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The origin and evolution of the novel insect wing remain enigmatic after a century-long discussion. The mechanism of wing development in hemimetabolous insects, in which the first functional wings evolved, is key to understand where and how insect wings evolutionarily originate. This study explored the developmental origin and the postembryonic dramatic growth of wings in the cricket Gryllus bimaculatus . We find that the lateral tergal margin, which is homologous between apterygote and pterygote insects, comprises a growth organizer to expand the body wall to form adult wing blades in Gryllus . We also find that Wnt, Fat-Dachsous, and Hippo pathways are involved in the disproportional growth of Gryllus wings. These data provide insights into where and how insect wings originate. Wings evolved from the pre-existing lateral terga of a wingless insect ancestor, and the reactivation or redeployment of Wnt/Fat-Dachsous/Hippo-mediated feed-forward circuit might have expanded the lateral terga. Here, the authors investigate wing development in cricket and find support for evolution of the novel insect wing from the pre-existing dorsal body wall of a wingless ancestor by activation of an evolutionarily conserved growth mechanism.

The sex determination gene doublesex ( dsx ) encodes a transcription factor with two domains, oligomerization domain 1 (OD1) and OD2, and is present throughout insects. Sex-specific Dsx splicing isoforms regulate the transcription of target genes and trigger sex differentiation in all Holometabola examined to date. However, in some hemimetabolous insects, dsx is not spliced sexually and its sequence is less conserved. Here, to elucidate evolutionary changes in dsx in domain organisation and regulation in termites, we searched genome and/or transcriptome databases for the dsx OD1 and OD2 in seven termite species and their sister group ( Cryptocercus woodroaches). Molecular phylogenetic and synteny analyses identified OD1 sequences of termites and C . punctulatus that clustered with dsx of Holometabola and regarded them as dsx orthologues. The Cryptocercus dsx orthologue containing OD2 was spliced sexually, as previously shown in other insects. However, OD2 was not found in all termite dsx orthologues. These orthologues were encoded by a single exon in three termites for which genome information is available; they were not alternatively spliced but transcribed in a male-specific manner in two examined species. Evolution of dsx regulation from sex-specific splicing to male-specific transcription may have occurred at an early stage of social evolution in termites.

Many scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex differentiation gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.

The Japanese rhinoceros beetle, Trypoxylus dichotomus , possesses large horns on its head and thorax, features whose biological significance has been explored across various fields, including evolutionary developmental biology, behavioral ecology, and materials science. To investigate the molecular basis of these characteristics, systemic larval RNA interference (RNAi) has been employed as a primary loss-of-function genetic tool. However, gain-of-function analyses and region-specific gene function assessments remain underdeveloped, thereby limiting the comprehensive understanding of the molecular mechanisms involved. To address this limitation, we developed an in vivo electroporation technique to introduce exogenous DNA vectors directly into the somatic tissues of T. dichotomus larvae to express the genes of interest. Additionally, we utilized the piggyBac transposon system to insert the exogenous DNA vectors into the host genome for stable gene expression. Our findings indicate that the T. dichotomus actin A3 gene promoter exhibits sufficient transcriptional activity in the early postembryonic stage of T. dichotomus via electroporation. Furthermore, we observed that this promoter functions effectively across a diverse range of insect species, including the harlequin ladybug, Harmonia axyridis and the silkworm, Bombyx mori , suggesting the broad applicability of the T. dichotomus actin A3 promoter in various insects.

The head horn of the Asian rhinoceros beetle develops as an extensively folded primordium before unfurling into its final 3D shape at the pupal molt. The information of the final 3D structure of the beetle horn is prefigured in the folding pattern of the developing primordium. However, the developmental mechanism underlying epithelial folding of the primordium is unknown. In this study, we addressed this gap in our understanding of the developmental patterning of the 3D horn shape of beetles by focusing on the formation of furrows at the surface of the primordium that become the bifurcated 3D shape of the horn. By gene knockdown analysis via RNAi, we found that knockdown of the gene Notch disturbed overall horn primordial furrow depth without affecting the 2D furrow pattern. In contrast, knockdown of CyclinE altered 2D horn primordial furrow pattern without affecting furrow depth. Our results show how the depth and 2D pattern of primordial surface furrows are regulated at least partially independently during beetle horn development, and how both can alter the final 3D shape of the horn.

