Knockdown of rotund gene through larval RNA interference affects genital and elytral morphology in the ground beetle Carabus maiyasanus (Coleoptera: Carabidae)

Abstract

Interspecific variation in genital morphology contributes to prezygotic reproductive isolation in many insect species. However, developmental genes affecting genital morphology are poorly understood. We undertook larval RNA interference (RNAi) experiments with rotund (rn) gene in the ground beetle Carabus (Ohomopterus) maiyasanus to examine the knockdown effects of this gene on genital development. We injected double‐stranded RNA (dsRNA) into third instar larvae of C. maiyasanus. The injection of rn dsRNA resulted in the absence or deformation of the copulatory piece in the male genitalia, which is a characteristic part playing a role in species‐specific genital coupling between the sexes. In addition, the rn dsRNA injection resulted in shortened elytra in both sexes, which is related to the known role of rn in forewing formation in Drosophila. Our study suggests that rn plays a previously underappreciated role in male genital development and potentially contributes to genital diversity in this beetle group.

Genetic basis of species-specific genitalia reveals role in species diversification

“…An elevated rn expression may be required for the extension of the copulatory piece. On the other hand, rn RNAi did not affect the development of female genitalia ( 27 ). This is consistent with the results of QTL mapping, which indicated that the genetic basis of the development of genital parts differs between the sexes.…”

“…Higher expression of these genes may be involved in elongation of the copulatory piece in C. maiyasanus . The rn transcription factor particularly is necessary for the development of various appendages and wings in Drosophila , and we found that the rn gene knockdown via larval RNA interference (RNAi) suppressed the formation of the copulatory piece in C. maiyasanus ( 27 ). An elevated rn expression may be required for the extension of the copulatory piece.…”

“…As we have not provided any evidence for the involvement of aforementioned genes, except rn , in the formation of species-specific genitalia, we must study their functions by knocking down candidate genes via larval RNAi. Larval RNAi is generally effective in beetles including Carabus ( 27 ) and is one of the only available methods for assessing the functions of focal genes in nonmodel beetles, which are difficult to breed in large numbers.…”

Gene expression during genital morphogenesis in the ground beetle Carabus maiyasanus

“…Therefore, it is important to identify genes that are differentially expressed among species during genital morphogenesis to understand the genetic and developmental basis of species‐specific genital morphologies. However, knowledge regarding gene expression and function during genital development in Ohomopterus species is limited (Fujimaki et al ., ; Sota et al ., ; Fujisawa et al ., ).…”

Heterochrony and growth rate variation mediate the development of divergent genital morphologies in closely related Ohomopterus ground beetles

“…The CP and VA in Ohomopterus ground beetles have been studied in behavioral and evolutionary contexts, including the identification of candidate genes or genomic regions associated with genital diversification (Fujimaki et al, 2014; Fujisawa et al, 2019; Nomura et al, 2019; Sasabe et al, 2007, 2010; Sota et al, 2018). An understanding of the morphogenesis of genitalia, including spatio–temporal differences between species, can provide a precise framework for analyses of the differential expression and functions of candidate genes.…”