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Members of the genus Rickettsiella are bacterial pathogens of insects and other arthropods. Recently, a novel facultative endosymbiont, “Candidatus Rickettsiella viridis,” was described in the pea aphid Acyrthosiphon pisum, whose infection causes a striking host phenotype: red and green genetic color morphs exist in aphid populations, and upon infection with the symbiont, red aphids become green due to increased production of green polycyclic quinone pigments. Here we determined the complete genome sequence of the symbiont. The 1.6-Mb circular genome, harboring some 1,400 protein-coding genes, was similar to the genome of entomopathogenic Rickettsiella grylli (1.6 Mb) but was smaller than the genomes of phylogenetically allied human pathogens Coxiella burnetii (2.0 Mb) and Legionella pneumophila (3.4 Mb). The symbiont’s metabolic pathways exhibited little complementarity to those of the coexisting primary symbiont Buchnera aphidicola, reflecting the facultative nature of the symbiont. The symbiont genome harbored neither polyketide synthase genes nor the evolutionarily allied fatty acid synthase genes that are suspected to catalyze the polycyclic quinone synthesis, indicating that the green pigments are produced not by the symbiont but by the host aphid. The symbiont genome retained many type IV secretion system genes and presumable effector protein genes, whose homologues in L. pneumophila were reported to modulate a variety of the host's cellular processes for facilitating infection and virulence. These results suggest the possibility that the symbiont is involved in the green pigment production by affecting the host’s metabolism using the secretion machineries for delivering the effector molecules into the host cells., IMPORTANCE Insect body color is relevant to a variety of biological aspects such as species recognition, sexual selection, mimicry, aposematism, and crypsis. Hence, the bacterial endosymbiont “Candidatus Rickettsiella viridis,” which alters aphid body color from red to green, is of ecological interest, given that different predators preferentially exploit either red- or green-colored aphids. Here we determined the complete 1.6-Mb genome of the symbiont and uncovered that, although the red-green color transition was ascribed to upregulated production of green polycyclic quinone pigments, the symbiont genome harbored few genes involved in the polycyclic quinone biosynthesis. Meanwhile, the symbiont genome contained type IV secretion system genes and presumable effector protein genes, whose homologues modulate eukaryotic cellular processes for facilitating infection and virulence in the pathogen Legionella pneumophila. We propose the hypothesis that the symbiont may upregulate the host’s production of polycyclic quinone pigments via cooption of secretion machineries and effector molecules for pathogenicity.

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.

Collection localities of the study species and statistical information reported in this study.

Additional information on trichomes induced in C. clematis and other study species

Supplementary methods, references, table, and figures.

Summary Diverse insect species harbor symbiotic bacteria, which play important roles such as provisioning nutrients and providing defense against natural enemies [1–6]. Whereas nutritional symbioses are often indispensable for both partners, defensive symbioses tend to be of a facultative nature [1–12]. The Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits Liberibacter spp. ( Alphaproteobacteria ), causing the devastating citrus greening disease or Huanglongbing [13, 14]. In a symbiotic organ called the bacteriome, D. citri harbors two distinct intracellular symbionts: a putative nutrition provider, Carsonella _DC ( Gammaproteobacteria ), and an unnamed betaproteobacterium with unknown function [15], for which we propose the name " Candidatus Profftella armatura." Here we report that Profftella is a defensive symbiont presumably of an obligate nature with an extremely streamlined genome. The genomes of Profftella and Carsonella _DC were drastically reduced to 464,857 bp and 174,014 bp, respectively, suggesting their ancient and mutually indispensible association with the host. Strikingly, 15% of the small Profftella genome encoded horizontally acquired genes for synthesizing a novel polyketide toxin. The toxin was extracted, pharmacologically and structurally characterized, and designated diaphorin. The presence of Profftella and its diaphorin-biosynthetic genes was perfectly conserved in the world's D. citri populations.

Some species of the aphid genus Nipponaphis (Nipponaphidini) form green, globular or fig-shaped galls on the evergreen Distylium racemosum, their primary host. Molecular phylogenetic analyses of aphid samples collected from both their galls and secondary hosts indicated the occurrence of four species in Japan: N. distychii, N. distyliicola, N. loochooensis and N. machilicola. The four species could also be discriminated from one another in morphology. The name N. litseae turned out to be a junior synonym of N. distychii. Galls formed by N. machilicola are reported for the first time in this paper. The life cycles of the four species are briefly reviewed.

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis.

