盐度对不同蜕皮时期罗氏沼虾生理生化及蜕皮相关基因表达的影响

Effects of salinity on Macrobrachium rosenbergii physiology, biochemistry and gene expression related to molting at different molting stages

  • 摘要: 【目的】分析盐度对不同蜕皮时期罗氏沼虾蜕皮生理生化指标、能量代谢酶活性及蜕皮相关基因表达的影响,为实际生产中罗氏沼虾培育提供理论参考。【方法】以300尾平均初始湿体质量4.28±0.53 g/尾的健康罗氏沼虾为对象,试验设0.2‰(对照)、 3.0‰、 6.0‰、 9.0‰、 12.0‰等5个盐度梯度,记录不同盐度条件下罗氏沼虾的蜕皮周期、每日蜕皮率和蜕皮增重率。按照不同蜕皮时期取肝胰腺、肌肉和鳃丝,使用生化试剂盒测定丙酮酸激酶 (PK)和琥珀酸脱氢酶(SDH)活力,并用实时荧光定量PCR(qRT-PCR)检测蜕皮抑制激素(MIH)、维甲酸X受体(RXR)、蜕皮激素受体(EcR)、保幼激素酯酶环氧水解酶(JHEH)基因的表达情况。【结果】罗氏沼虾的蜕皮周期随盐度的升高而不断延长,其中6.0‰盐度组蜕皮周期最长,显著高于0.2‰盐度组 (P<0.05);蜕皮增重率随盐度的升高总体呈下降趋势,在盐度6.0‰、 9.0‰和12.0‰组中的蜕皮增重率显著低于0.2‰盐度组。在5个盐度条件下,肝胰腺、鳃丝的PK和SDH的活力均在蜕皮后期B有最大值,随着蜕皮后时间的延长,到蜕皮前期D时,其活力呈现下降的趋势,表明肝胰腺、鳃丝组织在蜕皮后期时能量代谢酶活力达到最高水平。在4个蜕皮阶段下,随着盐度的升高,肝胰腺和鳃丝的PK和SDH活力总体呈先上升后下降的趋势。低盐度的刺激导致罗氏沼虾肌肉的PK和SDH活力升高,而在12.0‰盐度组活力下降。MIH基因在各盐度组的相对表达量均随蜕壳时间的延长而明显下降,在0.2‰和3.0‰盐度组蜕皮后期A与蜕皮后期B的相对表达量显著高于蜕皮间期C。RXR基因在蜕皮间期C的相对表达量最高,其他蜕皮时期均极显著低于0.2‰盐度组(P<0.001)。与蜕皮间期C相比,各盐度组JHEH基因均在蜕皮前期D的相对表达量最高,而在3.0‰、6.0‰和12.0‰盐度组时,鳃丝EcR基因的相对表达量在蜕皮前期D极显著高于蜕皮间期C (P<0.001)。【结论】低盐度对罗氏沼虾的生长蜕皮有较大影响,不仅导致蜕皮增重率下降,蜕皮周期变长,对生产实践产生负面影响,还可使罗氏沼虾肌肉和肝胰腺的PK和SDH活力升高,从而促使肝胰腺和肌肉中的糖酵解速率加快,三羧酸循环作用加强,但在高盐度 (12.0‰)条件下受到抑制。盐度胁迫下, MIH基因上调表达、 EcR基因下调表达会引发蜕皮前期D时间延迟,蜕皮困难,致使JHEHRXR基因表达下调,是导致罗氏沼虾生长缓慢的主要原因。罗氏沼虾养殖适宜的水体盐度为0.2‰~3.0‰。

     

    Abstract: 【Objective】To analyze effects of salinity on physiological and biochemical indexes,energy metabolism enzyme activity and gene expression related to molting of Macrobrachium rosenbergii,so as to provide reliable reference for M.rosenbergii cultivation in production.【Method】 300 healthy M.rosenbergii with an average initial wet body weight of 4.28±0.53 g/tail were taken as objects,and divided into 5 salinity groups of 0.2‰(control),3.0‰,6.0‰,9.0‰ and 12‰.Molting cycle,daily molting rate and molting weight gain rate of each shrimp were recorded.Pyruvate kinase(PK) activity and succinate dehydrogenase(SDH)activity of M.rosenbergii at different molting stages were determined using biochemical kits.Expression of ecdysone(MIH),retinoid X receptor(RXR),ecdysone receptor(EcR)and juvenile hormone lipase(JHEH)genes were detected by real-time quantitative PCR(qRT-PCR).【Result】 Molting period of M.rosenberii was prolonged with increasing salinity,and the molting time of 6‰ salinity group was the longest,which was significantly higher than that of the 0.2‰ salinity group(P<0.05).Rate of molting weight gain decreased with the increase of salinity,and the rate of molting weight gain in the salinity groups of 6.0‰,9.0‰ and 12.0‰ was significantly lower than that in the 0.2‰ salinity group.Under 5 salinity conditions,PK activity and SDH activity in hepatopancreas and gill filament reached the maximum at the late molting stage B,decreasing at the early molting stage D with the prolongation of the post-molting time.The results indicated that the energy metabolism enzyme activity of hepatopancreas and gill filaments reached the highest level in the late molting period.In the 4 molting stages,PK activity and SDH activity in hepatopancreas,gill filaments and muscle tissues increased first and then decreased with the increase of salinity.PK activity and SDH activity in muscle and hepatopancreas increased after stimulation with low salinity,while the enzyme activity decreased at 12.0‰ salinity.Relative expression of MIH gene in all salinity groups decreased significantly with the prolongation of molting.Relative expression of MIH gene in late molting stage A and late molting stage B in 0.2‰ and 3‰ salinity groups was significantly higher than that in intermolt stage C.Relative expression of RXR gene was the highest in the intermolt stage C,and was significantly lower than that in the 0.2‰ salinity group in the other molt stages(P<0.001).Compared with intermolt stage C,relative expression of JHEH gene in all salinity groups was the highest in premolt stage D,and relative expression of EcR gene in gill filament of 3.0‰,6.0‰ and 12.0‰ salinity groups was significantly higher in premolt stage D than that in intermolt stage C(P<0.001).【Conclusion】 Low salinity stress has great effects on molting of M.rosenbergii,leading to decreased weight gain rate and longer molting cycle,and it negatively influences production.Low salinity stress raises PK activity and SDH activity in muscle and hepatopancreas,so glycolysis rate and the tricarboxylic acid cycle in hepatopancreas and muscle are increased.However,PK activity and SDH activity are inhibited at 12.0‰ salinity group.Under salinity stress,the up-regulated expression of MIH gene and the down-regulated expression of EcR gene may lead to delay of premolt stage D and difficult molting,resulting in down-regulated expression of JHEH and RXR genes,which is the main reason for the slow growth of M.rosenbergii.The suitable water salinity for M.rosenbergii culture is 0.2‰-3.0‰.

     

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