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Microorganisms often live in symbiosis with their hosts, and some are considered mutualists, where all species involved benefit from the interaction. How free-living microorganisms have evolved to become mutualists is unclear. Here we report an experimental system in which non-symbiotic Escherichia coli evolves into an insect mutualist. The stinkbug Plautia stali is typically associated with its essential gut symbiont, Pantoea sp., which colonizes a specialized symbiotic organ. When sterilized newborn nymphs were infected with E. coli rather than Pantoea sp., only a few insects survived, in which E. coli exhibited specific localization to the symbiotic organ and vertical transmission to the offspring. Through transgenerational maintenance with P. stali, several hypermutating E. coli lines independently evolved to support the host’s high adult emergence and improved body colour; these were called ‘mutualistic’ E. coli. These mutants exhibited slower bacterial growth, smaller size, loss of flagellar motility and lack of an extracellular matrix. Transcriptomic and genomic analyses of ‘mutualistic’ E. coli lines revealed independent mutations that disrupted the carbon catabolite repression global transcriptional regulator system. Each mutation reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that single carbon catabolite repression mutations can make E. coli an insect mutualist. These findings provide an experimental system for future work on host–microbe symbioses and may explain why microbial mutualisms are omnipresent in nature.

Microbial mutualists are pivotal for insect adaptation, which often entails the evolution of elaborate organs for symbiosis. Addressing what mechanisms underpin the development of such organs is of evolutionary interest. Here we investigated the stinkbugPlautia staliwhose posterior midgut is transformed into a specialized symbiotic organ. Despite being a simple tube in newborns, it developed numerous crypts in four rows, whose inner cavity hosts a specific bacterial symbiont, during 1stto 2ndnymphal instar. Visualization of dividing cells revealed that active cell proliferation was coincident with the crypt formation, although spatial patterns of the proliferating cells did not reflect the crypt arrangement. Visualization of visceral muscles in the midgut, consisting of circular muscles and longitudinal muscles, uncovered that, strikingly, circular muscles exhibited a characteristic arrangement running between the crypts specifically in the symbiotic organ. Even in early 1stinstar when no crypts were seen, two rows of epithelial areas delineated by bifurcated circular muscles were identified. In 2ndinstar, crossing muscle fibers newly appeared and connected the adjacent circular muscles, whereby the midgut epithelium was divided into four rows of crypt-to-be areas. The crypt formation proceeded even in aposymbiotic nymphs, uncovering autonomous nature of the crypt development. We propose a mechanistic model of crypt formation wherein the spatial arrangement of muscle fibers and the proliferation of epithelial cells underpin the formation of crypts as midgut evaginations.IMPORTANCEDiverse organisms are associated with microbial mutualists, in which specialized host organs often develop for retaining the microbial partners. In the light of the origin of evolutionary novelties, it is important to understand what mechanisms underpin the elaborate morphogenesis of such symbiotic organs, which must have been shaped through interactions with the microbial symbionts. Using the stinkbugPlautia stalias a model, we demonstrated that visceral muscular patterning and proliferation of intestinal epithelial cells during early nymphal stages are involved in the formation of numerous symbiont-harboring crypts arranged in four rows in the posterior midgut to constitute the symbiotic organ. Strikingly, the crypt formation occurred normally even in symbiont-free nymphs, uncovering that the crypt development proceeds autonomously. These findings suggest that the crypt formation is deeply implemented into the normal development ofP. stali, which must reflect the considerably ancient evolutionary origin of the midgut symbiotic organ in stinkbugs.

A novel mechanism underlying butterfly wing colouration was discovered in two distantly related butterfly species, Siproeta stelenes (Nympalinae) and Philaethria diatonica (Heliconiinae). These butterflies have bright green wings, but the colour is not derived from pigments or nano-structures of the scales or the colour of the cuticular membrane, but from an ink-like liquid retained in the wings. Wing structure differs between the green and non-green areas. In the non-green region, the upper and lower cuticular membranes are attached to each other, whereas in the green region there is a space of 5–10 microns where liquid ink is held and living cells were observed. The green ink was composed of pigments similar to those in the hemolymph, but enriched with carotenoid lutein. This novel discovery sheds a new light on our understanding of the evolution of wing colouration in butterflies.

Significance As caterpillars metamorphose to butterflies, insects change their appearance dramatically through metamorphosis. Some insects have an immobile pupal stage for morphological remodeling (homometaboly). Other insects, such as cockroaches, have no pupal stage, and the juveniles and adults are morphologically similar (hemimetaboly). Notably, among the most-ancestral hemimetabolous insects, dragonflies drastically alter their appearance from aquatic nymphs to aerial adults. In dragonflies, we showed that transcription factors Kr-h1 and E93 are essential for regulating metamorphosis as in other insects, while broad , the master gene for pupation in holometabolous insects, regulates a number of both nymph-specific genes and adult-specific genes, providing insight into what evolutionary trajectory the key transcription factor broad has experienced before ending up with governing pupation and holometaboly.