Symbiosis often entails the emergence of novel adaptive traits in organisms. Microbial symbionts are indispensable for diverse insects via provisioning of essential nutrients, wherein novel host cells and organs for harboring the microbes, called bacteriocytes and bacteriomes, have evolved repeatedly. Molecular and developmental mechanisms underpinning the emergence of novel symbiotic cells and organs comprise an unsolved question in evolutionary developmental biology. Here, we report that a conserved homeotic gene, Ultrabithorax, plays a pivotal role in the bacteriocyte differentiation in a hemipteran insect Nysius plebeius. During embryonic development, six pairs of aggregated presumptive bacteriocytes appear on both sides of six abdominal segments, incorporate the symbiotic bacteria at the stage of germband retraction, and fuse into a pair of lateral bacteriomes at the stage of germband flip, where bacteriocyte-associated Ultrabithorax expression coincides with the symbiont infection process. Suppression of Ultrabithorax expression by maternal RNA interference results in disappearance of the bacteriocytes and the symbiont localization therein, suggesting that Ultrabithorax is involved in differentiation of the host cells for symbiosis. Suppression of other homeotic genes abdominal-A and Antennapedia disturbs integrity and positioning of the bacteriomes, affecting the configuration of the host organs for symbiosis. Our findings unveil the molecular and developmental mechanisms underlying the bacteriocyte differentiation, which may have evolved either via cooption of the transcription factors for inducing the novel symbiotic cells, or via revival of the developmental pathway for the bacteriocytes that had existed in the ancestral hemipterans.

Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens . A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris . These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae.

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.

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 9th to 12th, with the 11th instar (56.5%) and the 12th instar (24.2%) constituting the majority. From the 1st 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.

Male killing is a selfish reproductive manipulation caused by symbiotic bacteria, where male offspring of infected hosts are selectively killed. The underlying mechanisms and the process of their evolution are of great interest not only in terms of fundamental biology, but also their potential applications. The two bacterial Drosophila symbionts, Wolbachia and Spiroplasma, have independently evolved male-killing ability. This raises the question whether the underlying mechanisms share some similarities or are specific to each bacterial species. Here, we analyse pathogenic phenotypes of D. bifasciata infected with its natural male-killing Wolbachia strain and compare them with those of D. melanogaster infected with male-killing Spiroplasma. We show that male progeny infected with the Wolbachia strain die during embryogenesis with abnormal apoptosis. Interestingly, male-killing Wolbachia infection induces DNA damage and segregation defects in the dosage-compensated chromosome in male embryos, which are reminiscent of the phenotypes caused by male-killing Spiroplasma in D. melanogaster. By contrast, host neural development seems to proceed normally unlike male-killing Spiroplasma infection. Our results demonstrate that the dosage-compensated chromosome is a common target of two distinct male killers, yet Spiroplasma uniquely evolved the ability to damage neural tissue of male embryos.

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.

Pectin, an integral component of the plant cell wall, is a recalcitrant substrate against enzymatic challenges by most animals. In characterizing the source of a leaf beetle’s (Cassida rubiginosa) pectin-degrading phenotype, we demonstrate its dependency on an extracellular bacterium housed in specialized organs connected to the foregut. Despite possessing the smallest genome (0.27 Mb) of any organism not subsisting within a host cell, the symbiont nonetheless retained a functional pectinolytic metabolism targeting the polysaccharide’s two most abundant classes: homogalacturonan and rhamnogalacturonan I. Comparative transcriptomics revealed pectinase expression to be enriched in the symbiotic organs, consistent with enzymatic buildup in these structures following immunostaining with pectinase- targeting antibodies. Symbiont elimination results in a drastically reduced host survivorship and a diminished capacity to degrade pectin. Collectively, our findings highlight symbiosis as a strategy for an herbivore to metabolize one of nature’s most complex polysaccharides and a universal component of plant tissues.

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.

Bell-shaped ciliates of the subclass Peritrichia, such as Vorticella, Carchesium and Epistylis, are commonly found in freshwater and other aquatic environments, either solitary or colonial. Peritrichs attach to a substratum via a contractile or non-motile stalk, and collect food particles by water current using ciliary rows around the edge of the bell, called the peristome. Some peritrichs are epibiotic and ectocommensalistic associates of aquatic insects and other animals, settling on the surface of their specific hosts. Only a few peritrichs are known to establish a more internal association with their hosts, locating within the preoral cavity or esophagus of water beetles and presumably subsisting on food materials chewed and ingested by the insects. To date, no endoparasitic or endocommensalistic peritrichs have been reported from insects. Host insects reported to date have all been aquatic, and given the aquatic lifestyle of peritrichs, terrestrial hosts have been considered unlikely. In the present study, we report a dense population of bizarre microbes within the gut of a terrestrial insect, and histological, ultrastructural and molecular phylogenetic analyses identified it as a peritrich ciliate. The highly-developed hindgut of the stag beetle Aegus currani contained oval colonial peritrichs connected by branched stalks resembling grape clusters. Each zooid exhibited a reduced peristome without disc, a vestibulum with active ciliary movement inside, and an elongated macronucleus. These features are morphologically reminiscent of but distinct in some respects from those in Operculariella parasitica, known from the esophagus of dysticid diving beetles. Taxonomic, ecological and functional aspects of this gut-dwelling peritrich warrant future study.