Double-stranded RNA (dsRNA) inducing RNA interference (RNAi) is expected to be applicable to management of agricultural pests. In this study, we selected a ladybird beetle, Henosepilachna vigintioctopunctata , as a model target pest that devours vegetable leaves, and examined the effects of feeding the pest sterilized microbes highly accumulating a target dsRNA for RNAi induction. We constructed an efficient production system for diap1* -dsRNA, which suppresses expression of the essential gene diap1 (encoding death-associated inhibitor of apoptosis protein 1) in H. vigintioctopunctata , using an industrial strain of Corynebacterium glutamicum as the host microbe. The diap1* -dsRNA was overproduced in C. glutamicum by convergent transcription using a strong promoter derived from corynephage BFK20, and the amount of dsRNA accumulated in fermented cells reached about 75 mg per liter of culture. In addition, we developed a convenient method for stabilizing the dsRNA within the microbes by simply sterilizing the diap1* -dsRNA-expressing C. glutamicum cells with ethanol. When the sterilized microbes containing diap1* -dsRNA were fed to larvae of H. vigintioctopunctata , diap1 expression in the pest was suppressed, and the leaf-feeding activity of the larvae declined. These results suggest that this system is capable of producing stabilized dsRNA for RNAi at low cost, and it will open a way to practical application of dsRNA as an environmentally-friendly agricultural insecticide.

Fossil insects living some 300 million years ago show winglike pads on all thoracic and abdominal segments, which suggests their serial homology. It remains unclear whether winglike structures in nonwinged segments have been lost or modified through evolution. Here, we identified a ventral lateral part of the body wall on the first thoracic segment, the hypomeron, and pupal dorsolateral denticular outgrowths as wing serial homologs in the mealworm beetle Tenebrio molitor. Both domains transform into winglike structures under Hox RNA interference conditions. Gene expression and functional analyses revealed central roles for the key wing selector genes, vestigial and scalloped, in the hypomeron and the denticular outgrowth formation. We propose that modification, rather than loss, of dorsal appendages has provided an additional diversifying mechanism of insect body plan.

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. fat1/2/3 RNAi , fat4 RNAi and dachsous RNAi 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.

Background Lineage-specific adult structures form through modifications of pre-existing juvenile body parts during postembryonic development in insects. It remains unclear how these novel traits originate from ancestral structures within the constrained body plan. In the coffin-headed cricket Loxoblemmus equestris , an ancestral rounded head shape directly transforms into a flattened derived form in a sex-specific manner. To understand the origin of novel traits, we investigated the development of the adult head in L . equestris as a model of lineage-specific novelty. Results We found that head morphologies remained sexually monomorphic until the final molt, and the male-specific head shape emerged in the frons region during the transition to adulthood in L. equestris . Two- and three-dimensional morphological analyses revealed that the sexual dimorphism in the frons epithelial folding patterns appeared in the late final nymphal instar. These results suggest that the male-specific novel head development is linked to the final molt in L . equestris . We tested this hypothesis by knocking down the metamorphic gene network (MGN) comprised of Krüppel-homolog 1 ( Kr-h1 ), broad ( br ), and Ecdysone induced protein 93F ( E93 ). Despite the timing shifts of the nymph-to-adult transition caused by knockdown of the MGN, male-specific head structures are formed only after the final molt. Conclusions These results demonstrate that the novel male head structures are formed during the final molt through the formation of sex-specific epithelial patterns in L. equestris . This highlights the unique metamorphic lifecycle with the final molt as a driver that has created lineage- and sex-specific adult forms in insects.

How genetic information is modified to generate phenotypic variation within a species is one of the central questions in evolutionary biology. Here we focus on the striking intraspecific diversity of >200 aposematic elytral (forewing) colour patterns of the multicoloured Asian ladybird beetle, Harmonia axyridis , which is regulated by a tightly linked genetic locus h . Our loss-of-function analyses, genetic association studies, de novo genome assemblies, and gene expression data reveal that the GATA transcription factor gene pannier is the major regulatory gene located at the h locus, and suggest that repeated inversions and cis -regulatory modifications at pannier led to the expansion of colour pattern variation in H. axyridis . Moreover, we show that the colour-patterning function of pannier is conserved in the seven-spotted ladybird beetle, Coccinella septempunctata , suggesting that H. axyridis ’ extraordinary intraspecific variation may have arisen from ancient modifications in conserved elytral colour-patterning mechanisms in ladybird beetles. The harlequin ladybird beetle, Harmonia axyridis , has remarkable phenotypic diversity, with over 200 colour patterns. Here, Ando et al. show that this patterning is regulated by the transcription factor gene pannier and has diversified by repeated inversions and cis -regulatory modifications of pannier .