Significance Inotocin, the oxytocin/vasopressin-like peptide, is widely conserved in arthropods; however, little is known about its molecular function. Here, we show that, in ants, the expression levels of inotocin and its receptor are correlated with the age of workers and their behavior. We also demonstrate that inotocin signaling is involved in desiccation resistance by regulating the synthesis of cuticular hydrocarbons. We propose that the up-regulation of inotocin and its receptor as workers age and switch tasks from nursing to foraging is a key physiological adaption to survive drier environments outside of the nest., Oxytocin/vasopressin-like peptides are important regulators of physiology and social behavior in vertebrates. However, the function of inotocin, the homologous peptide in arthropods, remains largely unknown. Here, we show that the level of expression of inotocin and inotocin receptor are correlated with task allocation in the ant Camponotus fellah. Both genes are up-regulated when workers age and switch tasks from nursing to foraging. in situ hybridization revealed that inotocin receptor is specifically expressed in oenocytes, which are specialized cells synthesizing cuticular hydrocarbons which function as desiccation barriers in insects and for social recognition in ants. dsRNA injection targeting inotocin receptor, together with pharmacological treatments using three identified antagonists blocking inotocin signaling, revealed that inotocin signaling regulates the expression of cytochrome P450 4G1 (CYP4G1) and the synthesis of cuticular hydrocarbons, which play an important role in desiccation resistance once workers initiate foraging.

Significance When laying eggs, plataspid stinkbugs deposit small packets called “symbiont capsules.” Newborn stinkbugs suck the capsules to acquire a bacterial mutualist. Without the symbiont, the babies cannot grow and die. Due to the long-lasting symbiosis, the symbiont has experienced genome reduction and become uncultivable. Within the capsules, however, the symbiont can survive for over a week outside the host. Why and how? Here, we uncover a molecular secret implemented in the symbiont capsules. Mother stinkbugs massively produce a special intestinal secretion protein, PMDP, at the expense of their own survival. PMDP embeds the fragile symbiont cells and protects them within the capsules. The host-provisioned molecule for sustaining symbiosis may be utilized for cultivation and/or preservation of fastidious microorganisms., Microbial symbioses significantly contribute to diverse organisms, where long-lasting associations tend to result in symbiont genome erosion, uncultivability, extinction, and replacement. How such inherently deteriorating symbiosis can be harnessed to stable partnership is of general evolutionary interest. Here, we report the discovery of a host protein essential for sustaining symbiosis. Plataspid stinkbugs obligatorily host an uncultivable and genome-reduced gut symbiont, Ishikawaella. Upon oviposition, females deposit “capsules” for symbiont delivery to offspring. Within the capsules, the fragile symbiotic bacteria survive the harsh conditions outside the host until acquired by newborn nymphs to establish vertical transmission. We identified a single protein dominating the capsule content, which is massively secreted by female-specific intestinal organs, embedding the symbiont cells, and packaged into the capsules. Knockdown of the protein resulted in symbiont degeneration, arrested capsule production, symbiont transmission failure, and retarded nymphal growth, unveiling its essential function for ensuring symbiont survival and vertical transmission. The protein originated from a lineage of odorant-binding protein-like multigene family, shedding light on the origin of evolutionary novelty regarding symbiosis. Experimental suppression of capsule production extended the female’s lifespan, uncovering a substantial cost for maintaining symbiosis. In addition to the host’s guardian protein, the symbiont’s molecular chaperone, GroEL, was overproduced in the capsules, highlighting that the symbiont’s eroding functionality is compensated for by stabilizer molecules of host and symbiont origins. Our finding provides insight into how intimate host–symbiont associations can be maintained over evolutionary time despite the symbiont’s potential vulnerability to degeneration and malfunctioning.

Insect eggshells must meet various demands of developing embryos. These demands sometimes conflict with each other; therefore, there are tradeoffs between eggshell properties, such as robustness and permeability. To meet these conflicting demands, particular eggshell structures have evolved in diverse insect species. Here, we report a rare eggshell structure found in the eggshell of a cicada, Cryptotympana facialis. This species has a prolonged egg period with embryonic diapause and a trait of humidity-inducible hatching, which would impose severe demands on the eggshell. We found that in eggs of this species, unlike many other insect eggs, a dedicated cleavage site, known as a hatching line, was formed not in the chorion but in the serosal cuticle. The hatching line was composed of a fine furrow accompanied by ridges on both sides. This furrow-ridge structure formed in the terminal phase of embryogenesis through the partial degradation of an initially thick and nearly flat cuticle layer. We showed that the permeability of the eggshell was low in the diapause stage, when the cuticle was thick, and increased with degradation of the serosal cuticle. We also demonstrated that the force required to cleave the eggshell was reduced after the formation of the hatching line. These results suggest that the establishment of the hatching line on the serosal cuticle enables flexible modification of eggshell properties during embryogenesis, and we predict that it is an adaptation to maximize the protective role of the shell during the long egg period while reducing the barrier to emerging nymphs at the time of hatching. Supplementary Information The online version contains supplementary material available at 10.1186/s40851-021-00178-8.

