Abstract:Molting is a vital biological phenomenon of crustaceans and is closely related to the growth and leg regeneration of Chinese mitten crab (Eriocheir sinensis). In order to detect the mechanism of molting and growth of Chinese mitten crab, the concentration of ecdysteroid hormone (EH), the expression of ecdysone receptor gene (EcR) and molt-inhibiting hormone (MIH) gene at different molting stages were investigated by ELISA and qRT-PCR, respectively. Meanwhile, the correlation analysis was also conducted between growth traits and the concentration of EH, expression of EcR or MIH genes. The results showed that the concentration of EH fluctuated regularly in one molt cycle, and molting would started when EH content reached about 12 U/mL. The concentration of EH was significantly positive correlation with expression of EcR(P<0.01), but was significantly negative correlation with expression of MIH(P<0.01). The concentration of EH and expression of EcR at premolt stage were significant higher than that at postmolt stage (P<0.05), and they were the lowest at intermolt stage compared with that at other molting stages (P<0.05). However, the expression of MIH showed completely opposite trend compared with the concentration of EH and expression of EcR, indicating the highest at intermolt stage compared with that at other molting stages (P<0.05). There was a significantly negative correlation for the expression of EcR between intermolt and postmolt stage (P<0.05), and significantly positive correlation for the expression of MIH between premolt and intermolt stage, as well as between premolt and postmolt stage (P<0.05), while there was no significant correlation for the EcR and MIH genes at other molting stages (P>0.05), which indicated that the expression of MIH at premolt stage had a remarkably effect on the expression of MIH at postmolt and intermolt stage; while the expression of EcR at intermolt stage had a significant effect on the expression of EcR at postmolt stage. The correlation analysis showed that the concentration of EH was remarkably positively correlated with body weight at both premolt and intermolt stages (P<0.05), the expression of EcR at different molting stages was significantly positively correlated with body weight (P<0.05), and it was also notably positively correlated with carapace length and carapace width (P<0.05) at intermolt stage. However, the expression level of MIH was negatively correlated with body weight at postmolt stages (P<0.05), while remarkably negatively correlated at premolt and intermolt stages (P<0.05). and it was remarkably significantly negatively correlated with carapace length, carapace width at intermolt stage (P<0.01). Briefly, the results indicated that the concentration of EH had a significant effect on body weight of Chinese mitten crab at premolt and intermolt stage. The expression of EcR and MIH at different molting stages had significant effect on body weight, and their expression at intermolt stage also had significant effect on morphological traits. This study gives new insight into molecular mechanism of molting and growth of Chinese mitten crab, and provides a theoretically molecular and physiological basis for weight increment differences after molting in this crab.
崔晓雨, 朱冬发, 汤洁, 等. 2013. 三疣梭子蟹EcR基因的克隆及表达分析[J]. 水产学报, 37(11): 1645-1654. (Cui X Y, Zhu D F, Tang J, et al. 2013. Cloning and expression analysis of ecdysteroid receptor (EcR) in Portunus trituberculatus [J]. Journal of Fisheries of China, 37(11):1645-1654.)顾志敏, 何林岗.1991. 切除单侧眼柄对中华绒螯蟹蜕壳、生长、成熟的影响[J]. 淡水渔业, (5):10-13. (Gu Z M, He L G. 1991. Effect of unilateral eyestalk enucleation on ecdysis, growth and maturation of mitten crab (Eriocheir sinensis)[J]. Freshwater Fisheries, (5):10-13.)黄姝. 2014. 实验室条件下中华绒螯蟹成蟹的蜕壳、生长观察与蜕皮激素受体基因的克隆、表达分析[D]. 