Authorship：Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1111/ens.12555

Language：English   Publisher：THE JAPANESE FORESTRY SOCIETY  

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DOI： 10.11519/jfsc.134.0_1

CiNii Research

Language：English   Publisher：THE JAPANESE FORESTRY SOCIETY  

<p>[in Japanese]</p>

DOI： 10.11519/jfsc.134.0_4

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2022.1044771

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1264/jsme2.ME22042

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41564-022-01179-9

Language：English   Publisher：THE JAPANESE FORESTRY SOCIETY  

<p>[in Japanese]</p>

DOI： 10.11519/jfsc.133.0_159

CiNii Research

Language：English   Publisher：THE JAPANESE FORESTRY SOCIETY  

<p>[in Japanese]</p>

DOI： 10.11519/jfsc.133.0_198

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1073/pnas.2103957118

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.2108/zs200163

Language：English   Publishing type：Research paper (scientific journal)  

Diverse animals, including insects, harbor microbial symbionts within their gut, body cavity, or cells. The subsocial parastrachiid stinkbug Parastrachia japonensis is well-known for its peculiar ecological and behavioral traits, including its prolonged non-feeding diapause period and maternal care of eggs/nymphs in an underground nest. P. japonensis harbors a specific bacterial symbiont within the gut cavity extracellularly, which is vertically inherited through maternal excretion of symbiont-containing white mucus. Thus far, biological roles of the symbiont in the host lifecycle has been little understood. Here we sequenced the genome of the uncultivable gut symbiont "Candidatus Benitsuchiphilus tojoi." The symbiont has an 804 kb circular chromosome encoding 606 proteins and a 14.5 kb plasmid encoding 13 proteins. Phylogenetic analysis indicated that the bacterium is closely related to other obligate insect symbionts belonging to the Gammaproteobacteria, including Buchnera of aphids and Blochmannia of ants, and the most closely related to Ishikawaella, an extracellular gut symbiont of plataspid stinkbugs. These data suggested that the symbiont genome has evolved like highly reduced gamma-proteobacterial symbiont genomes reported from a variety of insects. The presence of genes involved in biosynthesis pathways for amino acids, vitamins, and cofactors in the genome implicated the symbiont as a nutritional mutualist, supplementing essential nutrients to the host. Interestingly, the symbiont's plasmid encoded genes for thiamine and carotenoid synthesis pathways, suggesting the possibility of additional functions of the symbiont for protecting the host against oxidative stress and DNA damage. Finally, possible involvement of the symbiont in uric acid metabolism during diapause is discussed.

DOI： 10.3389/fmicb.2020.00840

Language：English   Publishing type：Research paper (scientific journal)  

The majority of plant-sucking stinkbugs of the superfamily Pentatomoidea possess numerous crypts in the midgut as the symbiotic organ, where specific and beneficial symbiotic bacteria are harbored extracellularly. The host–symbiont relationships are co-speciating in the families Plataspidae, Acanthosomatidae and Urostylididae, but promiscuous in the families Pentatomidae and Cydnidae. As for the family Scutelleridae, only a few species have been examined for their gut symbiotic bacteria. Here, we comprehensively investigated Japanese scutellerid stinkbugs representing 6 genera, 8 species, and 24 individuals. Molecular phylogenetic analysis revealed that the scutellerid gut symbionts are polyphyletic, consisting of at least seven distinct phylogenetic groups in the Gammaproteobacteria. In four of the seven groups, the symbionts were closely related to each other, to the pentatomid gut symbionts, and to environmental bacteria of the genus Pantoea. These results strongly suggest that the scutellerid gut symbionts are of multiple evolutionary origins, presumably entailing repeated symbiont acquisitions, replacements and/or horizontal transfers in a promiscuous manner. Elimination of the gut symbionts from Lampromicra miyakona resulted in high mortality without adult emergence, confirming that the gut symbionts are of beneficial nature. In conclusion, the host–symbiont associations in the Scutelleridae seem to be similar to those in the Pentatomidae and Cydnidae.