Many plant-sucking stinkbugs possess a specialized symbiotic organ with numerous crypts in a posterior region of the midgut. In stinkbugs of the superfamily Pentatomoidea, specific γ-proteobacteria are hosted in the crypt cavities, which are vertically transmitted through host generations and essential for normal growth and survival of the host insects. Here we report the discovery of an exceptional gut symbiotic association in the saw-toothed stinkbug, Megymenum gracilicorne (Hemiptera: Pentatomoidea: Dinidoridae), in which specific γ-proteobacterial symbionts are not transmitted vertically but acquired environmentally. Histological inspection identified a very thin and long midgut symbiotic organ with two rows of tiny crypts whose cavities harbor rod-shaped bacterial cells. Molecular phylogenetic analyses of bacterial 16S rRNA gene sequences from the symbiotic organs of field-collected insects revealed that (i) M. gracilicorne is stably associated with Pantoea-allied γ-proteobacteria within the midgut crypts, (ii) the symbiotic bacteria exhibit a considerable level of diversity across host individuals and populations, (iii) the major symbiotic bacteria represent an environmental bacterial lineage that was reported to be capable of symbiosis with the stinkbug Plautia stali, and (iv) the minor symbiotic bacteria also represent several bacterial lineages that were reported as cultivable symbionts of P. stali and other stinkbugs. The symbiotic bacteria were shown to be generally cultivable. Microbial inspection of ovipositing adult females and their eggs and nymphs uncovered the absence of stable vertical transmission of the symbiotic bacteria. Rearing experiments showed that symbiont-supplemented newborn nymphs exhibit improved survival, suggesting the beneficial nature of the symbiotic association.

Insect color patterns are prominently diverse, serving significant physiological and ecological functions. However, the environments surrounding insects are not always constant, complicating attempts to set the most suitable color pattern as a fixed phenotype. While not ubiquitous, many insects possess a sophisticated mechanism that adjusts their surface color in response to external cues to cope with such variable environments. Compared with other well-known color changing-animals like vertebrates and aquatic invertebrates, insects face the challenge of flexible color change because they lack chromatophores like vertebrates but have a melanized exoskeleton. Instead, a variety of unique processes have been adopted in a wide range of insects. This chapter outlines the diverse mechanisms underlying various insect color changes by introducing physiological and biochemical research results and recent progress in molecular studies. Here, the term color change is considered in a broad sense; it covers not only color changes that take place within the life of a single insect, but also alternative color morphs resulting from adaptive developmental plasticity. This review highlights that ecologically relevant color changes are achieved by an elaborate system that requires the coordination of several dedicated pieces of machinery, including variable architectures, perception and integration systems for environmental cues, neuroendocrine signaling systems, and color-generating apparatuses.

Many plant-sucking stinkbugs possess a specialized symbiotic organ with numerous crypts in a posterior region of the midgut. In stinkbugs of the superfamily Pentatomoidea, specific γ-proteobacteria are hosted in the crypt cavities, which are vertically transmitted through host generations and essential for normal growth and survival of the host insects. Here we report the discovery of an exceptional gut symbiotic association in the saw-toothed stinkbug

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

The stinkbug Plautia stali Scott is a notorious agricultural pest whose posterior midgut hosts specific bacteria essential for its growth and survival, highlighted as an experimental model for symbiosis studies. Some symbiotic bacteria of P. stali are cultivable, found free-living in and acquired from the environment, and, furthermore, some free-living environmental bacteria are potentially capable of establishing symbiotic association with P. stali. In this context, it is expected that such environmental bacteria may occasionally contaminate and infect the experimental insects maintained in the laboratory, which could potentially affect the functional analyses of the symbiosis. Here we report that such contamination events do occur under a laboratory rearing conditions for P. stali. When symbiont-deprived newborn nymphs from surface-sterilized eggs were reared in sterilized plastic containers with autoclaved water, most of them died as nymphs presumably as a result of aposymbiosis, but only a small fraction could attain adulthood and the adult insects were all infected with γ-proteobacteria allied to Pantoea and Enterobacter. A variety of bacteria, mainly Bacillus and also Pantoea and Enterobacter, were detected from peanuts and soybeans provided as food for P. stali. Autoclaving of peanuts and soybeans eradicated these bacteria but negatively affected the host survival, whereas ethanol sterilization of peanuts and soybeans removed Pantoea and Enterobacter, but not Bacillus, without negative effects on the host survival. On the basis of these results, we established a practical procedure for aseptic rearing of P. stali, which will enable reliable and strict analyses of host–symbiont interactions in the model symbiotic system.