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.

Symbiotic bacteria are commonly associated with cells and tissues of diverse animals and other organisms, which affect hosts' biology in a variety of ways. Most of these symbionts are present in the cytoplasm of host cells and maternally transmitted through host generations. The paucity of paternal symbiont transmission is likely relevant to the extremely streamlined sperm structure: the head consisting of condensed nucleus and the tail made of microtubule bundles, without the symbiont-harboring cytoplasm that is discarded in the process of spermatogenesis. Here, we report a previously unknown mechanism of paternal symbiont transmission via an intrasperm passage. In the leafhopper Nephotettix cincticeps, a facultative Rickettsia symbiont was found not only in the cytoplasm but also in the nucleus of host cells. In male insects, strikingly, most sperm heads contained multiple intranuclear Rickettsia cells. The Rickettsia infection scarcely affected the host fitness including normal sperm functioning. Mating experiments revealed both maternal and paternal transmission of the Rickettsia symbiont through host generations. When cultured with mosquito and silkworm cell lines, the Rickettsia symbiont was preferentially localized within the insect cell nuclei, indicating that the Rickettsia symbiont itself must have a mechanism for targeting nucleus. The mechanisms underlying the sperm head infection without disturbing sperm functioning are, although currently unknown, of both basic and applied interest.

In insects, antimicrobial humoral immunity is governed by two distinct gene cascades, IMD pathway mainly targeting Gram-negative bacteria and Toll pathway preferentially targeting Gram-positive bacteria, which are widely conserved among diverse metazoans. However, recent genomic studies uncovered that IMD pathway is exceptionally absent in some hemipteran lineages like aphids and assassin bugs. How the apparently incomplete immune pathways have evolved with functionality is of interest. Here we report the discovery that, in the hemipteran stinkbug Plautia stali , both IMD and Toll pathways are present but their functional differentiation is blurred. Injection of Gram-negative bacteria and Gram-positive bacteria upregulated effector genes of both pathways. Notably, RNAi experiments unveiled significant functional permeation and crosstalk between IMD and Toll pathways: RNAi of IMD pathway genes suppressed upregulation of effector molecules of both pathways, where the suppression was more remarkable for IMD effectors; and RNAi of Toll pathway genes reduced upregulation of effector molecules of both pathways, where the suppression was more conspicuous for Toll effectors. These results suggest the possibility that, in hemipterans and other arthropods, IMD and Toll pathways are intertwined to target wider and overlapping arrays of microbes, which might have predisposed and facilitated the evolution of incomplete immune pathways.

Many insects possess endosymbiotic bacteria inside their body, wherein intimate interactions occur between the partners. While recent technological advancements have deepened our understanding of metabolic and evolutionary features of the symbiont genomes, molecular mechanisms underpinning the intimate interactions remain difficult to approach because the insect symbionts are generally uncultivable. The bean bug Riptortus pedestris is associated with the betaproteobacterial Burkholderia symbiont in a posterior region of the midgut, which develops numerous crypts harbouring the symbiont extracellularly. Distinct from other insect symbiotic systems, R. pedestris acquires the Burkholderia symbiont not by vertical transmission but from the environment every generation. By making use of the cultivability and the genetic tractability of the symbiont, we constructed a transgenic Burkholderia strain labelled with green fluorescent protein (GFP), which enabled detailed observation of spatiotemporal dynamics and the colonization process of the symbiont in freshly prepared specimens. The symbiont live imaging revealed that, at the second instar, colonization of the symbiotic midgut M4 region started around 6 h after inoculation (hai). By 24 hai, the symbiont cells appeared in the main tract and also in several crypts of the M4. By 48 hai, most of the crypts were colonized by the symbiont cells. By 72 hai, all the crypts were filled up with the symbiont cells and the symbiont localization pattern continued during the subsequent nymphal development. Quantitative PCR of the symbiont confirmed the infection dynamics quantitatively. These results highlight the stinkbug-Burkholderia gut symbiosis as an unprecedented model for comprehensive understanding of molecular mechanisms underpinning insect symbiosis.

