title: Identification and functional analyses of sex determination genes in the sexually dimorphic stag beetle Cyclommatus metallifer.
abstract: 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.
authors.affiliation: Department of Entomology, Washington State University, Pullman, WA, 99164, USA.|Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan.|Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi, 464-8601, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Gotoh
authors.affiliation: Department of Entomology, Washington State University, Pullman, WA, 99164, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Zinna
authors.affiliation: Department of Entomology, Washington State University, Pullman, WA, 99164, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Warren
authors.affiliation: Department of Zoology, Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: DeNieu
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi, 464-8601, Japan.|Division of Evolutionary Developmental Biology, National Institute for Basic Biology, Okazaki, Aichi, 444-8585, Japan.
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Niimi
authors.affiliation: Department of Zoology, Michigan State University, East Lansing, MI, 48824, USA.|Department of Biology, McMaster University, Hamilton, ONT, L8S 4K1, Canada.
authors.forename: Ian
authors.initials: I
authors.lastname: Dworkin
authors.affiliation: Division of Biological Sciences, University of Montana-Missoula, Missoula, MT, 59812, USA.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Emlen
authors.affiliation: Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Miura
authors.affiliation: Department of Entomology, Washington State University, Pullman, WA, 99164, USA. lavine@wsu.edu.
authors.forename: Laura C
authors.initials: LC
authors.lastname: Lavine
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001517:Coleoptera / Q000235:genetics* / Q000502:physiology; D005091:Exons; D005260:Female; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D017344:Genes, Insect; D008297:Male; D005810:Multigene Family; D020033:Protein Isoforms / Q000235:genetics; D034622:RNA Interference; D017423:Sequence Analysis, RNA; D019849:Sex Determination Processes; D012733:Sex Differentiation / Q000235:genetics*
chemical_list: D020033:Protein Isoforms
keywords: Cyclommatus metallifer; Sex determination; Sexual dimorphism; Sexual selection