Effects And The Osmoregulation Mechanisms Of Calcium And Magnesium Concentrations And Salinity Acclimatization On Serum Osmolality Of Juvenile Paralichthys Olivaceus

Japanese flounder, Paralichthys olivaceus, which is found in the coast of Northeast Asia, such as China, Japan and Korea, is a kind of high-value commercial fish and plays an important role in marine aquaculture. The author studies the effect and the osmoregulation mechanisms of calcium and magnesium concentrations and salinity acclimatization on serum osmolarity of juvenile Japanese flounder. The main results and conclusions from the studies are as follows:1. The effect and the osmoregulation mechanisms of calcium concentration on serum osmolarity of juvenile P. olivaceusThe osmoregulation mechanisms of juvenile Japanese flounder transferred directly from seawater (Ca2+,9.7 mM) to aritificial seawater with different calcium concentrations were studied for 216 hours and 60 days respectively. Changes in serum osmolarity, serum ions, gill, intestine and kidney Na+-K+-ATPase and Ca2+-ATPase activities, as well as the density, size and ultrastructure of chloride cell in gill filament were measured at 6,12,24,72,96,144,216 hour and 60 day. First, changes of experimental parameters were observed in low calcium concentration treatment (a1: 1.25 mM) for 216 h. Serum osmolarity increased significantly at 6 h, and returned to control group level after 12 h. Gill and intestine Na+-K+-ATPase and Ca+-ATPase activities showed transiently decreased at 6 h and 72 h respectively, and quickly returned to control group level at 12 h and 96 h separately. However, kidney Na+-K+-ATPase and Ca+-ATPase activities increased significantly, reached a peak value at 6 h, slightly decreased after 24 h and maintained a new steady state (P<0.05). Serum Na+, Cl- and K+ concentrations showed no significant change (P>0.05). Serum calcium concentration slightly decreased after 6 h and maintained a new steady state, while magnesium concentration increased significantly at 6 h and hold steady (P<0.05). Light microscope results showed that the density and size of chloride cell had no significant change at 216 h (P>0.05), while electron microscope results showed that chloride cell membranous tubules reduced at 6 h. Second, changes of experimental parameters were also observed in high calcium concentration treatments (a3:37.5, a4:75, a5:100 mM) for 216 h. The results showed that serum osmolarity maintained a steady state during the whole experiment (P>0.05). Gill, intestine and kidney Na+-K+-ATPase and Ca2+-ATPase activities increased at 6 h,72 h and 6 h respectively, and then decreased at 24 h,96 h and-24 h separately and hold steady, but higher than control treatment. Serum Na+, Cl- and K+ concentrations showed no significant change (P>0.05). Serum calcium concentration of as increased significantly after 72 h and maintained a new steady state (P<0.05), whlie calcium of a3 and a4 showed no change. Serum magnesium concentration of a4 and as decreased significantly after 6 h and maintained a new steady state (P<0.05), while magnesium of a3 showed no change. Light microscope results showed that the density of chloride cell of the three treatments had no significant change at 216 h (P>0.05), while the size of chloride cell of a5 increased significantly (P<0.05). Electron microscope results showed that the cristae of mitochondria of as were more stronger, but the tubular network became weaker at 6 h. Third, changes of experimental parameters were measured in low calcium concentration treatment (b1:5 mM) at 60 d. The results showed that all of experimental parameters recovered to normal levels except for the density of chloride cell which increased significantly (P<0.05). Last, changes of experimental parameters were also measured in high calcium concentration treatments (b3:20, b4:37.5, b5:75 mM) at 60 d. The results showed that serum osmolarity in the three treatments had no significant change (P>0.05). Gill, intestine and kidney Na+-K+-ATPase and Ca--ATPase activities in b3 and b4 recovered to control group level, while increased in b5. Serum Na+, Cl- and K+ concentrations showed no significant change (P>0.05). Serum calcium concentration was positive with ambient calcium concentration, while serum magnesium concentration was negative with ambient calcium concentration (P<0.05). Light microscope results showed that the density of chloride cell in the three treatments had no significant change (P>0.05), while the size of chloride cell of b5 increased significantly (P<0.05). Electron microscope results showed