| Freshwater crabs were the youngest members in the evolutionary history of Brachyura,which evolved from the ancestor of marine crabs and finally adapted to freshwater life.During the invasion of freshwater,freshwater crabs needed to face tremendous pressure to survive and they undergone dramatic changes of morphology,life history and physiological.The regulation of water and salt balance was one of the most critical challenges for freshwater crabs.Osmoregulation was mainly to maintain the homeostasis of inner environment by reducing the passive salt loss and discharging osmotic water from the body.The physiological anatomy and histology had been suggested that osmoregulation had formed a physiological adaptation for freshwater crabs during the evolutionary transition from a marine life to a freshwater habitat environment.However,this adaptive molecular mechanism remain poorly explored.Here,we choosed 7 candidate genes related to osmoregulation.First of all,Differential expression level of 7 candidate genes in gill of Sinopotamon yangtsekiense was examined by quantitative real-time PCR technique,which verified the correlation between these genes and osmoregulation of truly freshwater crabs represented by Sinopotamon.Secondly,We had successfully amplified 5 of 7 candidate genes in the previous studies as target genes,taked 18 genera and 21 species of freshwater crabs from 2 families in China as the research object,taked 11 genera and 17 species of marine crabs from 9 families as background groups.We compared the genes related to osmoregulation in freshwater crabs with their counterparts in marine crabs to test whether adaptive evolution occured on adapting to the freshwater habitats.7 genes related to osmoregulation encoded three kinds of functional proteins:Ion transporter,such as Na+/K+-ATPase a subunit,V-type proton ATPase,Cytoplasmic carbonic anydrase and Voltage-dependent anion-selective channel;heat shock protein,such as HSP 90 and HSP 70;and 14-3-3 protein.The experimental study showed that under the different salinity gradient(0,5‰ 10‰,15‰,20‰,25‰,30‰,35‰),7 genes related to osmoregulation in gill of Sinopotamon yangtsekiense were significant differences in expression level.With the increased of the salinity gradient,the expression level of Na+/K+-ATPase a subunit,V-type proton ATPase and VDAC encoding ion transport proteins showed a trend of increasing and then decreased slightly.However,the expression level of carbonic anydrase cytoplasmic had a downward trend.At the same time,the expression level of hsp 90、hsp 70 and 14-3-3 were significantly increased.In addition,this experiment showed that Sinopotamon yangtsekiense could be domesticated in equivalent of seawater salinity for 125 days,which implied that they had a strong ability to tolerate salt.The selective pressure analysis of PAML was carried out on 5 genes in this paper.The result showed that positive selection acting on Na+/K+-ATPase a subunit,VDAC,hsp 90 and 14-3-3.From the functional point of view,Na+/K+-ATPase was the key enzyme to maintain osmotic and ion balance.Voltage-dependent anion-selective channel regulated cell metabolism and ATP/ADP conversion.14-3-3 protein was related to the regulation of ion channels.The positive selection of them was related to the change of osmoregulatory mechanism of freshwater crabs.HSP 90 taked an important role in the stress response of organisms as a molecular chaperone,so the positive selection of hsp 90 was suggesting that the high expression of HSP 90 could contribute to the effective response to the new freshwater life.In addition,13 positively selected sites were detected by at least two ML methods.Among them,radical amino acids changes in 11 positively selected sites were distributed among branches of freshwater crabs,which suggested that adative evolution of genes related to osmoregulation generally appeared in the diversification of freshwater crabs.This study was the first to verify the differential expression level of 7 genes related to osmoregulation in truly freshwater crabs represented by And also revealed that 4 genes related to osmoregulation in freshwater crabs were positively selected.It is suggested the freshwater crabs ultimately developed an effective mechanism which could effectively adapt to the freshwater environment.The results of this study provide a new molecular evidence for the evolution mechanism of freshwater crabs adaptation to freshwater life. |