硕士学位论文, 上海海洋大学, 导师: 王成辉. pp. 22-28. (Huang S. 2014. Observation on molting and growth, analysis in molecular cloning and expression of ecdysone receptor (EcR) gene in adult Chinese mitten crab in the laboratory condition[D]. Thesis for M.S., Shanghai Ocean University, Supervisor: Wang C H. pp. 22-28.) 姜仁良, 谭玉钧, 吴嘉敏, 等. 1992. 中华绒螯蟹血淋巴中20-羟基蜕皮酮、17β-雌二醇和睾酮含量的变动[J]. 水产学报, 16(2):101-106. (Jiang R L, Tan Y J, Wu J M, et al. 1992. Changes of hemolymph 20-hydroxyecdysone, 17β-oestradiol and testosterone levels in Eriocheir sinensis[J]. Journal of Fisheries of China, 16(2):101-106.)焦满静. 2013. 中华绒螯蟹蜕皮机制与影响因素的研究[D]. 硕士学位论文, 河北大学, 导师: 康现江. pp. 2-34. (Jiao M J. 2013. Study on the mechanisms and influencing factors of the molting in Eriocheir sinensis[D]. Thesis for M.S., Heibei University, Supervisor: Kang X J. pp. 2-34.)蒲红双, 高祥刚, 侯红漫, 等. 2015. 甲壳动物蜕皮相关基因的研究进展[J]. 水产科学, 34(6):391-398. (Pu H S, Gao X G, Hou H M, et al. 2015. Advances in genes related to molting in crustacean[J]. Fisheries Science, 34(6):391-398.钱昭英. 2014. 凡纳滨对虾生长性能相关候选基因的分离鉴定、表达规律及调控功能研究[D]. 博士学位论文, 西北农林科技大学, 导师: 刘小林. pp.127-140.(Qian Z Y. 2014. Isolation, identification, expression and function studies of growth performance candidate genes in Litopenaeus vannamei[D]. Thesis for Ph.D., North West Agriculture and Forestry University, Supervisor: Liu X L. pp.127-140.)苏保锦. 2012. 家蚕蜕皮激素受体和超气门蛋白基因的表达调控研究[D]. 硕士学位论文, 苏州大学, 导师: 沈卫德. pp.3-12. (Su B J. 2012. The research of expressional regulation of ecdysone receptor and ultraspiracle genes in silkworm, Bombyx mori[D]. Thesis for M.S., Soochow University, Supervisor: Shen W D. pp. 3-12.)汪春建. 2012. 三疣梭子蟹蜕皮周期与第二次卵巢发育中MIH表达研究[D]. 硕士学位论文, 宁波大学, 导师: 朱冬发. pp.29-40.(Wang C J. 2012. Molt-inhibiting hormone mRNA levels during a molt cycle and the second ovarian development of Portunus trituberculatus[D]. Thesis for M.S., Ningbo University, Supervisor: Zhu D F. pp. 29-40.)汪春建, 朱冬发, 亓一舟, 等. 2013. 三疣梭子蟹蜕皮周期中 MIH基因 mRNA 水平与蜕皮激素浓度变化[J]. 水生生物学报, 37(1): 22-28.(Wang C J, Zhu D F, Bian Y Z, et al. 2013. Molt-inhibiting hormone levels and ecdysteroid titer during a molt cycle of Portunus trituberculatus[J]. Acta Hydrobiologica Sinica, 37(1): 22-28.)王武, 王成辉, 马旭洲. 2013. 河蟹生态养殖[M]. 中国农业出版社, 北京. pp.1-36.(Wang W, Wang C H, Ma X Z. 2013. Ecological aquaculture for mitten crab[M]. China Agriculture Press. Beijing. pp.1-36. )王瑶, 杨志刚, 沈城, 等. 2014. 中华绒螯蟹EcR基因全长 cDNA 克隆及表达分析[J]. 水产学报, 38(5): 651-661.(Wang Y, Yang Z G, Shen C, et al. 2014. The full length cDNA cloning and expression analysis of EcR from the Chinese mitten crab(Eriocheir sinensis)[J]. Journal of Fisheries of China, 38(5): 651-661.)魏振. 2012. 甲壳动物生长发育的激素调控研究概述[J].安徽农业科学, 40(16):8899-8900,8925.(Wei Z. 2012. Overview of the Studies on regulation of growth hormone in crustacean[J]. Journal of Agriculture Science, 40(16):8899-8900,8925.)姚俊杰, 赵云龙. 2006. 甲壳动物蜕皮的调节机制研究进展[J]. 水利渔业, 26(6): 8-10.(Yao J J, Zhao Y L. 2006. Regulation mechanism of molting of crustacean[J]. Reservoir Fisheries, 26(6): 8-10. )岳武成, 陈娇, 慈元吉, 等. 2016. 断肢再生对中华绒螯蟹蜕壳, 生长及相关基因表达的影响[J]. 浙江大学学报: 农业与生命科学版, 42(4): 502-508.(Yue W C, Chen J, Ci Y J, et al. 2016. Effects of limb regeneration on molt, growth and related gene expression in Chinese mitten crab (Eriocheir sinensis)[J]. Journal of Zhejiang University (Agriculture & Life Science), 42(4): 502-508.)朱小明, 李少菁. 2001. 甲壳动物幼体蜕皮的调控[J]. 水产学报, 25(4): 379-384.(Zhu X M, Li X J. 2001. Regulation of molting in crustacean larvae[J]. Journal of Fisheries of China[J]. 25(4): 379-384.)Asazuma H, Nagata S, Kono M, et al. 2007. Molecular cloning and expression analysis of ecdysone receptor and retinoid X receptor from the kuruma prawn, Marsupenaeus japonicus[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 148:139-150.Chang ES, Bruce MJ. 1980. Ecdysteroid titers of juvenile lobsters following molt induction[J]. Journal of Experimental Zoology, 214:157-160.Chang ES, Mykles DL. 2011. Regulation of crustacean molting: A review and our perspectives[J]. General and Comparative Endocrinology, 172:323-330.Das S, Durica DS. 2013. Ecdysteroid receptor signaling disruption obstructs blastemal cell