Here, we investigate the endosymbiotic microbiota of the Macrosteles leafhoppers M. striifrons and M. sexnotatus , known as vectors of phytopathogenic phytoplasmas. PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA genes identified two obligate endosymbionts, “ Candidatus Sulcia muelleri” and “ Candidatus Nasuia deltocephalinicola,” and five facultative endosymbionts, Wolbachia , Rickettsia , Burkholderia , Diplorickettsia , and a novel bacterium belonging to the Rickettsiaceae , from the leafhoppers. “ Ca . Sulcia muelleri” and “ Ca . Nasuia deltocephalinicola” exhibited 100% infection frequencies in the host species and populations and were separately harbored within different bacteriocytes that constituted a pair of coherent bacteriomes in the abdomen of the host insects, as in other deltocephaline leafhoppers. Wolbachia , Rickettsia , Burkholderia , Diplorickettsia , and the novel Rickettsiaceae bacterium exhibited infection frequencies at 7%, 31%, 12%, 0%, and 24% in M. striifrons and at 20%, 0%, 0%, 20%, and 0% in M. sexnotatus , respectively. Although undetected in the above analyses, phytoplasma infections were detected in 16% of M. striifrons and 60% of M. sexnotatus insects by nested PCR of 16S rRNA genes. Two genetically distinct phytoplasmas, namely, “ Candidatus Phytoplasma asteris,” associated with aster yellows and related plant diseases, and “ Candidatus Phytoplasma oryzae,” associated with rice yellow dwarf disease, were identified from the leafhoppers. These results highlight strikingly complex endosymbiotic microbiota of the Macrosteles leafhoppers and suggest ecological interactions between the obligate endosymbionts, the facultative endosymbionts, and the phytopathogenic phytoplasmas within the same host insects, which may affect vector competence of the leafhoppers.

SummaryThe smallest reported bacterial genome belongs to Tremblaya princeps, a symbiont of Planococcus citri mealybugs (PCIT). Tremblaya PCIT not only has a 139 kb genome, but possesses its own bacterial endosymbiont, Moranella endobia. Genome and transcriptome sequencing, including genome sequencing from a Tremblaya lineage lacking intracellular bacteria, reveals that the extreme genomic degeneracy of Tremblaya PCIT likely resulted from acquiring Moranella as an endosymbiont. In addition, at least 22 expressed horizontally transferred genes from multiple diverse bacteria to the mealybug genome likely complement missing symbiont genes. However, none of these horizontally transferred genes are from Tremblaya, showing that genome reduction in this symbiont has not been enabled by gene transfer to the host nucleus. Our results thus indicate that the functioning of this three-way symbiosis is dependent on genes from at least six lineages of organisms and reveal a path to intimate endosymbiosis distinct from that followed by organelles.

The aphid Ceratovacuna nekoashi and its allied species have been a taxonomically difficult group. They form peculiar “cat's-paw” galls (called “Nekoashi” in Japanese) on Styrax trees and also use Microstegium grasses as their secondary hosts. Through sampling aphids from both Styrax galls and Microstegium grasses in South Korea, Japan and Taiwan, and sequencing their DNA, we made it clear that four distinct species occur in these regions: C. nekoashi (Sasaki), C. oplismeni (Takahashi), C. orientalis (Takahashi) and C. subtropicana sp. nov. In Korea, C. nekoashi forms galls on both S. japonicus and S. obassia, whereas in Japan the species forms galls on the former but not on the latter; our molecular analyses unequivocally indicated the occurrence of a single species in South Korea and mainland Japan. Aphids of the four species on the secondary host were morphologically discriminated from one another. The identity of the primary- and secondary-host generations was also clarified for each species. All four species were found to produce second-instar sterile soldiers in their Styrax galls, and first-instar soldiers were found in colonies of C. subtropicana on the secondary host.