that chloride cell tubular network of b5 were greater development. We concluded that serum osmolarity of juvenile Japanese flounder had no significant change under short and long adaptive process when calcium concentration abruptly changed, but it could keep ions kinetic equilibrium of body fluid by adjusting Na+-K+-ATPase and Ca2+-ATPase activities in gill, intestine and kidney as well as serum calcium and magnesium concentrations.2. The effect and the osmoregulation mechanisms of calcium and magnesium total concentrations on serum osmolarity of juvenile P. olivaceusThe osmoregulation mechanisms of juvenile Japanese flounder transferred directly from seawater (Ca2++Mg2+:60 mM) to artificial seawater with different calcium and magnesium total concentrations were studied for 216 hours and 60 days respectively. Chages in serum osmolarity, serum ions, gill Na+-K+-ATPase and Ca2+-ATPase activities, as well as the density, size and ultrastructure of chloride cell in gill filament were measured at 6,12,24,48,72,96,144,216 hour and 60 day. First, changes of experimental parameters were measured in low calcium and magnesium total concentrations treatments (A1:7.5, A2:27 mM) for 216 h. Serum osmolarity of A1 and A2 increased significantly only at 72 h and 6 h respectively (P<0.05). Gill Na+-K+-ATPase and Ca2+-ATPase activities decreased at 6 h, but began to increase after 12 h, reached a peak vaule at 72 h, and ruturned to control group level after 96 h. Serum Na+, Cl- and K+ concentrations of A1 had no change (P>0.05), while serum Ca2+ concentration of A1 slightly decreased within 48 h, and returned to control group level after 72 h. Serum Mg2+ concentration of A1 significantly increased at 6 h and 144 h (P<0.05). Serum ions of A2 had no significant change (P>0.05). Light microscope results showed that the density and size of chloride cell of A1 and A2 had no significant change at 216 h (P>0.05). Electron microscope results showed that chloride cell tubular network became loose and the cristae of mitochondria became vague of A1 at 216 h. Second, changes of experimental parameters were also measured in high calcium and magnesium total concentrations treatments (A4:105, A5: 180 mM) for 216 h. Serum osmolarity of A4 significantly increased only at 72 h and 216 h (P<0.05), while serum osmolarity of A5 significantly increased after 24 h and hold steady (P<0.05). Serum ions of A4 had no significant change, while serum Na+ Cl-, and Ca2+ of A5 increased significantly at 48 h,24 h and 6 h respectively and maintained a new steady state (P<0.05). However, serum K+ concentration increased significantly only at 72 h and 96 h (P<0.05). Serum magnesium concentration significantly decreased at 6 h,12 h and maintained at a low state (P<0.05). Light microscope results showed that the density and size of chloride cell of A4 had no significant change, while the size of chloride cell of A5 significantly increased (P<0.05). Electron microscope results showed that the volum of mitochondriale of chloride cell were larger and the tubular network were greater development of A4, and the density of mitochondria were greater abundance of A5. Third, changes of experimental paramters were observed in low calcium and magnesium total concentrations treatments (B1:27, B2:45 mM) at 60 d. Both serum osmolarity and ions returned to normal levels, while gill Na+-K+-ATPase and Ca2+-ATPase activities significantly increased (P<0.05). Light microscope results showed that the density of chloride cell of B1 significantly increased (P<0.05), while the size of chloride cell had no change (P>0.05). Electron microscope results showed that chloride cell mitochondria were more abundance and the tubular network were greater development in B1. Last, changes of experimental parameters were also observed in high calcium and magnesium total concentrations treatments (B4:90, B5:105 mM) at 60 d. Serum osmolarity, serum ions and gill Na+-K+-ATPase and Ca2+-ATPase activities were all no significant change (P>0.05). Light microscope results showed that the density of chloride cell of B5 significantly decreased (P<0.05), while the size of chloride cell significantly increased (P<0.05). Electron microscope results showed that chloride cell mitochondria criatae were stronger, and the tubular network were greater development of B5. We concluded that serum osmolarity of juvenile Japanese flounder increased significantly both in low and high treatments when calcium and magnesium total concentrations changed shortly, but it returned to control group level after long adaptive process by adjusting