proliferation during limb regeneration in the fiddler crab, Uca pugilator[J]. Molecular and Cellular Endocrinology, 365:249-259.Durica DS, Chung AC-K, Hopkins PM. 1999. Characterization of EcR and RXR gene homologs and receptor expression during the molt cycle in the crab, Uca pugilator[J]. American Zoologist, 39:758-773Durica DS, Wu X, Anilkumar G, et al. 2002. Characterization of crab EcR and RXR homologs and expression during limb regeneration and oocyte maturation[J]. Molecular and Cellular Endocrinology, 189:59-76.Gong J, Yu K, Shu L, et al. 2015. Evaluating the effects of temperature, salinity, starvation and autotomy on molting success, molting interval and expression of ecdysone receptor in early juvenile mud crabs, Scylla paramamosain[J]. Journal of Experimental Marine Biology and Ecology, 464:11-17.Hopkins PM .1983. Patterns of serum ecdysteroids during induced and uninduced proecdysis in the fiddler crab, Uca pugilator[J]. General and comparative endocrinology, 52:350-356.Huang S, Chen X C, Wang J, et al. 2017. Selection of appropriate reference genes for qPCR in the Chinese mitten crab, Eriocheir sinensis (Decapoda, Varunidae)[J]. Crustaceana, 90(3):275-296. Huang S, Wang J, Yue W C, et al. 2015. Transcriptomic variation of hepatopancreas reveals the energy metabolism and biological processes associated with molting in Chinese mitten crab, Eriocheir sinensis[J]. Scientific Reports, 5:14015.Lachaise F. 1993. The molting gland of crustaceans: localization, activity, and endocrine control[J]. Journal of Crustacean Biology, 13(2):198-234.Lee KJ, Elton TS, Bej AK, et al. 1995. Molecular cloning of a cDNA encoding putative molt-inhibiting hormone from the blue crab, Callinectes sapidus[J]. Biochemical and Biophysical Research Communications, 209(3):1126-1131.Lee KJ, Watson RD, Roer RD. 1998. Molt-inhibiting hormone mRNA levels and ecdysteroid titer during a molt cycle of the blue crab, Callinectes sapidus[J]. Biochemical and Biophysical Research Communications 249:624-627.Li X L, Xin J J, Feng T T, et al. 2011. Single nucleotide polymorphism discovery of molt inhibiting hormone gene 3 exons and its association with growth traits in white shrimp (Litopenaeus vannamei)[J]. Journal of Animal and Veterinary Advances, 10(21): 2856-2858.Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2? ΔΔCT method[J]. Methods, 25:402-408.Martin Creuzburg D, Westerlund SA, Hoffmann KH. 2007. Ecdysteroid levels in Daphnia magna during a molt cycle: Determination by radioimmunoassay (RIA) and liquid chromatography-mass spectrometry (LC–MS)[J]. General and Comparative Endocrinology, 151:66-71.MELLON, JR D, GREER, E. 1987. Induction of precocous molting and claw transformation in alpheid shrimps by exogenous 20-hydroxyecdysone[J]. Marine Biological Laboratory, 172:350-356.Nakatsuji T, Sonobe H. 2004. Regulation of ecdysteroid secretion from the Y-organ by molt-inhibiting hormone in the American crayfish, Procambarus clarkii[J]. General and Comparative Endocrinology, 135:358-364.Ohira T, Watanabe T, Nagasawa H, et al. 1997. Molecular Cloning of a molt-inhibiting hormone cDNA from the Kuruma Prawn, Penaeus japonicus[J]. Zoological Science 14:785-789.Shyamal S, Anilkumar G, Bhaskaran R, et al. 2015. Significant fluctuations in ecdysteroid receptor gene (EcR) expression in relation to seasons of molt and reproduction in the grapsid crab, Metopograpsus messor (Brachyura: Decapoda)[J]. General and Comparative Endocrinology, 211:39-51.Skinner DM .1985. Molting and regeneration[M]. Academic Press. Florida. pp.43-146.Tennessen Jason M, Thummel Carl S. 2011. Coordinating rrowth and maturation-insights from Drosophila[J]. Current Biology, 21:R750-R757Thanh NM, Barnes AC, Mather PB, et al. 2010. Single nucleotide polymorphisms in the actin and crustacean hyperglycemic hormone genes and their correlation with individual growth performance in giant freshwater prawn Macrobrachium rosenbergii[J]. Aquaculture, 301:7-15.