The processes and mechanisms underlying the diversification of host-microbe endosymbiotic associations are of evolutionary interest. Here we investigated the bacteriocyte-associated primary symbionts of weevils wherein the ancient symbiont Nardonella has experienced two independent replacement events: once by Curculioniphilus symbiont in the lineage of Curculio and allied weevils of the tribe Curculionini, and once by Sodalis-allied symbiont in the lineage of grain weevils of the genus Sitophilus. The Curculioniphilus symbiont was detected from 27 of 36 Curculionini species examined, the symbiont phylogeny was congruent with the host weevil phylogeny, and the symbiont gene sequences exhibited AT-biased nucleotide compositions and accelerated molecular evolution. These results suggest that the Curculioniphilus symbiont was acquired by an ancestor of the tribe Curculionini, replaced the original symbiont Nardonella, and has co-speciated with the host weevils over evolutionary time, but has been occasionally lost in several host lineages. By contrast, the Sodalis-allied symbiont of Sitophilus weevils exhibited no host-symbiont co-speciation, no AT-biased nucleotide compositions and only moderately accelerated molecular evolution. These results suggest that the Sodalis-allied symbiont was certainly acquired by an ancestor of the Sitophilus weevils and replaced the original Nardonella symbiont, but the symbiotic association must have experienced occasional re-associations such as new acquisitions, horizontal transfers, replacements and/or losses. We detected Sodalis-allied facultative symbionts in populations of the Curculionini weevils, which might represent potential evolutionary sources of the Sodalis-allied primary symbionts. Comparison of these newcomer bacteriocyte-associated symbiont lineages highlights potential evolutionary trajectories and consequences of novel symbionts after independent replacements of the same ancient symbiont.

Background Diverse animals are intimately associated with microbial symbionts. How such host–symbiont associations have evolved is a fundamental biological issue. Recent studies have revealed a variety of evolutionary relationships, such as obligatory, facultative, and free-living, of gut bacterial symbiosis within the stinkbug family Pentatomidae, although the whole evolutionary picture remains elusive. Results Here we investigated a comprehensive assembly of Japanese pentatomid stinkbugs representing 28 genera, 35 species, and 143 populations. Polymerase chain reaction (PCR), cloning, and sequencing of bacterial 16S rRNA gene from their midgut symbiotic organ consistently detected a single bacterial species from each of the insect samples, indicating a general tendency toward monosymbiotic gut association. Bacterial sequences detected from different populations of the same species were completely or nearly identical, indicating that the majority of the gut symbiotic associations are stably maintained at the species level. Furthermore, bacterial sequences detected from different species in the same genus tended to form well-supported clades, suggesting that host–symbiont associations are often stable even at the genus level. Meanwhile, when we compared such sequences with published sequences available in DNA databases, we found a number of counter-examples to such stable host–symbiont relationships; i.e., symbionts from different host species in the same genus may be phylogenetically distant, and symbionts from the same host species may be phylogenetically diverse. Likewise, symbionts of diverse pentatomid species may be closely related to symbionts of other stinkbug families, and symbionts of diverse pentatomid species may even be allied to free-living bacteria. Molecular evolutionary analyses revealed that higher molecular evolutionary rates, higher AT nucleotide compositions, and smaller genome sizes tended to be associated with the pentatomid symbionts constituting the stable lineages, whereas these traits were rarely observed in the pentatomid symbionts of promiscuous type. Conclusions These results indicate that gut symbiotic bacteria have evolved repeatedly and dynamically in the stinkbug family Pentatomidae, which have plausibly entailed frequent symbiont acquisitions, losses, replacements and transfers, while establishing a number of relatively stable host-symbiont associations. The diverse host-symbiont relationships observed in the Pentatomidae will provide an ideal arena for investigating the evolution of symbiosis experimentally and theoretically. Electronic supplementary material The online version of this article (doi:10.1186/s40851-016-0061-4) contains supplementary material, which is available to authorized users.

Recent studies have suggested that gut symbionts modulate insect development and reproduction. However, the mechanisms by which gut symbionts modulate host physiologies and the molecules involved in these changes are unclear. To address these questions, we prepared three different groups of the insect Riptortus pedestris: Burkholderia gut symbiont-colonized (Sym) insects, Burkholderia-non-colonized (Apo) insects, and Burkholderia-depleted (SymBurk-) insects, which were fed tetracycline. When the hemolymph proteins of three insects were analyzed by SDS-PAGE, the hexamerin-α, hexamerin-β and vitellogenin-1 proteins of Sym-adults were highly expressed compared to those of Apo- and SymBurk--insects. To investigate the expression patterns of these three genes during insect development, we measured the transcriptional levels of these genes. The hexamerin-β gene was specifically expressed at all nymphal stages, and its expression was detected 4-5 days earlier in Sym-insect nymphs than that in Apo- and SymBurk--insects. However, the hexamerin-α and vitellogenin-1 genes were only expressed in adult females, and they were also detected 6-7 days earlier and were 2-fold higher in Sym-adult females than those in the other insects. Depletion of hexamerin-β by RNA interference in 2nd instar Sym-nymphs delayed adult emergence, whereas hexamerin-α and vitellogenin-1 RNA interference in 5th instar nymphs caused loss of color of the eggs of Sym-insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production by regulating production of these three hemolymph storage proteins.

Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner., Symbiotic bacteria are able to interfere with host reproduction in ways that are detrimental to the host organism. Here the authors show that Spiroplasma induces DNA damage on the male X chromosome in Drosophila, causing sex-specific apoptosis.

Relative rate tests of 16S rRNA gene sequences of the cultivable and uncultivable stinkbug gut symbionts in comparison with allied free-living bacteria. (DOCX 152Â kb)

Relative rate tests of 16S rRNA gene sequences of the gut symbionts of pentatomid stinkbugs in comparison with allied free-living bacteria. (DOCX 163Â kb)

Bacterial taxa and their relevant parameters analyzed in Fig. 2. (DOCX 136 kb)

WO is a temperate bacteriophage that infects Wolbachia, a maternally inherited endosymbiont of arthropods. WO has lysogenic and lytic cycles, the latter of which is an important process for the spread of WO infection. In this study, we measured the lytic activities of two WO phages, WOCauB2 and WOCauB3, infecting a Wolbachia strain, wCauB. In the lytic cycle of WO, both ends of the prophage are ligated to create a junction sequence called attP in the phage genome. We performed real-time quantitative polymerase chain reaction to measure the amounts of attP sequences produced by WOCauB2 and WOCauB3 in wCauB-infected Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) and wCauB-infected insect cell lines. WOCauB2 produced the phage genome more actively than WOCauB3 in E. kuehniella, whereas WOcauB3 was more active than WOCauB2 in the cell lines, suggesting that the environment of host cells in which Wolbachia is harbored affects the lytic activity of WO phages. The lytic activity was constantly very low: the amounts of attP relative to the prophages were lower than 1 × 10−3 in all measurements, which was discussed in conjunction with the intracellular life of Wolbachia.

Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia . Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius , 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus . Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius , including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation “ Candidatus Lariskella arthropodarum” is proposed for the endosymbiont clade.

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 green rice leafhopper Nephotettix cincticeps (Uhler) is a commonly distributed pest of rice in East Asia. Early histological studies describe the presence of two bacteriome-associated symbionts and a rickettsial microorganism in N. cincticeps, but their microbiological affiliations have been elusive. We identified these bacterial symbionts using modern microbiological techniques. Cloning and sequencing of the 16S ribosomal RNA gene from dissected bacteriomes yielded two major and a minor bacterial sequences: a major sequence was placed in the Bacteroidetes clade of Sulcia muelleri, an ancient symbiont lineage associated with diverse hemipteran insects; another major sequence was allied to a β-proteobacterial sequence from a leafhopper Matsumuratettix hiroglyphicus; the minor sequence fell in the α-proteobacterial genus Rickettsia. In situ hybridization and transmission electron microscopy showed that the Sulcia symbiont and the β-proteobacterial symbiont are harbored within different types of bacteriocytes that constitute the outer and inner regions of the bacteriome, respectively. Oral administration of tetracycline to nymphal N. cincticeps resulted in retarded growth, high mortality rates, and failure in adult emergence, suggesting important biological roles of the symbionts for the host insect. The designation Candidatus Nasuia deltocephalinicola is proposed for the β-proteobacterial symbiont clade associated with N. cincticeps and allied leafhoppers of the subfamily Deltocephalinae.

Development of insecticide resistance has been a serious concern worldwide, whose mechanisms have been attributed to evolutionary changes in pest insect genomes such as alteration of drug target sites, up-regulation of degrading enzymes, and enhancement of drug excretion. Here, we report a previously unknown mechanism of insecticide resistance: Infection with an insecticide-degrading bacterial symbiont immediately establishes insecticide resistance in pest insects. The bean bug Riptortus pedestris and allied stinkbugs harbor mutualistic gut symbiotic bacteria of the genus Burkholderia , which are acquired by nymphal insects from environmental soil every generation. In agricultural fields, fenitrothion-degrading Burkolderia strains are present at very low densities. We demonstrated that the fenitrothion-degrading Burkholderia strains establish a specific and beneficial symbiosis with the stinkbugs and confer a resistance of the host insects against fenitrothion. Experimental applications of fenitrothion to field soils drastically enriched fenitrothion-degrading bacteria from undetectable levels to >80% of total culturable bacterial counts in the field soils, and >90% of stinkbugs reared with the enriched soil established symbiosis with fenitrothion-degrading Burkholderia . In a Japanese island where fenitrothion has been constantly applied to sugarcane fields, we identified a stinkbug population wherein the insects live on sugarcane and ≈8% of them host fenitrothion-degrading Burkholderia . Our finding suggests the possibility that the symbiont-mediated insecticide resistance may develop even in the absence of pest insects, quickly establish within a single insect generation, and potentially move around horizontally between different pest insects and other organisms.

