MOLT-INHIBITING HORMONE LEVELS AND ECDYSTEROID TITER DURING A MOLT CYCLE OF PORTUNUS TRITUBERCULATUS
MOLT-INHIBITING HORMONE LEVELS AND ECDYSTEROID TITER DURING A MOLT CYCLE OF PORTUNUS TRITUBERCULATUS
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发表时间：
2013
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通讯作者：
Wang Chun
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作者：
Wang Chun
三疣梭子蟹（Portunus trituberculatus）是我国沿海重要的水产养殖蟹类，是我国重要的食用蟹类，其眼柄X器官窦腺（X-organ-sinus gland，XO-SG）分泌的多肽--蜕皮抑制激素（molt-inhibiting hormone，MIH）已经提出通过抑制Y器官（YO）蜕皮甾体的合成来调节蜕皮建立了实时荧光定量PCR（qRT-PCR）相对定量检测中华绒螯蟹体内MIH mRNA表达水平的方法，结果表明，MIH mRNA在XO-SG中的表达水平最高，以D 0期后期为对照，用2 ΔΔCt法分析了蜕皮周期中MIH mRNA的表达水平，结果表明：在蜕皮周期中，MIH mRNA的表达下调0.42± 0.08，而在蜕皮周期中，MIH mRNA的表达上调0.42± 0.08（A、B、C期分别为1.09± 0.09、1.35±0.16倍，后期D1、D2分别下调0.78± 0.07、0.27± 0.08、0.20±0.04倍，此外，我们还采用高效液相色谱-电喷雾串联质谱法（LC-MS/MS）完成了对蜕皮激素浓度的测定过程（20-羟基蜕皮激素，结果表明，蜕皮激素浓度在蜕皮后期（A/B）低于仪器检测限0.33pg，蜕皮间期（A/B）低于仪器检测限0.33pg，蜕皮激素浓度在蜕皮后期（A/B）低于仪器检测限0.33pg，蜕皮中期（B/B）低于仪器检测限0.33pg。蜕皮期（C），蜕皮激素的浓度逐渐恢复到（1.666±0.762）ng/mL，蜕皮激素滴度在蜕皮前期逐渐升高，（4.047±1.5133），（6.756±4.928），在D 0、D1和D2后期，蜕皮激素滴度分别为（8.609±3.827）ng/mL，并逐渐升高，在D1后期达到峰值，在D2后期，蜕皮激素滴度为（8.609±3.827）ng/mL（19.534±4.799）ng/mL，D4后期下降到11.616 ng/mL。血淋巴中MIH的表达量与蜕皮激素呈负相关，表明MIH可能通过抑制YO合成蜕皮激素来调控蜕皮的发生和进行。
Portunus trituberculatus as a popular table delicacy is one of the most important fishery and aquaculture species of crab around the coast of China.In crustaceans,molt-inhibiting hormone(MIH),a polypeptide secreted by the X-organ–sinus gland(XO-SG) of the eyestalks,had been proposed to regulate molting by inhibiting the synthesis of ecdysteroids from Y-organs(YO).The method for determining the levels of MIH mRNA in the swimming crab had been developed using relative quantification of quantitative real-time PCR(qRT-PCR).We found the expression level of MIH mRNA was the highest in the XO-SG.By taking surstage D0as the control group,the levels of MIH mRNA were analyzed by 2 ΔΔCt in a molt cycle,and the results showed that MIH transcripts down-regulated 0.42±0.08,increased(1.09±0.09,increased 1.35±0.16 fold in stage A,B,C,respectively,and down-regulated 0.78±0.07,down-regulated 0.27±0.08,down-regulated 0.20±0.04 fold in surstage D1,D2,D3/4,respectively.In addition,we used the method of high performance liquid chromatography-e1ectrospray ionization tandem mass spectrometry(LC-MS/MS) to complete the process of measuring the consistency of portunus molting ecdysteroid(20-hydroxyecdysone,20E) in hemolymph.The results showed that the consistency of ecdysone was below the instrument detection limit of 0.33 pg in the post molt stage(A/B).In the inter-molt period(C),the consistency of ecdysone gradually returned to(1.666±0.762) ng/mL.In the pre-molt ecdysteroid titer increased gradually to(4.047±1.5133),(6.756±4.928) and(8.609±3.827) ng/mL in surstage D0,D1and D2,respectively.The ecdysteroid titer increased steadily to a peak of(19.534±4.799) ng/mL in the surstage D3,then dropped to 11.616 ng/mL in surstage D4.These stage-specific expression changes in MIH mRNA levels were accompanied by significant fluctuations in hemolymph ecdysteroid titer.During a molt cycle of the swimming crab,the expression of MIH exhibited a negative correlation with ecdysone in hemolymph,and this correlationindicated that MIH regulated the occurrence and advance of molt by inhibiting the ecdysteroid synthesis from YO.