gill Na+-K+-ATPase and Ca2+-ATPase activities as well as serum ions to keep ions kinetic equilibrium of body fluid.3. The effect of calcium and magnesium concentrations on the number, ultrastructure and secretion of gill mucous cells of juvenile P. olivaceusThis part included 5 experiments. Exp.1, the effect of calcium and magnesium total concentrations on the number of gill arch mucous cells of juvenile Japanese flounder at 9 d. The results showed that the number of mucous cells of A1 (7.5 mM) and A2 (27 mM) increased slightly, but had no significant change comparing with control group (P>0.05). The number of mucous cells of A4(105 mM) and A5 (180 mM) decreased slightly, and it decreased significantly in A5 (P<0.05). Exp.2, the effect of calcium and magnesium total concentrations on the number of gill arch mucous cells of juvenile Japanese flounder at 60 d. The results showed that the number of mucous cells increased significantly in B1 (27 mM) (P<0.05), while decreased in B3 (105 mM). Exp.3, the effect of calcium concentration on the number of gill arch mucous cells of juvenile Japanese flounder at 9 d. The results showed that the number of mucous cells increased in C1 (1.25 mM), and decreased in C3(37.5 mM), C4 (75 mM) and C5 (100 mM), but there was no significant change among different groups (P>0.05). Exp.4, the effect of calcium concentration on the number of gill arch mucous cells of juvenile Japanese flounder at 60 d. The results showed that the number of mucous cells of D1 (5 mM) increased, but had no significant change comparing with control group (P>0.05). The number of mucous cells of D3 (37.5 mM) and D4 (75 mM) decreased, and the D4 were significantly lower than D1 (P<0.05). Exp.5, the effect of high calcium concentration stress on the number, ultrastructure and secretion of mucous cells in the gill of juvenile Japanese flounder. The rusults showed that the number and ultrastructure of mucous cells had no obvious change between stress group and control group. The secretion of mucous cells was same between the two experimental groups, both belonging to apocrine secretion. The mucous cells secretory activity of stress group was vigorous with discharging massive mucus, while it was little in control group. We concluded that low calcium and magnesium total concentraitons or calium concentration could stimulate the proliferation of gill mucous cells, while high calcium and magnesium total concentraitons or calcium concentration'could suppress the proliferation of gill mucous cells. Besides, juvenile Japanese flounder had strong tolerance of high calcium concentration stress.4. The effect of salinity acclimatization on serum osmolarity and gill Na--K--ATPase activity of juvenile P. olivaceusThe osmoregulation mechanisms of juvenile Japanese flounder transferred from seawater (33‰) to high salinity (47‰) and low salinity (5‰) with different (3.5,7, 14) day salinity variations and during adaptive process were studied. Chages in serum osmolarity and gill Na+-K+-ATPase activity were measured every two days in acclimatization and at 1,2,4,8 d in adaptive process. (1) Changes of serum osmolarity were observed. The results showed that serum osmolarity was obviously positive with salinity in acclimatization. Besides, in adaptive process, serum osmolarity significantly decreased in high salinity with 7 day variation and low salinity with 7 and 14 day variations, and reached a steady state at 2 d,2 d and 4 d respevtively. However, serum osmolarity showed increased tendency in high salinity with 14 day variation. (2) Changes of gill Na+-K+-ATPase activity were also observed. The results showed that gill Na+-K+-ATPase activity showed increased tendency both in high and low salinity acclimatization with 3.5 day variation, while it showed no change with 7 and 14 day variation. In adapted process, gill Na+-K+-ATPase activity significantly increased at 2 d and maintained a new steady state in high salinity with 7 day variation, and the same phenomenon was observed in low salinity with 7 day variation. Gill Na+-K+-ATPase activity showed increase-decrease tendency in high salinity with 14 day variation, but there was no significant change. Gill Na+-K+-ATPase activity showed decrease-increase-decrease tendency in low salinity with 14 day variation. We concluded that gill Na+-K+-ATPase activity of juvenile Japanese flounder was lowest in seawater which it natural lived while increased both in high and low salinity acclimatization. Besides, the adaptive time of juvenile Japanese flounder was reduced during adapted process by 7 day variaiton.