DOI： 10.1007/s13355-019-00630-4

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1073/pnas.1712857114

Language：English   Publishing type：Research paper (scientific journal)  

A number of phytophagous stinkbugs are associated with specific bacterial symbionts in their alimentary tracts. The sloe bug Dolycoris baccarum (Linnaeus), a notorious pest of diverse crops, possesses a number of sac-like tissues, called crypts, in a posterior section of the midgut, wherein a specific bacterial symbiont colonizes. Here we characterized the symbiotic bacterium of D. baccarum by histological analysis, molecular phylogeny, and diagnostic PCR with a specific primer set. The cloning and sequencing analyses of bacterial 16S rRNA genes and fluorescent in situ hybridization demonstrated that the sloe bug is associated with a single species of Gammaproteobacteria in the midgut crypts. Molecular phylogenetic analysis strongly suggested that the symbiont should be placed in the genus Pantoea of the Enterobacteriaceae. Diagnostic PCR and egg surface sterilization with formalin indicated the stinkbug vertically transmits the Pantoea symbiont via egg-smearing. The sterilization-produced aposymbiotic nymphs showed high mortality and no insects reached adulthood. In addition, the Pantoea symbiont was uncultivable outside the insect host, indicating an obligate and intimate host-symbiont association.

DOI： 10.1007/s13355-016-0453-0

Language：English   Publishing type：Research paper (scientific journal)  

Global warming impacts diverse organisms not only directly but also indirectly via other organisms with which they interact. Recently, the possibility that elevated temperatures resulting from global warming may substantially affect biodiversity through disrupting mutualistic/parasitic associations has been highlighted. Here we report an experimental demonstration that global warming can affect a pest insect via suppression of its obligate bacterial symbiont. The southern green stinkbug Nezara viridula depends on a specific gut bacterium for its normal growth and survival. When the insects were reared inside or outside a simulated warming incubator wherein temperature was controlled at 2.5°C higher than outside, the insects reared in the incubator exhibited severe fitness defects (i.e., retarded growth, reduced size, yellowish body color, etc.) and significant reduction of symbiont population, particularly in the midsummer season, whereas the insects reared outside did not. Rearing at 30°C or 32.5°C resulted in similar defective phenotypes of the insects, whereas no adult insects emerged at 35°C. Notably, experimental symbiont suppression by an antibiotic treatment also induced similar defective phenotypes of the insects, indicating that the host’s defective phenotypes are attributable not to the heat stress itself but to the suppression of the symbiont population induced by elevated temperature. These results strongly suggest that high temperature in the midsummer season negatively affects the insects not directly but indirectly via the heat-vulnerable obligate bacterial symbiont, which highlights the practical relevance of mutualism collapse in this warming world.

DOI： 10.1128/mBio.01578-16

Language：English   Publishing type：Research paper (scientific journal)  

Recent resurgence of the bedbug Cimex lectularius is a global problem on the public health. On account of the worldwide rise of insecticide-resistant bedbug populations, exploration of new approaches to the bedbug control and management is anticipated. In this context, gene silencing by RNA interference (RNAi) has been considered for its potential application to pest control and management, because RNAi enables specific suppression of target genes and thus flexible selection of target traits to be disrupted. In this study, in an attempt to develop a control strategy targeting reproduction of the bedbug, we investigated RNAimediated gene silencing of vitellogenin (Vg), a major yolk protein precursor essential for oogenesis. From the bedbug transcriptomes, we identified a typical Vg gene and a truncated Vg gene, which were designated as ClVg and ClVg-like, respectively. ClVg gene was highly expressed mainly in the fat body of adult females, which was more than 100 times higher than the expression level of ClVg-like gene, indicating that ClVg gene is the primary functional Vg gene in the bedbug. RNAi-mediated suppression of ClVg gene expression in adult females resulted in drastically reduced egg production, atrophied ovaries, and inflated abdomen due to hypertrophied fat bodies. These phenotypic consequences are expected not only to suppress the bedbug reproduction directly but also to deteriorate its feeding and survival indirectly via behavioral modifications. These results suggest the potential of ClVg gene as a promising target for RNAi-based population management of the bedbug.

DOI： 10.1371/journal.pone.0153984

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1128/mBio.01732-15

Language：English   Publishing type：Research paper (scientific journal)  

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 Pimelocerusperforatus (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.

DOI： 10.1007/s13355-015-0326-y

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1128/AEM.04057-14

Language：English   Publishing type：Research paper (scientific journal)  

Maternal 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 specializedmidgut 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 eggcovering 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.

DOI： 10.1016/j.cub.2014.08.065

Language：English   Publishing type：Research paper (scientific journal)  

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 w Cle 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.

