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Background: Cis-regulatory mutations often underlie phenotypic evolution. However, because identifying the locations of promoters and enhancers in non-coding regions is challenging, we have fewer examples of identified causative cis-regulatory mutations that underlie naturally occurring phenotypic variations than of causative amino acid-altering mutations. Because cis-regulatory elements have epigenetic marks of specific histone modifications, we can detect cis-regulatory elements by mapping and analyzing them. Here, we investigated histone modifications and chromatin accessibility with cleavage under targets and tagmentation (CUT&Tag) and assay for transposase-accessible chromatin-sequencing (ATAC-seq)., Results: Using the threespine stickleback (Gasterosteus aculeatus) as a model, we confirmed that the genes for which nearby regions showed active marks, such as H3K4me1, H3K4me3, and high chromatin accessibility, were highly expressed. In contrast, the expression levels of genes for which nearby regions showed repressive marks, such as H3K27me3, were reduced, suggesting that our chromatin analysis protocols overall worked well. Genomic regions with peaks of histone modifications showed higher nucleotide diversity within and between populations. By comparing gene expression in the gills of the marine and stream ecotypes, we identified several insertions and deletions (indels) with transposable element fragments in the candidate cis-regulatory regions., Conclusions: Thus, mapping and analyzing histone modifications can help identify cis-regulatory elements and accelerate the identification of causative mutations in the non-coding regions underlying naturally occurring phenotypic variations., (© 2024. The Author(s).)

Intrinsic postzygotic isolation typically appears as reduced viability or fertility of interspecific hybrids caused by genetic incompatibilities between diverged parental genomes. Dobzhansky-Muller interactions among individual genes, and chromosomal rearrangements causing problems with chromosome synapsis and recombination in meiosis, have both long been considered as major mechanisms behind intrinsic postzygotic isolation. Recent research has, however, suggested that the genetic basis of intrinsic postzygotic isolation can be more complex and involves, for example, overall divergence of the DNA sequence or epigenetic changes. Here, we review the mechanisms of intrinsic postzygotic isolation from genic, chromosomal, genomic, and epigenetic perspectives across diverse taxa. We provide empirical evidence for these mechanisms, discuss their importance in the speciation process, and highlight questions that remain unanswered., (Copyright © 2023 Cold Spring Harbor Laboratory Press; all rights reserved.)

Many insects engage in stable nutritional symbioses with bacteria that supplement limiting essential nutrients to their host. While several plant sap-feeding Hemipteran lineages are known to be simultaneously associated with two or more endosymbionts with complementary biosynthetic pathways to synthesize amino acids or vitamins, such co-obligate symbioses have not been functionally characterized in other insect orders. Here, we report on the characterization of a dual co-obligate, bacteriome-localized symbiosis in a family of xylophagous beetles using comparative genomics, fluorescence microscopy, and phylogenetic analyses. Across the beetle family Bostrichidae, most investigated species harbored the Bacteroidota symbiont Shikimatogenerans bostrichidophilus that encodes the shikimate pathway to produce tyrosine precursors in its severely reduced genome, likely supplementing the beetles' cuticle biosynthesis, sclerotisation, and melanisation. One clade of Bostrichid beetles additionally housed the co-obligate symbiont Bostrichicola ureolyticus that is inferred to complement the function of Shikimatogenerans by recycling urea and provisioning the essential amino acid lysine, thereby providing additional benefits on nitrogen-poor diets. Both symbionts represent ancient associations within the Bostrichidae that have subsequently experienced genome erosion and co-speciation with their hosts. While Bostrichicola was repeatedly lost, Shikimatogenerans has been retained throughout the family and exhibits a perfect pattern of co-speciation. Our results reveal that co-obligate symbioses with complementary metabolic capabilities occur beyond the well-known sap-feeding Hemiptera and highlight the importance of symbiont-mediated cuticle supplementation and nitrogen recycling for herbivorous beetles., (© 2023. The Author(s).)

