Transcriptomic and functional screening of weapon formation genes implies significance of cell adhesion molecules and female-biased genes in broad-horned flour beetle

For understanding the evolutionary mechanism of sexually selected exaggerated traits, it is essential to uncover its molecular basis. By using broad-horned flour beetle that has male-specific exaggerated structures (mandibular horn, head horn and gena enlargement), we investigated the transcriptomic and functional characters of sex-biased genes. Comparative transcriptome of male vs. female prepupal heads elucidated 673 sex-biased genes. Counter-intuitively, majority of them were female-biased (584 genes), and GO enrichment analysis showed cell-adhesion molecules were frequently female-biased. This pattern motivated us to hypothesize that female-biased transcripts (i.e. the transcripts diminished in males) may play a role in outgrowth formation. Potentially, female-biased genes may act as suppressors of weapon structure. In order to test the functionality of female-biased genes, we performed RNAi-mediated functional screening for top 20 female-biased genes and 3 genes in the most enriched GO term (cell-cell adhesion, fat1/2/3, fat4 and dachsous). Knockdown of one transcription factor, zinc finger protein 608 (zfp608) resulted in the formation of male-like gena in females, supporting the outgrowth suppression function of this gene. Similarly, knockdown of fat4 induced rudimental, abnormal mandibular horn in female. fat1/2/3RNAi, fat4RNAi and dachsousRNAi males exhibited thick and/or short mandibular horns and legs. These cell adhesion molecules are known to regulate tissue growth direction and known to be involved in the weapon formation in Scarabaeoidea beetles. Functional evidence in phylogenetically distant broad-horned flour beetle suggest that cell adhesion genes are repeatedly deployed in the acquisition of outgrowth. In conclusion, this study clarified the overlooked functions of female-biased genes in weapon development. A diverse range of animals have evolved the male sexual weapons that provide an advantage in male-male competition. Understanding the genetic mechanisms behind the sexually dimorphic expression of these weapons is crucial for comprehending their evolution. However, this area of study remains largely unexplored. By comparing the gene expression in the heads of male and female prepupae, we identified 673 sex-biased genes, with 584 genes being highly expressed in females. Through knockdown experiments targeting 23 female-biased genes, we discovered that four genes play a role in determining the size and expression of male-exaggerated traits. Intriguingly, two of these genes, including a transcription factor and a cell adhesion molecule, were found to suppress weapon development in females, implying that down-regulation of these genes in males enhances weapon development. These findings illuminate the often-overlooked function of female-biased genes in male weapon development and propose the idea that the evolution of weapons involves the down-regulation of certain genes in males.

Automated summary

This study investigates the molecular basis of sexually selected exaggerated traits in broad-horned flour beetles. By analyzing the transcriptomic and functional characteristics of sex-biased genes, the researchers found that female-biased genes play a role in suppressing weapon structure development. Knockdown experiments targeting these genes further supported their involvement in the formation of male-exaggerated traits. These findings highlight the overlooked functions of female-biased genes in weapon development and suggest that the evolution of such traits involves the down-regulation of certain genes in males.