SHORT COMMUNICATION Knockdown of rotund gene through larval RNA interference affects genital and elytral morphology in the ground beetle Carabus maiyasanus (Coleoptera: Carabidae) Teiji SOTA 1 , Hisashi SUGAWARA 1 , Tomochika FUJISAWA 1 , Kotaro FUJIMAKI 1,2 and Teruyuki NIIMI 3 1 Department of Zoology, Graduate School of Science, Kyoto University, Kyoto, Japan, 2 Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, USA and 3 Division of Evolutionary Developmental Biology, National Institute for Basic Biology, Okazaki, Japan 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 (Oho- mopterus) 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 characteris- tic 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 for- mation 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. Key words: copulatory piece, Ohomopterus, vaginal appendix. The diversity of genital morphology in animals that reproduce by internal fertilization is a remarkable aspect of animal evolution (Eberhard 1985). The shape and size of the genital parts, particularly male ones, dif- fer even among closely related species, and these differ- ences are effective for prezygotic reproductive isolation, although interspecific differences might have arisen through intraspecific selection. Consequently, the diver- sity of genitalia is related to the species diversity of ani- mals. Numerous studies have examined the selection of genital morphology within species or among closely related species (Langerhans et al. 2016), but the genetic basis of species-specific genital morphology has been studied in only a few species. Genes involved in the development of genitalia in insects have been studied mostly in Drosophila (e.g., Sánchez & Guerrero 2001), and recent studies have found candidate genes causing inter- and intraspecific variation in genital morphology of Drosophila species (Masly et al. 2011; Takahara & Takahashi 2015; Tanaka et al. 2015). Other than Dro- sophila, developmental genes for genitalia have been studied in only a few hemipteran and coleopteran spe- cies (Aspiras et al. 2011; Macagno & Moczek 2015). The genetic basis of interspecific differences in genital morphology has been explored in ground beetles of the genus Carabus, subgenus Ohomopterus. This group shows species-specific differences in male and female genital parts, the copulatory piece and vaginal appen- dix (Fig. 1; Ishikawa 1987); species-specific matching of these parts between the sexes is effective for repro- ductive isolation between species. In one lineage of this subgenus, these parts show marked interspecific varia- tion, and the genetic background has been investigated in quantitative genetic studies (Sasabe et al. 2007, 2010) and a comparative transcriptome study (Fujimaki et al. 2014). However, the genes involved in the development and variation in the genitalia of Oho- mopterus beetles are unknown. Correspondence: Teiji Sota, Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan. Email: sota@terra.zool.kyoto-u.ac.jp Received 8 March 2018; accepted 7 June 2018. © 2018 The Entomological Society of Japan Entomological Science (2018) doi: 10.1111/ens.12330