| Chinese mitten crab(Eriocheir sinensis)is one of the most valuable species of crustaceans,which is of high amounts by cultivation in aquaculture in China.The sex-regulation mechanism and development in molecular genetic breeding and production practice have important guiding significance.Although the cultivation technology is mature,it is still unclear about the development mechanism.The study of developmental and sex dimorphism is important in theoretical research and application.Nowadays,many aquatic animals through sex control technology to achieve mono-sex farming to increase production,improve quality,and increase the economic benefits.In this study,we use E.sinensis as the research object,analyzing the conservative Hox genes in E.sinensis,which play a key role in the formation and development of biological somite process of evolution.The study will contribute to an understanding of E.sinensis morphological development mechanism.We cloned fruitless gene in E.sinensis,and identified two isoforms which one associated with female-specific character development and the other associated with male-specific character development.Further research will be performed in fruitless gene involved in sex regulation pathways.The main progress is as following:By searching the transcriptomes of E.sinensis,we found 187 unigenes named Hox.We Blast all the unigenes with D.melanogaster,and found in E.sinensis there are 10 Hox genes named EslabcEspb、EsHox3、EsDfd、EsScr、Esftz、EsAntp、EsUbx、EsAbdA、EsAbdB.By analyzing of different developmental stages of E.sinensis,we infer the Hox genes play an important role in morphological development.In E.sinensis,we found the 10 Hox genes all contained a YPWM motif and a hemeodomain which are of high similarity.Compared with D.melanogaster.we found that although the two species are far relatives in evolution,the HOX protein sequences are conserved.By analyzing the 7 different developmental stages from zoeal to juvenial,we found the Hox genes perform a different expression patterns in different developmental stages,which is relative to morphological changes.When we search in the spices of the early developmental stage samples.Eslab and Espb are involved in head character development.After that,EsDfd,EsScr and EsUbx are involved in abdomen development and changes,especially in stage megalopa to juvenile.When the crabs endure brachyurization,the gene Eslab,Espb,EsHox3,EsDfd,EsScr and EsUbx change expression between the two processes.The fruitless(fru)gene plays an important role in sex determination pathway and courtship behavior of Drosophila melanogaster.In the present study,two fru isoforms(Esfru1 and Esfru2)were identified from the Chinese mitten crab E.sinensis.Sequence analysis showed that Esfrul and Esfru2 were encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA.Esfrul had all introns completely spliced out,while Esfru2 retained an additional sequence(78 bp)in intron 1.They both contained a conserved BTB domain but lacked a C-terminal C2H2 zinc-finger region reported in D.melanogaster.Analysis on temporal expression profiles of Esfru1-2 and Esfiu2 showed that they expressed similar pattern in embryonic stages but different in larval stages.The expression of Esfru1-2 was dropped gradually from zoea Ⅲ stage,however,Esfru2 increased from zoea IV stage and reached the peak at megalopa stage.Tissue distribution and in situ hybridization analysis revealed that they showed sexually dimorphic expression in gonads,hepatopancreas and brains.Esfru1-2 showed significantly higher expression level in female gonads and hepatopancreas than in males,while was highly expressed in male brains than that in females.Interestingly,Esfru2 displayed male-specific manner in each tissue,especially in gonads and hepatopancreas.Our results indicate Esfru1 might be involved in both sexual brain neuronal structures development and related to female-specific character development.Esfi-u2 might be participated in male-specific character development.This is the first reported spliced variants of fru transcripts in crustaceans and provide basic information for further functional studies of crab sex-determination mechanism. |