Diverse insects are intimately associated with microbial symbionts, which play a variety of biological roles in their adaptation to and survival in the natural environment. Such insects often possess specialized organs for hosting the microbial symbionts. What developmental processes and mechanisms underlie the formation of the host organs for microbial symbiosis is of fundamental biological interest but poorly understood. Here we investigate the morphogenesis of the midgut symbiotic organ and the process of symbiont colonization therein during the developmental course of the stinkbug Plautia stali. Upon hatching, the midgut is a simple and smooth tube. Subsequently, symbiont colonization to the posterior midgut occurs, and thickening and folding of the midgut epithelium proceed during the first instar period. By the second instar, rudimentary crypts have formed, and their inner cavities are colonized by the symbiotic bacteria. From the second instar to the fourth instar, while the alimentary tract grows and the posterior midgut is established as the symbiotic organ with numerous crypts, the anterior midgut and the posterior midgut are structurally and functionally isolated by a strong constriction in the middle. By the early fifth instar, the midgut symbiotic organ attains the maximal length, but toward the mid fifth instar, the basal region of each crypt starts to constrict and narrow, which deforms the midgut symbiotic organ as a whole into a shorter, thicker and twisted shape. By the late fifth instar to adulthood, the crypts are constricted off, by which the symbiotic bacteria are confined in the crypt cavities and isolated from the midgut main tract, and concurrently, the strong midgut constriction in the middle becomes loose and open, by which the food flow from the anterior midgut to the posterior midgut recovers. This study provides the most detailed and comprehensive descriptions ever reported on the morphogenesis of the symbiotic organ and the process of symbiont colonization in an obligatory insect-bacterium gut symbiotic system. Considering that P. stali is recently emerging as a useful model system for experimentally studying the intimate insect-microbe gut symbiosis, the knowledge obtained in this study establishes the foundation for the further development of this research field.

In phytophagous insects, in spite of some general advantages of oviposition on a vital part of their host food plants, certain species prefer dead tissues for oviposition. In the present study, we examined oviposition-related behaviors of a cicada, Cryptotympana facialis (Walker), which lays eggs exclusively into dead twigs. From behavioral observation of females experimentally assigned to live or dead plant material, we found that egg laying into freshly cut live twigs is abandoned in two phases, i.e., before and after initiation of egg nest-creating behavior with the ovipositor. Behavioral sequence analyses revealed that oviposition was generally preceded by rubbing with the rostrum tip and brief stylet-penetration behavior, suggesting that oral assessment may play a primary role in decision-making of oviposition in the earlier discriminating phase. From the similarity in behavioral flows of this assessment to vital tissue-seeking behaviors, cicada females are presumed to judge a twig as dead by sensing the absence of vital cues. These findings contribute to understanding a behavioral basis of dead plant recognition for oviposition, potentially giving an insight into ecological and evolutionary aspects of diverse oviposition preferences.

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.

Significance Cicadas are dependent on the essential bacterial symbionts Sulcia and Hodgkinia. The symbiont genomes are extremely streamlined for provisioning of essential amino acids and other nutrients. In some cicada lineages, Hodgkinia genomes are fragmented into numerous minicircles, which may represent a critical stage of genomic erosion close to collapse. What would happen subsequently? Our survey of the Japanese cicada diversity revealed that while Sulcia is conserved among all species, the majority of them have lost Hodgkinia and instead harbor yeast-like fungal associates. The fungal symbionts are phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, indicating recurrent symbiont replacements by entomopathogens in cicadas and providing insights into the mechanisms underlying the parasitism-symbiosis evolutionary continuum, compensation of symbiont genome erosion, and diversification of host-symbiont associations., Diverse insects are associated with ancient bacterial symbionts, whose genomes have often suffered drastic reduction and degeneration. In extreme cases, such symbiont genomes seem almost unable to sustain the basic cellular functioning, which comprises an open question in the evolution of symbiosis. Here, we report an insect group wherein an ancient symbiont lineage suffering massive genome erosion has experienced recurrent extinction and replacement by host-associated pathogenic microbes. Cicadas are associated with the ancient bacterial co-obligate symbionts Sulcia and Hodgkinia, whose streamlined genomes are specialized for synthesizing essential amino acids, thereby enabling the host to live on plant sap. However, our inspection of 24 Japanese cicada species revealed that while all species possessed Sulcia, only nine species retained Hodgkinia, and their genomes exhibited substantial structural instability. The remaining 15 species lacked Hodgkinia and instead harbored yeast-like fungal symbionts. Detailed phylogenetic analyses uncovered repeated Hodgkinia-fungus and fungus-fungus replacements in cicadas. The fungal symbionts were phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, identifying entomopathogenic origins of the fungal symbionts. Most fungal symbionts of cicadas were uncultivable, but the fungal symbiont of Meimuna opalifera was cultivable, possibly because it is at an early stage of fungal symbiont replacement. Genome sequencing of the fungal symbiont revealed its metabolic versatility, presumably capable of synthesizing almost all amino acids, vitamins, and other metabolites, which is more than sufficient to compensate for the Hodgkinia loss. These findings highlight a straightforward ecological and evolutionary connection between parasitism and symbiosis, which may provide an evolutionary trajectory to renovate deteriorated ancient symbiosis via pathogen domestication.

