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

Abstract

BackgroundGenes 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.ResultsWe 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.ConclusionsTaken 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 materialThe online version of this article (doi:10.1186/s12864-016-2522-8) contains supplementary material, which is ...

The function of appendage patterning genes in mandible development of the sexually dimorphic stag beetle

“…Recently, the male-specific mandible enlargement in the sexually dimorphic stag beetle Cyclommatus metallifer was found to be controlled by both nutrition-dependent juvenile hormone (JH) effects and through the sex-specific sex determination cascade (Gotoh et al 2011(Gotoh et al , 2014(Gotoh et al , 2016. Males of this species exhibit extremely enlarged mandibles and larger males have disproportionally larger mandibles than smaller males (Fig.…”

doublesexregulates sexually dimorphic beetle horn formation by integrating spatial and temporal developmental contexts in the Japanese rhinoceros beetleTrypoxylus dichotomus

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“…castaneum; [44], the stag beetle Cyclommatus metallifer; [45]). Therefore, the function of Tra and Tra2 as sex determination factors targeting dsx, and the function of Tra2 as an essential factor progressing normal development during larval-pupal development may be conserved among Polyphaga, the coleopteran suborder including the above three beetles.…”

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