DOI： 10.1073/pnas.1409284111

Language：English   Publishing type：Research paper (scientific journal)  

Invasive species may rapidly spread throughout new areas once introduced, which may potentially lead to serious damage to local fauna and flora. Information on geographical origins, introduction routes, and biology in native regions of such invasive species is of critical importance in identifying means of transport, preventing reintroduction, and establishing control/eradication methods. The plataspid stinkbug Megacopta cribraria, known as kudzu bug, recently invaded North America and now has become not only an agricultural pest of soybean but also a nuisance pest. Here we investigate the geographical origin of the invasive M. cribraria populations. Phylogeographical analyses based on 8.7 kb mitochondrial DNA sequences of the introduced and East Asian native Megacopta populations identified a well-supported clade consisting of the introduced populations and M. punctatissima populations in the Kyushu region of Japan, which strongly suggests that the invading M. cribraria populations are derived from a M. punctatissima population in the Kyushu region. Therefore, the region is proposed as a promising source of natural enemies for biological control of the invasive pest. Based on the phylogenetic information, relationship and treatment of the two Megacopta species are discussed.

DOI： 10.1371/journal.pone.0089107

Language：English   Publishing type：Research paper (scientific journal)  

Stinkbugs of the genus Antestiopsis, so-called antestia bugs or variegated coffee bugs, are notorious pests of coffee plants in Africa. We investigated the symbiotic bacteria associated with Antestiopsis thunbergii, a major coffee plant pest in Rwanda. PCR, cloning, sequencing, and phylogenetic analysis of bacterial genes identified four distinct bacterial lineages associated with A. thunbergii: A gammaproteobacterial gut symbiont and symbionts representing the genera Sodalis, Spiroplasma, and Rickettsia. In situ hybridization showed that the gut symbiont densely occupied the lumen of midgut crypts, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont sparsely and sporadically infected various cells and tissues. Diagnostic PCR survey of 154 A. thunbergii individuals collected at 8 localities in Rwanda revealed high infection frequencies (100% for the gut symbiont, 51.3% for the Sodalis symbiont, 52.6% for the Spiroplasma symbiont, and 24.0% for the Rickettsia symbiont). These results suggest that the gut symbiont is the primary symbiotic associate of obligate nature for A. thunbergii, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont are the secondary symbiotic associates of facultative nature. We observed high coinfection frequencies, i.e., 7.8% of individuals with quadruple infection with all the symbionts, 32.5% with triple infections with the gut symbiont and two of the secondary symbionts, and 39.6% with double infections with the gut symbiont and any of the three secondary symbionts, which were statistically not different from the expected coinfection frequencies and probably reflected random associations. The knowledge of symbiotic microbiota in A. thunbergii will provide useful background information for controlling this devastating coffee plant pest.

DOI： 10.1128/AEM.00554-14

Language：English   Publishing type：Research paper (scientific journal)  

Sociality may affect symbiosis and vice versa. Many plant-sucking stinkbugs harbor mutualistic bacterial symbionts in the midgut. In the superfamily Pentatomoidea, adult females excrete symbiont-containing materials from the anus, which their offspring ingest orally and establish vertical symbiont transmission. In many stinkbug families whose members are mostly non-social, females excrete symbiont-containing materials onto/beside eggs upon oviposition. However, exceptional cases have been reported from two subsocial species representing the closely related families Cydnidae and Parastrachiidae, wherein females remain nearby eggs for maternal care after oviposition, and provide their offspring with symbiont-containing secretions at later stages, either just before or after hatching. These observations suggested that sociality of the host stinkbugs may be correlated with their symbiont transmission strategies. However, we found that cydnid stinkbugs of the genus Adomerus, which are associated with gammaproteobacterial gut symbionts and exhibit elaborate maternal care over their offspring, smear symbiont-containing secretions onto eggs upon oviposition as many non-social stinkbugs do. Surface sterilization of the eggs resulted in aposymbiotic insects of slower growth, smaller size and abnormal body coloration, indicating vertical symbiont transmission via egg surface contamination and presumable beneficial nature of the symbiosis. The Adomerus symbionts exhibited AT-biased nucleotide compositions, accelerated molecular evolutionary rates and reduced genome size, while these degenerative genomic traits were less severe than those in the symbiont of a subsocial parastrachiid. These results suggest that not only sociality but also other ecological and evolutionary aspects of the host stinkbugs, including the host-symbiont co-evolutionary history, may have substantially affected their symbiont transmission strategies. (250 words).