To develop high capacity electrode materials for lithium-ion battery (LIB), dissimilar materials are mixed and, as a result, carbon nanofibers containing silicon (Si) nanoparticles and its components are successfully created by electrospinning method and some heat treatments. Tetraethoxysilane (TEOS) and Si nanoparticles are adopted as additives of carbon nanofibers because of their huge potential for obtaining high capacity. In this research, therefore, we develop TEOS/Si hybrid carbon nanofibers. Consequently, some samples obtain much higher charging/discharging capacity than the theoretical capacity for graphite (372 mAh/g, LiC6) even after second cycle.

Many weevils are known as notorious devastating agricultural pests and generally associated with symbiotic bacteria. Here, we investigated the following pest and non-pest weevils collected in Japan for their bacterial associates: the banana stem weevil Odoiporus longicollis (Olivier); the Asiatic palm weevil Rhabdoscelus lineaticollis (Heller); the red palm weevil Rhynchophorus ferrugineus (Olivier); the Japanese giant weevil Sipalinus gigas (Fabricius); the olive weevil Pimelocerus perforatus (Roelofs); the black hard weevil Pachyrhynchus infernalis Fairmaire; and the Yonaguni hard weevil Metapocyrtus yonagunianus Chujo. Bacterial 16S rRNA gene was amplified by a polymerase chain reaction (PCR) from all the weevils, and genotyping and sequencing of the PCR products revealed that Nardonella, an ancient weevil-associated endosymbiont lineage, is the dominant bacterial associate for them. Molecular phylogenetic analyses based on bacterial 16S rRNA and groEL gene sequences showed that the weevil endosymbionts are placed within the Nardonella clade in the γ-Proteobacteria. The phylogenetic relationship of the Nardonella endosymbionts was concordant with the systematics of the weevil hosts, favoring the hypothesis of weevil-Nardonella co-speciation over evolutionary time. In situ hybridization visualized localization of the Nardonella endosymbionts in the larval bacteriome at the foregut-midgut junction in R. ferrugineus and S. gigas, and in the ovarial tips of adult females in O. longicollis. Our results highlight the general relevance of the Nardonella endosymbionts to the biology, control and management of these and other pest weevils.

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.

Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle’s cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella’s sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host’s cuticle formation and hardening. Notably, Nardonella’s tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host’s aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host’s final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host–symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

Obligate insect-bacterium nutritional mutualism is among the most sophisticated forms of symbiosis, wherein the host and the symbiont are integrated into a coherent biological entity and unable to survive without the partnership. Originally, however, such obligate symbiotic bacteria must have been derived from free-living bacteria. How highly specialized obligate mutualisms have arisen from less specialized associations is of interest. Here we address this evolutionary issue by focusing on an exceptional insect-Wolbachia nutritional mutualism. Although Wolbachia endosymbionts are ubiquitously found in diverse insects and generally regarded as facultative/parasitic associates for their insect hosts, a Wolbachia strain associated with the bedbug Cimex lectularius, designated as wCle, was shown to be essential for host's growth and reproduction via provisioning of B vitamins. We determined the 1,250,060-bp genome of wCle, which was generally similar to the genomes of insect-associated facultative Wolbachia strains, except for the presence of an operon encoding the complete biotin synthetic pathway that was acquired via lateral gene transfer presumably from a coinfecting endosymbiont Cardinium or Rickettsia. Nutritional and physiological experiments, in which wCle-infected and wCle-cured bedbugs of the same genetic background were fed on B-vitamin-manipulated blood meals via an artificial feeding system, demonstrated that wCle certainly synthesizes biotin, and the wCle-provisioned biotin significantly contributes to the host fitness. These findings strongly suggest that acquisition of a single gene cluster consisting of biotin synthesis genes underlies the bedbug-Wolbachia nutritional mutualism, uncovering an evolutionary transition from facultative symbiosis to obligate mutualism facilitated by lateral gene transfer in an endosymbiont lineage.