Insects comprise over half of the described species, and the acquisition of metamorphosis must have contributed to their diversity and prosperity. The order Odonata (dragonflies and damselflies) is among the most-ancestral insects with drastic morphological changes upon metamorphosis, in which understanding of the molecular mechanisms will provide insight into the evolution of incomplete and complete metamorphosis in insects. In order to identify metamorphosis-related genes in Odonata, we performed comprehensive RNA-sequencing of the blue-tailed damselfly Ischnura senegalensis at different developmental stages. Comparative RNA-sequencing analyses between nymphs and adults identified eight nymph-specific and seven adult-specific transcripts. RNA interference (RNAi) of these candidate genes demonstrated that three transcription factors, Krüppel homolog 1 ( Kr-h1 ), broad , and E93 play important roles in metamorphosis of both I. senegalensis and a phylogenetically distant dragonfly, Pseudothemis zonata E93 is essential for adult morphogenesis, and RNAi of Kr-h1 induced precocious metamorphosis in epidermis via up-regulation of E93 Precocious metamorphosis was also induced by RNAi of the juvenile hormone receptor Methoprene-tolerant ( Met ), confirming that the regulation of metamorphosis by the MEKRE93 (Met-Kr-h1-E93) pathway is conserved across diverse insects including the basal insect lineage Odonata. Notably, RNAi of broad produced unique grayish pigmentation on the nymphal abdominal epidermis. Survey of downstream genes for Kr-h1 , broad , and E93 uncovered that unlike other insects, broad regulates a substantial number of nymph-specific and adult-specific genes independently of Kr-h1 and E93 These findings highlight the importance of functional changes and rewiring of the transcription factors Kr-h1 , broad , and E93 in the evolution of insect metamorphosis., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The order Odonata (dragonflies and damselflies) comprises diurnal insects with well-developed vision, showing diverse colors in adult wings and bodies. It is one of the most ancestral winged insect groups. Because Odonata species use visual cues to recognize each other, color patterns have been investigated from ecological and evolutionary viewpoints. Here we review the recent progress on molecular mechanisms of pigmentation, especially focused on light-blue coloration. Results from histology and pigment analysis showed that ommochrome pigments on the proximal layer and pteridine pigments on the distal layer of the epidermis are essential for light-blue coloration. We also summarize genes involved in the biosynthesis of three major insect pigments conserved across insects and discuss that gene-functional analysis deserves future studies., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Odonata species display a remarkable diversity of colour patterns, including intrasexual polymorphisms. In the damselfly ( Ischnura senegalensis ), the expression of a sex-determining transcription factor, the doublesex ( Isdsx ) gene is reportedly associated with female colour polymorphism (CP) (gynomorph for female-specific colour and andromorph for male-mimicking colour). Here, the function of Isdsx in thoracic coloration was investigated by electroporation-mediated RNA interference (RNAi). RNAi of the Isdsx common region in males and andromorphic females reduced melanization and thus changed the colour pattern into that of gynomorphic females, while the gynomorphic colour pattern was not affected. By contrast, RNAi against the Isdsx long isoform produced no changes, suggesting that the Isdsx short isoform is important for body colour masculinization in both males and andromorphic females. When examining the expression levels of five genes with differences between sexes and female morphs, two melanin-suppressing genes, black and ebony , were expressed at higher levels in the Isdsx RNAi body area than a control area. Therefore, the Isdsx short isoform may induce thoracic colour differentiation by suppressing black and ebony , thereby generating female CP in I. senegalensis. These findings contribute to the understanding of the molecular and evolutionary mechanisms underlying female CP in Odonata.

The order Odonata (dragonflies and damselflies) is among the most ancestral groups of winged insects with drastic morphological changes upon metamorphosis, and thus important for understanding evo-devo aspects of insects. However, basic developmental descriptions of Odonata have been scarce. In an attempt to establish the foundation of developmental and experimental biology of Odonata, we present an unprecedentedly comprehensive survey of dragonflies and damselflies, in total 158 larvae representing 49 species and 14 families, wherein morphological changes of all the final and/or penultimate instar larvae were photographed and monitored everyday. Although their morphology and development were diverse, we consistently identified two visually recognizable morphogenetic events in the final larval instar, namely start of wing expansion and onset of melanization on the wing sheaths, thereby categorizing the final instar into three stages. While the duration of the first stage ranged 4-66 days across diverse Odonata species, the second or third stages exhibited relatively small variation ranging 3-22 days or 1-8 days, respectively, probably reflecting the steady and irreversible metamorphosis process after stage 2. We also described other characteristic morphological changes during the larval development, although they were observed only in some Odonata species and lineages.

