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Background  Genes in the sex determination pathway are important regulators of sexually dimorphic animal traits, including the elaborate and exaggerated male ornaments and weapons of sexual selection. In this study, we identified and functionally analyzed members of the sex determination gene family in the golden metallic stag beetle  Cyclommatus metallifer,  which exhibits extreme differences in mandible size between males and females.    Results  We constructed a  C. metallifer  transcriptomic database from larval and prepupal developmental stages and tissues of both males and females. Using Roche 454 pyrosequencing, we generated a  de novo  assembled database from a total of 1,223,516 raw reads, which resulted in 14,565 isotigs (putative transcript isoforms) contained in 10,794 isogroups (putative identified genes). We queried this database for  C. metallifer  conserved sex determination genes and identified 14 candidate sex determination pathway genes. We then characterized the roles of several of these genes in development of extreme sexual dimorphic traits in this species. We performed molecular expression analyses with RT-PCR and functional analyses using RNAi on three  C. metallifer  candidate genes –  Sex-lethal  ( CmSxl ) , transformer-2  ( Cmtra2 ), and  intersex  ( Cmix ). No differences in expression pattern were found between the sexes for any of these three genes. In the RNAi gene-knockdown experiments, we found that only the  Cmix  had any effect on sexually dimorphic morphology, and these mimicked the effects of  Cmdsx  knockdown in females. Knockdown of  CmSxl  had no measurable effects on stag beetle phenotype, while knockdown of  Cmtra2  resulted in complete lethality at the prepupal period. These results indicate that the roles of  CmSxl  and  Cmtra2  in the sex determination cascade are likely to have diverged in stag beetles when compared to  Drosophila.  Our results also suggest that  Cmix  has a conserved role in this pathway. In addition to those three genes, we also performed a more complete functional analysis of the  C. metallifer dsx  gene ( Cmdsx ) to identify the isoforms that regulate dimorphism more fully using exon-specific RNAi. We identified a total of 16 alternative splice variants of the  Cmdsx  gene that code for up to 14 separate exons. Despite the variation in RNA splice products of the  Cmdsx  gene, only four protein isoforms are predicted. The results of our exon-specific RNAi indicated that the essential CmDsx isoform for postembryonic male differentiation is CmDsxB, whereas postembryonic female specific differentiation is mainly regulated by CmDsxD.    Conclusions  Taken together, our results highlight the importance of studying the function of highly conserved sex determination pathways in numerous insect species, especially those with dramatic and exaggerated sexual dimorphism, because conservation in protein structure does not always translate into conservation in downstream function.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-016-2522-8) contains supplementary material, which is available to authorized users.

Identification and functional analyses of sex determination genes in the sexually dimorphic stag beetle Cyclommatus metallifer