We previously reported regarding an ecdysone-inducible angiotensin-converting enzyme (ACE) gene. We found another four ACE genes in the Bombyx genome. The present study was undertaken to clarify the evolutionally changed function of the ACE of Bombyx mori. Core regions of deduced amino acid sequences of ACE genes were compared with those of other insect ACE genes. Five Bombyx genes have the conserved Zn2+-binding-site motif (HEXXH); however, BmAcer4 has only one and BmAcer3 has no catalytic ligand. BmAcer1 and BmAcer2 were expressed in several organs. BmAcer3 was expressed in testes, and BmAcer4 and BmAcer5 were expressed in compound eyes; however, the transcription levels of these three genes were very low. Quantitative RT-PCR and Western analysis were conducted to determine the tissue distribution and developmental expression of BmAcer1and BmAcer2. Transcripts of BmAcer1 and BmAcer2 were found in the reproductive organs during the larval and pupal stages. BmAcer1 was dominant in fat bodies during the feeding stage and showed high expression in the epidermis, wing discs, and pupal wing tissues after the wandering stage. Its expression patterns in epidermis, wing discs, and wing tissues resembled the hemolymph ecdysteroid titer in the larval and pupal stages. Acer1 was observed in the hemolymph at all stages, appearing to be the source of it are fat bodies, wings, and epidermis, and functioning after being secreted into the hemolymph. BmAcer2 was abundant in the midgut during the feeding stage and after the wandering stage and in silk glands after the pupal stage. We conclude that the evolution of BmAcer occurred through duplication, and, thereafter, functional diversification developed.

The bedbug Cimex lectularius is notorious as a blood-feeding exoparasite of human and other warm-blooded animals. In addition to its medical and hygienic importance, C. lectularius is known for its unique biological traits, including obligate nutritional mutualism with a vitamin-provisioning Wolbachia endosymbiont and a peculiar mating habit called traumatic insemination wherein male sperm is injected into the female body cavity, migrates to the ovary, and fertilizes eggs therein. For these unique traits, novel insect organs, the bacteriome for endosymbiosis and the spermalege for traumatic insemination, have evolved in the lineage of bedbugs. We constructed cDNA libraries of the bacteriome and the spermalege of C. lectularius, and performed expressed sequence tag (EST) analyses of these peculiar insect organs. In total 4480 ESTs were compiled, which were assembled into 2989 unique sequences (USs). The following ESTs and USs were identified from the different organs: 1151 ESTs and 901 USs from the female whole body; 1098 ESTs and 879 USs from the female bacteriome; 1145 ESTs and 783 USs from the male bacteriome; and 1086 ESTs and 920 USs from the female spermalege. These EST data will provide a valuable genetic resource for understanding the developmental and evolutionary aspects of these novel insect organs.

The hormonal regulation of cuticle protein genes is a good model to study the molecular mechanism of signaling by ecdysteroids, which initiates each of the major developmental transitions in insects. This study was conducted to clarify the regulation of the expression of an ecdysone-inducible cuticle protein gene, BMWCP10 . Induction of the BMWCP10 transcript by ecdysone was partly inhibited in the presence of cycloheximide, which implies that the BMWCP10 promoter is directly and indirectly activated by ecdysone. Using electrophoretic mobility shift analysis and a competition experiment, we identified a putative ecdysone response element (EcRE1) located at positions −93 to −81 relative to the transcription start site. Site-directed mutagenesis of this site, followed by introduction into wing discs, dramatically abolished the reporter activity. This EcRE1 is necessary for the activation of the promoter by 20-hydroxyecdysone (20E) in the wing disc, since the mutation of EcRE1 caused loss of responsiveness to 20E. Collectively, the data obtained in our current and previous work indicate that ecdysone receptor and Broad-Complex Z2 (BR-Z2) are required for maximal BMWCP10 expression in wing disc.

In two Japanese cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata, with different habitat distributions, fully developed embryos hatch in response to high humidity due to rainfall. Despite the advantage of hatching on rainy days, this trait burdens embryos with an extra period of desiccation until the unpredictable advent of rain. We compared the ability of the fully developed embryos of these cicadas to endure periods of low humidity. Eggs were exposed to a combination of different humidities (43% and 75% relative humidity, RH) and durations (0–15 days), and then transferred to an environment with 100% RH to stimulate hatching. In both species, total hatching rates decreased as duration increased, although there was no significant effect of humidity. In C. facialis, a considerable proportion of the eggs hatched during the desiccation period, and the hatching rate was higher at 75% RH than at 43% RH. After transfer to 100% RH, most hatching occurred within a day regardless of the desiccation level. In G. nigrofuscata, no nymphs hatched during the desiccation periods. However, more eggs required more than a day after transfer to 100% RH to hatch after desiccation at 43% RH than at 75% RH. Consequently, the overall proportion of timely hatching of eggs (eggs hatching within a day of moisture supply) was higher after desiccation at 43% RH in C. facialis, but it was higher after desiccation at 75% RH in G. nigrofuscata. These different physiological responses of the two species may reflect adaptation to habitat dryness.

