the volumes of gill MRCs in water with salinity 20 and control group were significantly larger than that in water with salinities 5 and 10(P<0.05). So the results indicated that the structure of gill MRCs changed when the osmolalities of surroundings changed. 3. The research of scanning electronmicroscopic(SEM) observations and immunofluorescence staining gill mitochondrion-rich cells(MRCs) under different salinitiesTo study the changes in apical openings of gill mitochondrion-rich cells(MRCs) and the immunofluorescence staining NKA, NKCC and NHE3,E. coioides juveniles relayed in the seawater with salinity of 30 were directly transferred to water with salinities at 0,5, 10, 20, and 30 for 48 h, while those in seawater with salinity of 30 were taken as the control group. SEM observations revealed that there was a special apical opening in gill MRCs. The apical surfaces of gill MRCs in water with salinities 0 and 5 were generally flat or a little concave with dense microvilli-like projections. And in water with salinity 10 was a convex apical surface with dense microvilli-like projections. In contrast, the apical surfaces of gill MRCs in water with salinity 20 and the control group exhibited deeply invaginated surfaces with smaller orifices without projections. The NKA-immunoreactive cells distributed on the gill filament especially at the base, and the fluorescence intensity strengthened with the increasing of water salinity. NKCC-immunoreactive cells started to be discovered in water with salinity 10, and then the numbers and fluorescence intensity of NKCC-immunoreactive cells increased in water with salinity 20 and control group. In contrast, the numbers and fluorescence intensity of NHE3-immunoreactive cells were the most in water with salinity 0, and decreased with the rise of water salinity. At last, NHE3-immunoreactive cells disappeared in water with salinity 20 and the control group. In the light of this study, in order to adapt to the change of environmental salinity, the apical surfaces of gill MRCs changed. In the osmoregulation of gill MRCs, NKA, NKCC and NHE3 regulate osmotic balance through different mechanisms. |
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