| Chinese mitten crab(Eriocheir sinensis), also has been considered as river crab,is a very important aquatic economic animal in China. However, the precocious problem in this crab has plagued the healthy development of its aquaculture industry and given rise to huge losses in economy. In this paper, a sibling individuals were used from larval to juvenile stages to conduct analysis of growth characteristics, morphological development and gene expression, including molt-inhibiting hormone(MIH), ecdysteroid receptor(Ec R), retinoid X receptor(RXR), crustacean hyperglycemi hormone(CHH), gonad-inhibiting hormone(GIH) and Vitellogenin(Vg). Meanwhile, the comparative transcriptome analysis was conducted between the normal and precocious crabs. The aims of this paper are to illustrate the molecular mechanism of precocious problem in mitten crab by integrating the growth, morphological change and gene expression. The results are presented as follows:Part1. Analysis of growth and the expression of molting-related genes in larva Chinese mitten crabs from different rearing environmentThere is huge growth difference in Chinese mitten crab(Eriocheir sinensis) resulting from different culture environments. In order to study the influence of environmental conditions on growth of larval crab, and to understand the temporal and spatial change of gene expression and their interaction with environments, the growth and gene expression characteristics were investigated for megalopas and larval crab employed from one family reared in outdoor ponds and indoor closet. Expression profiles of five molting-related genes using quantitative real-time PCR(RT-PCR) technology were investigated, including crustacean hyperglycemi hormone(CHH); molt-inhibiting hormone(MIH); ecdysteroid receptor(Ec R); retinoid X receptor(RXR) and gonad-inhibiting hormone(GIH). The results indicated average weight of larva crabs reared in outdoor pond condition was significantly higher than that of crab reared in indoor closet(P<0.05). The environmental conditions made no significant influence to the expression of MIH and RXR genes, but significant difference was found in CHH, Ec R and GIH genes between the two environments(P<0.01). There was significant expression difference between all the five genes( RXR, CHH, Ec R, GIH and RXR) among different stages of larval crab. Meanwhile, significant interaction effects(P<0.01) between rearing environments and development stages were found in the four genes(RXR, CHH, Ec R and GIH), but not found in MIH gene(P>0.05). Furthermore, the positive co-expression were detected for the genes of MIH, GIH, CHH genes, but negative expression were found between the RXR and GIH genes, which regulate molting and growth in crab.Part 2. Correlation analysis between growth traits and expression of growth and development-related genes in juvenile Chinese mitten crabPrecocious is one special phenomenon in cultivation of juvenile Chinese mitten crab, however, its molecular mechanism researches has been poorly conducted. In this study, a sibling individuals from larval to juvenile staged(during June to October) were used to investigate the growth, morphological change and expression profiles in molting and gonad development genes. The results showed that the maximum growth rate of juvenile crab was found in July, and then declined monthly. The precocious crabs were first observed in August, and reached the peak in October. The highest expression profile for MIH gene was found in June and then gradually decreased in the remaining months. However, the expression levels of CHH, Ec R and RXR genes were significantly higher in October than those in the other months. There were significant positive correlations(P<0.01) among MIH, CHH, Ec R and RXR genes, and between CHH and GIH genes. The body weight of juvenile crab had significant positive correlation(P<0.01) with the expression levels of GIH and Vg genes, while significant negative correlation(P<0.05) between the expression of MIH, CHH and RXR genes. There were significant difference(P<0.01) for Vg gene and marginally significant difference(P=0.056) for GIH gene between Precocious crabsand normal individuals, indicating that GIH and Vg genes had tight correlation with sexual precocity in mitten crab. Part 3. Comparative transcriptome analysis between precious and normal juvenile mitten crabsPrecocity is a common problem in aquaculture of juvenile Chinese mitten crab, which has given rise to obvious effect on the quality and production of juvenile crabs. However, the molecular mechanism of precocity remains unclear. In this study, comparative transcriptome study was conducted between precocious and normal crabs in eye and hepatopancreas tissues. A total of 36,573 transcripts were assembled, with 17,352 annotated transcripts. Transcriptomic comparison revealed 896 and 17 genes differentially expressed in eye and hepatopancreas, respectively. Compared with normal crabs, ribosomal protein, heat shock protein and mitochondrial related genes were highly expressed in precocious crabs. All these highly expressed genes may directly induce the ovary development of female crabs. Ribosomal proteins are essential reasons for motivating ovary development, while precocious female crabs are able to survive in high temperature environment may due to the highly expressed heat shock protein genes. Meantime, metabolism is highly activated in precocious crabs for the reason of highly expressed mitochondrial genes which may cause storage of nutrient substance in vivo providing foundation for the ovary development. In summary, this study provided not only preliminary molecular mechanism of precocity in mitten crab, but also guidelines for the prevention and detection of precocious crabs in aquaculture. |
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