The population of the cicada Cryptotympana facialis began to increase in Osaka, Japan, during the late 20th century. Climate warming is considered a major cause, although the relationship between temperature and the cicada population increase remains unclear. By examining cold tolerance in overwintering eggs of C. facialis in relation to another cicada, Graptopsaltria nigrofuscata, whose population has recently decreased in Osaka, we tested the hypothesis that warming has caused the population increase of C. facialis by decreasing egg mortality due to winter temperatures. A short-term (24 h) cold exposure experiment demonstrated that the half-lethal temperatures (LT50) of C. facialis and G. nigrofuscata were −23.3°C and −28.9°C, respectively, although these extreme low temperatures never occurred in Osaka during the 20th century. Prolonged exposure to −5°C for up to 30 days had no harmful effects on the hatching rate in either species. Overwintering mortality was also assessed under naturally fluctuating conditions by transferring eggs to cooler elevated sites that mimicked the environment prior to the current warming. Eggs of C. facialis that overwintered at the cooler site exhibited similar hatching rates to those maintained at the original site. The results of these experiments consistently indicated that overwintering eggs of C. facialis possess adequate tolerance to the low temperatures of the 20th century. Therefore, we rejected our initial hypothesis that recent increases in the C. facialis population have been caused by warming-related reductions in overwintering egg mortality.

The seasonal timing mechanism of egg hatching was examined in two cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata, with different but overlapping geographical distributions. These species lay eggs in summer, and nymphs hatch in the summer of the following year after egg durations of 10-12 months. When eggs were maintained at 25 degrees C from oviposition, both the species entered embryonic diapause within 60 days irrespective of photoperiod, but at different developmental stages between the two species. The optimal temperature for diapause development was approximately 15 degrees C in both the species. The development rate for postdiapause morphogenesis increased linearly with temperature in the range of 20-27.5 degrees C in C. facialis, and of 15-25 degrees C in G. nigrofuscata. The lower development threshold and the sum of effective temperatures were computed as 14.3 degrees C and 715.3 day-degrees in C. facialis and 12.1 degrees C and 566.6 day-degrees in G. nigrofuscata, respectively. The hatching dates predicted by these large thermal constants accorded with the hatching dates observed in the field, i.e., late June and mid-July in G. nigrofuscata and C. facialis, respectively. Therefore, the high thermal requirements for postdiapause development compel the cicadas to hatch in summer.

Endosymbiotic bacteria of the genus Wolbachia represent the most successful symbiotic bacteria in the terrestrial ecosystem. The success of Wolbachia has been ascribed to its remarkable phenotypic effects on host reproduction, such as cytoplasmic incompatibility, whereby maternally inherited bacteria can spread in their host populations at the expense of their host’s fitness. Meanwhile, recent theoretical as well as empirical studies have unveiled that weak and/or conditional positive fitness effects may significantly facilitate invasion and spread of Wolbachia infections in host populations. Here, we report a previously unrecognized nutritional aspect, the provision of riboflavin (vitamin B2), that potentially underpins the Wolbachia-mediated fitness benefit to insect hosts. A comparative genomic survey for synthetic capability of B vitamins revealed that only the synthesis pathway for riboflavin is highly conserved among diverse insect-associated Wolbachia strains, while the synthesis pathways for other B vitamins were either incomplete or absent. Molecular phylogenetic and genomic analyses of riboflavin synthesis genes from diverse Wolbachia strains revealed that, in general, their phylogenetic relationships are concordant with Wolbachia’s genomic phylogeny, suggesting that the riboflavin synthesis genes have been stably maintained in the course of Wolbachia evolution. In rearing experiments with bedbugs (Cimex lectularius) on blood meals in which B vitamin contents were manipulated, we demonstrated that Wolbachia’s riboflavin provisioning significantly contributes to growth, survival, and reproduction of the insect host. These results provide a physiological basis upon which Wolbachia-mediated positive fitness consequences are manifested and shed new light on the ecological and evolutionary relevance of Wolbachia infections., IMPORTANCE Conventionally, Wolbachia has been regarded as a parasitic bacterial endosymbiont that manipulates the host insect’s reproduction in a selfish manner, which tends to affect a host’s fitness negatively. Meanwhile, some theories predict that, at the same time, Wolbachia can directly affect the host’s fitness positively, which may potentially reconcile the negative effect and facilitate spread and stability of the symbiotic association. Here we demonstrate, by using comparative genomic and experimental approaches, that among synthetic pathways for B vitamins, the synthetic pathway for riboflavin (vitamin B2) is exceptionally conserved among diverse insect-associated Wolbachia strains, and Wolbachia’s riboflavin provisioning certainly contributes to growth, survival, and reproduction in an insect. These findings uncover a nutritional mechanism of a Wolbachia-mediated fitness benefit, which provides empirical evidence highlighting a “Jekyll and Hyde” aspect of Wolbachia infection.

