| Gymnocypris przewalskii, commonly known as scale-less fish, is an important economic fish in Lake Qinghai. Because of climate changes and human activities, the amount of G. przewalskii is decreasing. It has been listed as an endangered fish. G.przewalskii lives in Lake of Qinghai that has a low concentration of dissolved oxygen,low-calcium, high pH and high salinity level. G. przewalskii has a strong tolerance with low-calcium. The main objective of this study is to understand the response of juvenile G. przewalskii exposed to low ambient calcium through studying calcium distribution,energy metabolism. In addition, semi-quantitative and quantitative PCR Research were conducted to analyze CaBPs, PMCA and NCX mRNA expression in gills and kidney.These results will provide helpful knowledge on G. przewalskii resource protection.In accordance with natural habitat of G. Przewalskii, the water salinity was set on10. The Ca2+concentration in control group is 113.46mg·L-1 and Ca2+ concentration in treatments are from 0.38 mg·L-1 to 11.35 mg·L-1. The results are as follows:1. To explore energy metabolism and growth performance of G.przewalskii juvenile in low calcium water environment, the weight daily gain, specific growth rate(SGR) and five indicators of energy budget values were measured. The results showed:(1) Daily gain and SGR decreased with the lower calcium concentration in water environment, in which the daily gain and SGR were significantly different from the control group(P<0.05).(2)With the decrease of calcium concentration in water, growth and fecal energy reduced accordingly, in which growth and fecal energy of the low calcium were significantly different from the control group(P<0.05); For metabolism,differences between low calcium treatments were significant(P<0.05), and the lowest calcium group were significant different from the control group. These showed that the concentration of calcium in the water environment is one of the reasons that causes slow growth of G.przewalskii.2. To observe the calcium tissue distribution of G.przewalskii in low calcium water environment, the calcium concentration in blood plasma, tissue, bait and fecal were measured. The results are as follows:(1)Ca2+, Mg2+, Cl- decreased significantly with the decrease of water calcium concentrations(P<0.05). With the decrease of calcium concentration in water environment, blood Ca2+, Mg2+ in 1/300 group were significant different from the control group(P<0.05), and it was approximately equal to 1/2 fold;Blood Cl- in 1/300 group was significantly lower than control group(P<0.05), but there was no significant difference between the low calcium group; Blood Na+ of 1/300 group was higher than the control group; there was no differences among the experimental groups for K+.(2)The Ca2+ concentrations in the otoliths, operculum and muscle tissue of the G.przewalskii juvenile had no significant differences among the treatments; the spine in 1/300 low calcium group was significantly lower than the control group(P<0.05), with 1.4 folds decrease.(3)The intake and excretion of Ca2+ between the low calcium group and the control group were significantly different(P<0.05), but there is no difference among the low calcium groups. These showed that the tissue distribution of calcium in the G.przewalskii was affected by calcium levels in the water environment.3. The quantitative PCR were conducted to test the mRNA expression of CaBPs,PMCA and NCX genes in the gill and kidney of G.przewalskii exposed to low ambient calcium concentrations. The results showed that:(1)G.przewalskii gill’s CaBPs expression level increased with decreasing calcium ion concentration in water, and a maximum value appears in the 60 d, 5 times higher than control group; kidney’s CaBPs expression appeared decreasing with the decrease of calcium ion concentration in water,with the 3.7-fold decreased at 60 days.(2)G.przewalskii gill’s PMCA expression appeared increasing at first and then decreasing with the low calcium treatment going on. At 30 d, gill’s PMCA expression reached its maximum in the lowest calcium group,which is 2.6-fold higher than the control group(P<0.05), while at 60 d, gill’s PMCA expression significantly reduced 1.9-fold than the control group(P<0.05); kidney’s PMCA expression did not change significantly among the groups.(3) G.przewalskii gill’s NCX expression appeared increasing at first and then decreasing with the calcium ion concentration decreased. At 30 d, gill’s NCX expression reached the maximum in the lowest calcium group, increased by 2.5-fold(P<0.05); At 60 d, gill’s NCX expressionsignificantly reduced 3-fold than the control group(P<0.05); kidney’s NCX expression did not change significantly among treatments. It is illustrated that G.przewalskii CaBPs,PMCA and NCX may play an important role in calcium balance during low calcium stress.4. The Ca2+-ATP enzyme kit was used to the test of Ca2+-ATPase activity. The results showed that:(1)During 60 d low calcium water environmental stress, the gill Ca2+-ATP enzyme activity was decreasing with the calcium concentration decreased,especially in the lowest calcium concentrations treatment, it is about 64.6 percent of the control group.(2)G.przewalskii juvenile kidney’s Ca2+-ATP enzyme activity was increased at first and decreased with the low calcium concentration. The lowest calcium concentration is more obvious, about 58.6 percent of the control group. The lower content of calcium ion in water environment reduced the calcium in body, naked carp regulated the expression of gills, kidney PMCA to change the Ca2+-ATPase enzyme activity, thereby regulating the body Ca2+ intake and reduce cell Ca2+ efflux and maintenance of intracellular calcium homeostasis. |
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