Light production by organisms, or bioluminescence, has fascinated not only scientists but also ordinary people all over the world, and it has been especially so in Japan. Here we review the biological information available to date for all luminous terrestrial animals known from Japan, particularly focusing on their diversity and systematics, their biology and ecology in Japan, and putative function and biochemistry of their luminescence. In total 58 luminous terrestrial animals have been described from Japan, which consist of 50 fireflies (Coleoptera: Lampyridae), one glowworm beetle (Coleoptera: Phengodidae), two fungus gnats (Diptera: Keroplatidae), one springtail (Collembola), one millipede (Diplopoda), one centipede (Chilopoda) and two earthworms (Oligochaeta). For all except some firefly species, the DNA "barcode" sequences of a cytochrome oxidase subunit I region are provided. We also introduce how intricately the seasonal appearance and glimmering of luminous insects, in particular those of fireflies, have been interwoven into the culture, art, literature and mentality of Japanese people.

The cabbage bugs Eurydema rugosa Motschulsky and Eurydema dominulus (Scopoli) (Heteroptera: Pentatomidae: Strachiini) possess a number of crypts in a posterior region of the midgut, which are filled with bacterial symbiont cells. Here we characterized the gut symbionts of Eurydema stinkbugs using molecular phylogenetic and histological techniques. Specific gammaproteobacteria were consistently identified from the posterior midgut of E. rugosa representing nine populations and E. dominulus representing six populations, respectively. The bacterial 16S rRNA gene sequences were identical within the species but slightly different (98.2% sequence identity) between the species. Molecular phylogenetic analysis revealed that the Eurydema symbionts formed a well-defined monophyletic group in the Gammaproteobacteria. The symbionts were phylogenetically distinct from the gut symbionts of the stinkbug families Acanthosomatidae, Plataspidae, Parastrachiidae, Scutelleridae, and other pentatomid species, suggesting multiple evolutionary origins of the gut symbiotic bacteria among diverse stinkbugs. In situ hybridization confirmed that the symbiont is located in the cavity of the midgut crypts. Aposymbiotic insects of E. rugosa, which were produced by egg surface sterilization, were viable but suffered retarded growth, reduced body weight, and abnormal body color, suggesting the biological importance of the symbiont for the host.

The southern green stinkbug Nezara viridula (Linnaeus) has a number of sac-like outgrowths, called crypts, in a posterior section of the midgut, wherein a specific bacterial symbiont is harbored. In previous studies on N. viridula from Hawaiian populations, experimental elimination of the symbiont caused few fitness defects in the host insect. Here we report that N. viridula from Japanese populations consistently harbors the same gammaproteobacterial gut symbiont, but, in contrast with previous work, experimental sterilization of the symbiont resulted in severe nymphal mortality, indicating an obligate host–symbiont relationship. Considering worldwide host–symbiont association and these experimental data, we suggest that N. viridula is generally and obligatorily associated with the gut symbiont, but that the effect of the symbiont on host biology may be different among geographic populations. Possible environmental factors that may affect the host–symbiont relationship are discussed.

We investigated seed bugs of the genus Nysius (Insecta: Hemiptera: Lygaeidae) for their symbiotic bacteria. From all the samples representing 4 species, 18 populations and 281 individuals, specific bacterial 16S rRNA gene sequences were consistently identified, which formed a distinct clade in the Gammaproteobacteria. In situ hybridization showed that the bacterium was endocellularly localized in a pair of large bacteriomes that were amorphous in shape, deep red in color, and in association with gonads. In the ovary of adult females, the endosymbiont was also localized in the ‘infection zone' in the middle of each germarium and in the ‘symbiont ball' at the anterior pole of each oocyte, indicating vertical transmission of the endosymbiont through the ovarial passage. Phylogenetic analyses based on bacterial 16S rRNA, groEL and gyrB genes consistently supported a coherent monophyly of the Nysius endosymbionts. The possibility of a sister relationship to ‘Candidatus Kleidoceria schneideri', the bacteriome-associated endosymbiont of a lygaeid bug Kleidocerys resedae, was statistically rejected, indicating independent evolutionary origins of the endosymbionts in the Lygaeidae. The endosymbiont genes consistently exhibited AT-biased nucleotide compositions and accelerated rates of molecular evolution, and the endosymbiont genome was only 0.6 Mb in size. The endosymbiont phylogeny was congruent with the host insect phylogeny, suggesting strict vertical transmission and host–symbiont co-speciation over evolutionary time. Based on these results, we discuss the evolution of bacteriomes and endosymbionts in the Heteroptera, most members of which are associated with gut symbiotic bacteria. The designation ‘Candidatus Schneideria nysicola' is proposed for the endosymbiont clade.