Developmental switching from growth to metamorphosis in imaginal primordia is an essential process of adult body planning in holometabolous insects. Although it is disciplined by a sequential action of the ecdysteroid, molecular mechanisms linking to cell proliferation are poorly understood. In the present study, we investigated the expression control of cell cycle-related genes by the ecdysteroid using the wing disc of the final-instar larvae of the silkworm, Bombyx mori. We found that the expression level of c-myc was remarkably elevated in the post-feeding cell proliferation phase, which coincided with a small increase in ecdysteroid titer. An in vitro wing disc culture showed that supplementation of the moderate level of the ecdysteroid upregulated c-myc expression within an hour and subsequently increased the expression of cell cycle core regulators, including A-, B-, D-, and E-type cyclin genes, Cdc25 and E2F1. We demonstrated that c-myc upregulation by the ecdysteroid was not inhibited in the presence of a protein synthesis inhibitor, suggesting a possibility that the ecdysteroid directly stimulates c-myc expression. Finally, results from the administration of a c-Myc inhibitor demonstrated that c-Myc plays an essential role in 20E-inducible cell proliferation. These findings suggested a novel pathway for ecdysteroid-inducible cell proliferation in insects, and it is likely to be conserved between insects and mammals in terms of steroid hormone regulation.

Introduction Urbanization converts animal habitats into globally homogeneous environments. Consequently, urban communities have low diversity and are often dominated by a few species. However, proximate environmental factor(s) causing community degradation have rarely been identified among diverse and co-varying urban parameters. Results The present study addresses the recent loss of cicada diversity in Osaka, Japan, where cicada communities are overwhelmed by a single species, Cryptotympana facialis. A field survey across an urban-forest gradient revealed that the trend of decreasing cicada diversity toward the urban core was mostly associated with the soil hardness among the environmental variables examined. Simultaneously, the proportion of C. facialis increased with soil hardness, although this effect was partially mitigated in forest patches. Newly hatched nymphs of C. facialis exhibited greater burrowing ability than that in other native species. Conclusions These findings identify soil compaction due to urbanization as a possible cause of cicada diversity loss, as it impedes the passage of nymphs to underground nests. This impact of urban soil compaction may influence ecosystem functioning of soil-dwelling arthropods and their trophically associated animals. Electronic supplementary material The online version of this article (doi:10.1186/s40851-015-0022-3) contains supplementary material, which is available to authorized users.

We have found that polysaccharide polymers such as sodium alginate and agar can be convenient dispersion agents for vaper grown carbon fiver (VGCF). VGCF/sodium alginate composite films and VGCF/SiC composite ceramics with good uniformity and dispersion were prepared by utilizing the resulted dispersion solution. The dispersed composition of VGCF into SiC led an increase in flexural strength, fracture toughness and electric conductivity of SiC. In addition to these results, the magnetic properties of metal ions (Fe3+, Gd3+) bound to poly (acrylic) acid-modified multiwalled carbon nanotube (MWCNT) were discussed.

SummaryMaternal investment for offspring’s growth and survival is widespread among diverse organisms [1–3]. Vertical symbiont transmission via maternal passage is also pivotal for offspring’s growth and survival in many organisms [4–6]. Hence, it is expected that vertical symbiont transmission may coevolve with various organismal traits concerning maternal investment in offspring. Here we report a novel phenotypic syndrome entailing morphological, histological, behavioral, and ecological specializations for maternal investment and vertical symbiont transmission in stinkbugs of the family Urostylididae [7–9]. Adult females develop huge ovaries exaggerated for polysaccharide excretion, possess novel ovipositor-associated organs for vertical transmission of a bacterial symbiont (“Candidatus Tachikawaea gelatinosa”), and lay eggs covered with voluminous symbiont-supplemented jelly. Newborns hatch in midwinter, feed solely on the jelly, acquire the symbiont, and grow during winter. In spring, the insects start feeding on plant sap, wherein the symbiont localizes to a specialized midgut region and supplies essential amino acids deficient in the host’s diet. The reduced symbiont genome and host-symbiont cospeciation indicate their obligate association over evolutionary time. Experimental deprivation of the jelly results in nymphal mortality, whereas restoration of the jelly leads to recovered nymphal growth, confirming that the jelly supports nymphal growth in winter. Chemical analyses demonstrate that the galactan-based jelly contains a sufficient quantity of amino acids to sustain nymphal growth to the third instar. The versatile biological roles of the symbiont-containing egg-covering jelly highlight intricate evolutionary interactions between maternal resource investment and vertical symbiont transmission, which are commonly important for offspring’s growth, survival, and ecological adaptation.