DOI： 10.1371/journal.pone.0065081

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1007/s13355-012-0112-z

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Symbiotic bacteria associated with midgut crypts of stinkbugs of the family Cydnidae, representing seven species and 13 populations, were investigated. All of the symbionts were species specific, and constituted at least four distinct lineages in the Gammaproteobacteria, indicating multiple evolutionary origins of the gut symbionts among the burrower bugs.

DOI： 10.1128/AEM.00867-12

Language：English   Publishing type：Research paper (scientific journal)  

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 resistancemay 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.

DOI： 10.1073/pnas.1200231109

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Bat flies of the family Nycteribiidae are known for their extreme morphological and physiological traits specialized for ectoparasitic blood-feeding lifestyle on bats, including lack of wings, reduced head and eyes, adenotrophic viviparity with a highly developed uterus and milk glands, as well as association with endosymbiotic bacteria. We investigated Japanese nycteribiid bat flies representing 4 genera, 8 species and 27 populations for their bacterial endosymbionts. From all the nycteribiid species examined, a distinct clade of gammaproteobacteria was consistently detected, which was allied to endosymbionts of other insects such as Riesia spp. of primate lice and Arsenophonus spp. of diverse insects. In adult insects, the endosymbiont was localized in specific bacteriocytes in the abdomen, suggesting an intimate host-symbiont association. In adult females, the endosymbiont was also found in the cavity of milk gland tubules, which suggests uterine vertical transmission of the endosymbiont to larvae through milk gland secretion. In adult females of Penicillidia jenynsii, we discovered a previously unknown type of symbiotic organ in the Nycteribiidae: a pair of large bacteriomes located inside the swellings on the fifth abdominal ventral plate. The endosymbiont genes consistently exhibited adenine/thymine biased nucleotide compositions and accelerated rates of molecular evolution. The endosymbiont genome was estimated to be highly reduced, ∼0.76 Mb in size. The endosymbiont phylogeny perfectly mirrored the host insect phylogeny, indicating strict vertical transmission and host-symbiont co-speciation in the evolutionary course of the Nycteribiidae. The designation Candidatus Aschnera chinzeii is proposed for the endosymbiont clade.

DOI： 10.1038/ismej.2011.125

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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.

DOI： 10.1038/ismej.2011.103

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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.

DOI： 10.1007/s13355-011-0081-7

Language：English   Publishing type：Research paper (scientific journal)  

In a subsocial stinkbug Parastrachia japonensis, mature adult females lay both fertile eggs and trophic eggs in an underground nest, guard the egg mass for 15-20. days, and collect and provide food fruits for the nymphs. We investigated the vertical transmission of this species' obligate gut symbiont '. Candidatus Benitsuchiphilus tojoi' from mother to offspring. We observed that the mothers started to excrete a copious amount of symbiont-containing white mucus from the anus onto the egg mass at around 45. min before egg hatching; the excretion continued for approximately 40. min, and about 5. min later, fertile eggs in the egg mass started to hatch synchronously. The newborn nymphs immediately ingested the mucous secretion and the trophic eggs, which were completely consumed within 24. h. Mother removal and anal-sealing experiments confirmed that vertical symbiont transmission was mediated by the nymphs' exploitation of the mother-derived mucous secretion. Mother removal also resulted in asynchronous egg hatching, suggesting maternal involvement in the egg-hatching synchrony. These results indicate that vertical symbiont transmission in P. japonensis is finely tuned to the timing of egg hatching through intricate mother-offspring behavioural interactions, and highlight how sociality can evolutionarily affect symbiosis and vice versa.

DOI： 10.1016/j.anbehav.2011.11.006

Language：English   Publishing type：Research paper (scientific journal)  