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

Cockroaches are commonly found in human residences and notorious as hygienic and nuisance pests. Notably, however, no more than 30 cockroach species are regarded as pests, while the majority of 4,500 cockroaches in the world are living in forest environments with little relevance to human life. Why some cockroaches have exceptionally adapted to anthropic environments and established pest status is of interest. Here we investigated the German cockroach Blattella germanica , which is a cosmopolitan pest species, and the forest cockroach Blattella nipponica , which is a wild species closely related to B. germanica . In contrast to easy rearing of B. germanica , laboratory rearing of B. nipponica was challenging-several trials enabled us to keep the insects for up to three months. We particularly focused on the distribution patterns of specialized cells, bacteriocytes, for harboring endosymbiotic Blattabacterium , which has been suggested to contribute to host's nitrogen metabolism and recycling, during the postembryonic development of the insects. The bacteriocytes were consistently localized to visceral fat bodies filling the abdominal body cavity, where a number of single bacteriocytes were scattered among the adipocytes, throughout the developmental stages in both females and males. The distribution patterns of the bacteriocytes were quite similar between B. germanica and B. nipponica , and also among other diverse cockroach species, plausibly reflecting the highly conserved cockroach- Blattabacterium symbiotic association over evolutionary time. Our study lays a foundation to experimentally investigate the origin and the processes of urban pest evolution, on account of possible involvement of microbial associates.

Many animals change their body color for visual signaling and environmental adaptation. Some dragonflies show wax-based color change and ultraviolet (UV) reflection, but the biochemical properties underlying the phenomena are totally unknown. Here we investigated the UV-reflective abdominal wax of dragonflies, thereby identifying very long-chain methyl ketones and aldehydes as unique and major wax components. Little wax was detected on young adults, but dense wax secretion was found mainly on the dorsal abdomen of mature males of Orthetrum albistylum and O. melania , and pruinose wax secretion was identified on the ventral abdomen of mature females of O. albistylum and Sympetrum darwinianum . Comparative transcriptomics demonstrated drastic upregulation of the ELOVL17 gene, a member of the fatty acid elongase gene family, whose expression reflected the distribution of very long-chain methyl ketones. Synthetic 2-pentacosanone, the major component of dragonfly's wax, spontaneously formed light-scattering scale-like fine structures with strong UV reflection, suggesting its potential utility for biomimetics., Competing Interests: RF, YY, MK, DI, RK, SY, TH An international patent on the synthesis method and application of very long chain methyl ketones and aldehydes was applied as PCT/JP2018/019559. NM, GO, YH, KY, TF No competing interests declared, (© 2019, Futahashi et al.)

In an attempt to establish an experimental dragonfly model, we developed a laboratory rearing system for the blue-tailed damselfly, Ischnura senegalensis. Adoption of multi-well plastic plates as rearing containers enabled mass-rearing of isolated larvae without cannibalism and convenient microscopic monitoring of individual larvae. Feeding Artemia brine shrimps to younger larvae and Tubifex worms for older larvae resulted in low mortality, synchronized ecdysis, and normal development of the larvae. We continuously monitored the development of 118 larvae every day, of which 49 individuals (41.5%) reached adulthood. The adult insects were fed with Drosophila flies in wet plastic cages, attained reproductive maturity in a week, copulated, laid fertilized eggs, and produced progeny. The final larval instar varied from 9 th to 12 th , with the 11 th instar (56.5%) and the 12 th instar (24.2%) constituting the majority. From the 1 st instar to the penultimate instar, the duration of each instar was relatively short, mainly ranging from three to 11 days. Afterwards, the duration of each instar was prolonged, reaching 7-25 days for the penultimate instar and 14-28 days for the final instar. Some larvae of final, penultimate and younger instars were subjected to continuous and close morphological examinations, which enabled developmental staging of larvae based on size, shape, and angle of compound eyes and other morphological traits. This laboratory rearing system may facilitate the understanding of physiological, biochemical, and molecular mechanisms underlying metamorphosis, hormonal control, morphogenesis, body color polymorphism, and other biological features of dragonflies.

