Growth Performance Evaluation And Gene Expression Profile Analysis On Limb Regeneration Of Chinese Mitten Crab

Chinese mitten crab(Eriocheir sinensis) has a powerful regeneration ability after legs amputated that is similar with other crabs,which make it as a good model in Regenerative Biology. In this study, culture biology, molecular biology and bioinformatics methods were conducted to investigate the growth performances and transcriptome profiles of Chinese mitten crab during their limb regeneration period. The results are presented as follows: Part1. Effects of limb regeneration on molt, growth and related gene expression in Chinese mitten crabIn this study, the molting, growth and related gene expression characteristics of larva Chinese mitten crab were investigated after their four legs(the first and third legs in the left, the second and forth legs in the right) were artificially removed. The relative analysis showed that leg regeneration rate was only 7.6% after the first molting and increased 91.6% after the second molting. the survive rates between intact crabs and amputated crabs have no significant difference(P>0.05); The inter-molting days in the leg amputated crabs was significant longer(P<0.05) after the first molting and significant shorter(P<0.05) after the second molting than that in the intact crabs, but the weight-gaining rate was significant higher(P<0.05) after the first molting and no significant difference(P=0.05) after the second molting compared to the intact crabs. There was no significant difference between them at the average inter-molting days. The related gene expression analysis indicated that there were no significant differences(P>0.05) between the leg amputated and intact crabs in insulin-like growth factor-2(IGF2), retinoid X receptor(RXR) molt-inhibiting hormone(MIH). However, there were significant higher expression in the molting hormone receptor(Ec R) and lower expression in the myostatin(MSTN) in the leg amputated crabs(P<0.05) than the intact crabs. In conclusion, Chinese mitten crab could regenerate a new legs in two molt cycle, there were no significant difference on survival rate, weight-gaining rate and molt cycles between the leg amputated and intact crabs. Ec R and MSTN genes played the important role in promoting limb regeneration. The leg amputated crab can be used for later aquaculture. Part2. Growth performance evaluation, genetic and physiological analysis on leg amputated Chinese mitten crabGrowth performance, molting hormone and related gene expression were investigated in Chinese mitten crab with different leg removed numbers(two legs: the forth leg of two bilateral; three legs: the forth leg of two bilateral and the third legs in the right) reared d in Outdoor concrete ponds for three molting cycles. The results indicated that there were significant difference in survival rate ranging from 24.3%(removed two legs crab), 41.0%(removed three legs crab) to 50.8%(intact crab); The increment rates in the traits of body weight, carapace length and carapace width was the highest in the intact crab, followed by the remove two legs crab, and the lowest in the removed three legs crab(P<0.05); the conditional factors of leg amputated crabs was significant lower than intact crabs at the third molting cycle(P<0.05). however, there was no significant difference of molting hormone level in blood among the leg amputated and intact crabs(P>0.05). The related gene expression profiles showed that there was no significant difference(P>0.05) between the leg amputated and intact crabs in insulin-like growth factor-2(IGF2) and molt-inhibiting hormone(MIH), but significantly lower expression in the myostatin(MSTN) in the leg amputated crabs(P<0.05) than the intact crabs.. In conclusion, although a compensatory growth was observed in leg amputated crab, it would be a disadvantaged effect on the growth when the legs were amputated in two years old Chinese mitten crab. it was suggested that vigorous and leg intact crabs should be chosen in aquaculture. Part3. Comparative transcriptome analysis on different stages of limb regeneration in Chinese mitten crabIn this study, comparative transcriptome analysis was conducted in three development stage(Stageâ… : Scab Formation, Stageâ…¡: Limb Bud Formation and stageâ…¢: New Leg Development) of limb regeneration in Chinese mitten crab. A total of 66333 transcripts were assembled, in which 30949 transcripts were annotated in NCBI-nr date base, and 26030 in Uniprot date base. Transcriptomic comparison analysis showed that there were 539 genes upregulated and 44 genes downregulated in the first stage; 1806 genes upregulated and 456 genes downregulated in the second stage; and 167 genes upregulated and 74 genes downregulated in the third stage. Gene function enrichment analysis indicated that there were different expression pattern among the three development stage, stage â… was an immune response process, stageâ…¡was a morphogenesis process, stageâ…¢represent a new leg growth. In conclusion, limb regeneration is a complex biological processes in Chinese mitten crab, gene expression pattern in different development stage of limb regeneration process showed an immune, morphogenesis and new leg growth process. Besides, the gene signal pathways and functional genes were detected in Chinese mitten crab, which associated with limb regeneration would be a reference date.