Wolbachia endosymbionts are widespread among insects and other arthropods, often causing cytoplasmic incompatibility and other reproductive phenotypes in their hosts. Recently, possibilities of Wolbachia-mediated pest control and management have been proposed, and the bean beetles of the subfamily Bruchinae are known as serious pests of harvested and stored beans worldwide. Here we investigated Wolbachia infections in bean beetles from the world, representing seven genera, 20 species and 87 populations. Of 20 species examined, Wolbachia infections were detected in four species, Megabruchidius sophorae, Callosobruchus analis, C. latealbus and C. chinensis. Infection frequencies were partial in M. sophorae but perfect in the other species. In addition to C. chinensis described in the previous studies, C. latealbus was infected with two distinct Wolbachia strains. These Wolbachia strains from the bean beetles were phylogenetically not closely related to each other. Among world populations of C. chinensis, some Taiwanese populations on a wild leguminous plant, Rhynchosia minima, exhibited a peculiar Wolbachia infection pattern, suggesting the possibility that these populations comprise a distinct host race or a cryptic species.

The alydid stinkbug Riptortus pedestris is specifically associated with a beneficial Burkholderia symbiont in the midgut crypts. Exceptional among insect-microbe mutualistic associations, the Burkholderia symbiont is not vertically transmitted but orally acquired by nymphal insects from the environment every generation. Here we experimentally investigated the process of symbiont acquisition during the nymphal development of R. pedestris . In a field population, many 2nd instar nymphs were Burkholderia free, while all 3rd, 4th, and 5th instar nymphs were infected. When reared on soil-grown potted soybean plants, Burkholderia acquisition occurred at a drastically higher frequency in the 2nd instar than in the other instars. Oral administration of cultured Burkholderia cells showed that 2nd and 3rd instar nymphs are significantly more susceptible to the symbiont infection than 1st, 4th, and 5th instar nymphs. Histological observations revealed rudimentary midgut crypts in the 1st instar, in contrast to well-developed midgut crypts in the 2nd and later instars. These results indicate that R. pedestris acquires the Burkholderia symbiont from the environment mainly during the 2nd instar period and strongly suggest that the competence for the symbiont infection is developmentally regulated by the host side. Potential mechanisms involved in infection competence and possible reasons why the infection preferentially occurs in the 2nd instar are discussed.

During the maturation of insect cuticle, protein-protein and protein-chitin crosslinkages are formed by the action of diphenoloxidases. Two types of diphenoloxidases, laccases and tyrosinases, are present in the insect cuticle. In coleopteran and hymenopteran insects, laccase2 gene has been identified as encoding an enzyme principally responsible for cuticular pigmentation and hardening, whereas biological roles of laccase genes in hemimetabolous insects remain to be established. Here we identified laccase2 genes from three hemipteran stinkbugs, Riptortus pedestris (Alydidae), Nysius plebeius (Lygaeidae) and Megacopta punctatissima (Plataspidae). In R. pedestris, laccase2 gene was highly expressed in epidermal tissues prior to molting. When the gene expression was suppressed by an RNA interference technique, cuticular pigmentation after molting were blocked depending on the dose of injected double-stranded RNA targeting the laccase2 gene. Similar results were obtained for N. plebeius and M. punctatissima. In all the stinkbug species, injecting 20 ng of double-stranded RNA was sufficient to prevent the cuticular maturation. These results indicate that laccase2 gene is generally required for cuticular pigmentation in different stinkbug families, highlighting its conserved biological function across diverse insect taxa.

Microbiological characterization of gut symbiotic bacteria in a limited number of stinkbugs of the families Acanthosomatidae, Plataspidae, Pentatomidae, Scutelleridae, Parastrachiidae, Alydidae and Pyrrhocoridae has shown symbiotic association with midgut bacteria to be common in phytophagous taxa of these heteropteran insects. Here we investigated the midgut bacterial symbiont of Eucorysses grandis, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified in insects from five different geographic origins. The bacterium was detected in 64 of 64 insects sampled from three host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, neither closely related to the gut symbiont of another scutellerid stinkbug, Cantao ocellatus, nor to gut symbionts of other stinkbugs. Diagnostic PCR, in situ hybridization and electron microscopy demonstrated that the bacterium is located extracelluarly, in the midgut fourth section, which possesses crypts. These results indicate that the primary gut symbionts have multiple evolutionary origins in the Scutelleridae. A Sodalis-allied facultative symbiont was also identified in some insects from natural populations. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.