The saw-toothed grain beetle, Oryzaephilus surinamensis (Silvanidae), is a cosmopolitan stored-product pest. Early studies on O. surinamensis in the 1930s described the presence of peculiar bacteriomes harboring endosymbiotic bacteria in the abdomen. Since then, however, the microbiological nature of the symbiont has been elusive. Here we investigated the endosymbiotic system of O. surinamensis in detail. In the abdomen of adults, pupae, and larvae, four oval bacteriomes were consistently identified, whose cytoplasm was full of extremely elongated tubular bacterial cells several micrometers wide and several hundred micrometers long. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes , in which the symbiont was the most closely related to the endosymbiont of a grain pest beetle, Rhyzopertha dominica (Bostrichidae). The symbiont was detected in developing embryos, corroborating vertical symbiont transmission through host generations. The symbiont gene showed AT-biased nucleotide composition and accelerated molecular evolution, plausibly reflecting degenerative evolution of the symbiont genome. When the symbiont infection was experimentally removed, the aposymbiotic insects grew and reproduced normally, but exhibited a slightly but significantly more reddish cuticle and lighter body mass. These results indicate that the symbiont of O. surinamensis is not essential for the host's growth and reproduction but contributes to the host's cuticle formation. Symbiont genome sequencing and detailed comparison of fitness parameters between symbiotic and aposymbiotic insects under various environmental conditions will provide further insights into the symbiont's biological roles for the stored-product pest. IMPORTANCE Some beetles notorious as stored-product pests possess well-developed symbiotic organs called bacteriomes for harboring specific symbiotic bacteria, although their biological roles have been poorly understood. Here we report a peculiar endosymbiotic system of a grain pest beetle, Oryzaephilus surinamensis , in which four oval bacteriomes in the abdomen are full of extremely elongated tubular bacterial cells. Experimental symbiont elimination did not hinder the host's growth and reproduction, but resulted in emergence of reddish beetles, uncovering the symbiont's involvement in host's cuticle formation. We speculate that the extremely elongated symbiont cell morphology might be due to the degenerative symbiont genome deficient in bacterial cell division and/or cell wall formation, which highlights an evolutionary consequence of intimate host-symbiont coevolution., (Copyright © 2017 Hirota et al.)

Background: The lesser grain borer Rhyzopertha dominica (Coleoptera: Bostrichidae) is a stored-product pest beetle. Early histological studies dating back to 1930s have reported that R. dominica and other bostrichid species possess a pair of oval symbiotic organs, called the bacteriomes, in which the cytoplasm is densely populated by pleomorphic symbiotic bacteria of peculiar rosette-like shape. However, the microbiological nature of the symbiont has remained elusive., Results: Here we investigated the bacterial symbiont of R. dominica using modern molecular, histological, and microscopic techniques. Whole-mount fluorescence in situ hybridization specifically targeting symbiotic bacteria consistently detected paired bacteriomes, in which the cytoplasm was full of pleomorphic bacterial cells, in the abdomen of adults, pupae and larvae, confirming previous histological descriptions. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont constituted a distinct bacterial lineage allied to a variety of insect-associated endosymbiont clades, including Uzinura of diaspidid scales, Walczuchella of giant scales, Brownia of root mealybugs, Sulcia of diverse hemipterans, and Blattabacterium of roaches. The symbiont gene exhibited markedly AT-biased nucleotide composition and significantly accelerated molecular evolution, suggesting degenerative evolution of the symbiont genome. The symbiotic bacteria were detected in oocytes and embryos, confirming continuous host-symbiont association and vertical symbiont transmission in the host life cycle., Conclusions: We demonstrate that the symbiont of R. dominica constitutes a novel bacterial lineage in the Bacteroidetes. We propose that reductive evolution of the symbiont genome may be relevant to the amorphous morphology of the bacterial cells via disruption of genes involved in cell wall synthesis and cell division. Genomic and functional aspects of the host-symbiont relationship deserve future studies.

The adult honeybee (Apis mellifera L.) mushroom bodies (MBs, a higher center in the insect brain) comprise four subtypes of intrinsic neurons: the class-I large-, middle-, and small-type Kenyon cells (lKCs, mKCs, and sKCs, respectively), and class-II KCs. Analysis of the differentiation of KC subtypes during metamorphosis is important for the better understanding of the roles of KC subtypes related to the honeybee behaviors. In the present study, aiming at identifying marker genes for KC subtypes, we used a cDNA microarray to comprehensively search for genes expressed in an MB-preferential manner in the honeybee brain. Among the 18 genes identified, we further analyzed three genes whose expression was enriched in the MBs: phospholipase C epsilon (PLCe), synaptotagmin 14 (Syt14), and discs large homolog 5 (dlg5). Quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of PLCe, Syt14, and dlg5 was more enriched in the MBs than in the other brain regions by approximately 31-, 6.8-, and 5.6-fold, respectively. In situ hybridization revealed that expression of both Syt14 and dlg5 was enriched in the lKCs but not in the mKCs and sKCs, whereas expression of PLCe was similar in all KC subtypes (the entire MBs) in the honeybee brain, suggesting that Syt14 and dlg5, and PLCe are available as marker genes for the lKCs, and all KC subtypes, respectively. In situ hybridization revealed that expression of PLCe is already detectable in the class-II KCs at the larval fifth instar feeding stage, indicating that PLCe expression is a characteristic common to the larval and adult MBs. In contrast, expression of both Syt14 and dlg5 became detectable at the day three pupa, indicating that Syt14 and dlg5 expressions are characteristic to the late pupal and adult MBs and the lKC specific molecular characteristics are established during the late pupal stages.