Transcriptome Analysis Of Different Developmental Stages Of Daphnia Similoides Sinensis And Screening Of Reproductive Switching And Male Sex Determination Candidate Genes

Daphnia species are common freshwater cladoceran and play an important role in aquatic food chain.Cladocera has two reproductive modes in its life history,namely parthenogenesis and sexual reproduction.Usually,when environmental conditions deteriorated,some eggs produced by parthenogenetic females may hatch males,and then carry out sexual reproduction.This special reproductive pattern makes cladoceran become an important group on studying the mechanism of reproductive switching and sex determination.However,there are few studies on the molecular mechanism of reproductive switching and male sex determination in Daphnia.In this study,we sequenced the transcriptome of different developmental stages of Daphnia similoides sinensis life cycle,and screened out candidate genes related to reproductive switching and male sex determination.Our results will provide valuable information for the study of functional genes identification and molecular regulation mechanism in Daphnia species.RNA-Seq was performed on the four developmental stages,juvenile female(JF),parthenogenetic female(PF),sexual female(SF)and male(M),to construct the transcriptome database of D.similoides sinensis.A total of 110437 transcripts were obtained,which were assembled into 22996 unigenes.The unigenes were matched with NCBI non-redundant(Nr)protein database.The highest percentage of matching bases(70.5%)in species was to D.magna.In JF,PF,SF and M,the number of unique unigenes are respectively 2863,445,437 and 586,and the number of common unigenes is 9708.The differentially expressed genes(DEGs)between two developmental stages(PF vs JF,SF vs JF,PF vs SF,M vs JF,M vs PF and M vs SF)were obtained by pairwise comparison of JF,PF,SF and M.The GO gene enrichment analysis showed that the up-regulated genes in both SF and M were enriched in hydrolase activity and peptidase activity.Compared with other three developmental stages(SF vs JF,SF vs PF and SF vs M),top thirty differentially up-regulated genes in SF were selected for RT-q PCR.Twenty-five candidate genes related to reproductive switching were screened,which significantly higher expression(P<0.01)in SF were observed than those in the other three developmental stages,,including some genes related to the ephippial formation in SF,such as cuticular protein genes(Cluster-5789.221: cuticular protein,Cluster-5789.225: cuticular protein),resilin gene(Cluster-5789.207: pro-resilin)and carboxypeptidase gene(Cluster-5789.11727: zinc carboxypeptidase A1),and other genes related to resting eggs formation in SF,such as serine protease genes(Cluster-5789.7107: putative serine protein nudel,Cluster-5789.8755: serine protease inhibitor dipetalogastin-like protein)and oocyte and embryo development related genes(Cluster-5789.1075: prostaglandin D2 synthase-like protein,Cluster-5789.6612:putative cytoplasmic polyadenylation element-binding protein 1,Cluster-5789.13610:peroxinectin-like protein).Compared with other three stages(M vs JF,M vs PF and M vs SF),top thirty differentially up-regulated genes in M were selected for RT-q PCR.Thirty-eight candidate genes related to male sex determination,which significantly higher expression(P<0.05)in M were obersved than those in the other three stages,were screened,including doublesex gene(Cluster-5789.12340: doublesex2)which was involved in sex differentiation of other Daphnia species,laminin gene(Cluster-5789.8159: laminin subunit gamma-3)which possibly related to the development of male gonads in D.similoides sinensis,trypsin genes(Cluster-5789.9553: trypsin-7-like protein,Cluster-5789.3677: putative trypsin-7,Cluster-5789.9554: putative trypsin-7,Cluster-5789.11655: trypsin alpha like,Cluster-5789.7668: trypsin like isoform X1)and serine protease genes(Cluster-5789.2039: serine protease ami-like)which may be related to sperm activation,and chitin synthase gene(Cluster-5789.11830: chitin synthase 2)and chitinase genes(Cluster-5789.6658: chitinase 3,Cluster-5789.5191: chitinase 5,Cluster-5789.7417: chitinase 8)which showed sexually dimorphic expression in D.similoides sinensis.In addition,some identified genes related to reproductive switching(CSP2,CSP3,dsx1,dsx2,Tra)and male sex determination(dsx1,Tra,DMRT93 B,antp)in other Daphnia species were also found in D.similoides sinensis.In this study,the expression levels of dsx1 and dsx2 in D.similoides sinensis male were significantly higher than those in three female stages,and the expression level of antp in male was significantly higher than those in two female adult(PF,SF)stages,implying that these genes were also involved in the process of male sex determination in D.similoides sinensis.