Obligate endocellular symbiotic bacteria of insects and other organisms generally exhibit drastic genome reduction. Recently, it was shown that symbiotic gut bacteria of some stinkbugs also have remarkably reduced genomes. Here, we report the complete genome sequence of such a gut bacterium Ishikawaella capsulata of the plataspid stinkbug Megacopta punctatissima. Gene repertoire and evolutionary patterns, including AT richness and elevated evolutionary rate, of the 745,590 bp genome were strikingly similar to those of obligate c-proteobacterial endocellular insect symbionts like Buchnera in aphids and Wigglesworthia in tsetse flies. Ishikawaella was suggested to supply essential amino acids for the plant-sucking stinkbug as Buchnera does for the host aphid. Although Buchnera is phylogenetically closer to Wigglesworthia than to Ishikawaella, in terms of gene repertoire Buchnera was similar to Ishikawaella rather than to Wigglesworthia, providing a possible case of genome-level convergence of gene content. Meanwhile, several notable differences were identified between the genomes of Ishikawaella and Buchnera, including retention of TCA cycle genes and lack of flagellum-related genes in Ishikawaella, which may reflect their adaptation to distinct symbiotic habitats. Unexpectedly, Ishikawaella retained fewer genes related to cell wall synthesis and lipid metabolism than many endocellular insect symbionts. The plasmid of Ishikawaella encoded genes for arginine metabolism and oxalate detoxification, suggesting the possibility of additional Ishikawaella roles similar to those of human gut bacteria. Our data highlight strikingly similar evolutionary patterns that are shared between the extracellular and endocellular insect symbiont genomes.

DOI： 10.1093/gbe/evr064

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1007/s13355-011-0066-6

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1128/AEM.00358-11

Language：English   Publishing type：Research paper (scientific journal)  

Various animals produce inviable eggs or egg-like structures called trophic eggs, which are presumed to be an extended maternal investment for the offspring. However, there is little knowledge about the ecological or physiological constraints associated with their evolutionary origin. Trophic eggs of the seminivorous subsocial burrower bug (Canthophorus niveimarginatus) have some unique characteristics. Trophic eggs are obligate for nymphal survival, and firstinstar nymphs die without them. To identify the cause of nymphal death, we hypothesized that first-instar nymphs starve to death because they cannot feed on anything but trophic eggs. Although first-instar nymphs fed on artificially exposed endosperm did survive, nymphs that were provided with intact seed were not able to penetrate the seed vessel and starved to death. Another hypothesis that trophic eggs play a role in transferring the midgut symbiont, essential for survival in heteropteran bugs, from mother to offspring was rejected because almost all nymphs had retained the symbiont without feeding on trophic eggs. These results suggest that poor feeding capacity of the offspring is the cause of nymphal death, and the important constraint that promotes the evolution of the curious trophic egg system in C. niveimarginatus.

DOI： 10.1098/rsbl.2010.0707

Language：English   Publishing type：Research paper (scientific journal)  

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host-symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host-symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host-symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed.

DOI： 10.1038/ismej.2010.150

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.2108/zsj.28.169

Language：English   Publishing type：Research paper (scientific journal)  

Weevils constitute the most species-rich animal group with over 60,000 described species, many of which possess specialized symbiotic organs and harbor bacterial endosymbionts. Among the diverse microbial associates of weevils, Nardonella spp. represent the most ancient and widespread endosymbiont lineage, having co-speciated with the host weevils for over 125 million years. Thus far, however, no empirical work on the role of Nardonella for weevil biology has been reported. Here we investigated the biological role of the Nardonella endosymbiont for the West Indian sweet potato weevil, Euscepes postfasciatus. This insect is an experimentally tractable pest insect that can easily be reared on a natural diet of sweet potato root as well as on an agar-based artificial diet. By larval feeding on an antibiotic-containing artificial diet, Nardonella infection was effectively eliminated from the treated insects. The antibiotic-treated insects exhibited significantly lighter body weight and lower growth rate than the control insects. Then, the antibiotic-treated insects and the control insects were respectively allowed to mate and oviposit on fresh sweet potatoes without the antibiotic. The offspring of the antibiotic-treated insects, which were all Nardonella-negative, exhibited significantly lighter body weight, smaller body size, lower growth rate and paler body color in comparison with the offspring of the control insects, which were all Nardonellapositive. In conclusion, the Nardonella endosymbiont is involved in normal growth and development of the host weevil. The biological role of the endosymbiont probably underlies the long-lasting host-symbiont co-speciation in the evolutionary course of weevils.

DOI： 10.1371/journal.pone.0013101

Language：English   Publishing type：Research paper (scientific journal)  

The stinkbug Parastrachia japonensis (Hemiptera: Parastrachiidae) is known for its prolonged prereproductive nonfeeding period, maternal care of eggs in an underground nest, and maternal collection and provisioning of food (fruits) for nymphs. A previous study suggested that a bacterial symbiont is involved in uric acid recycling in this insect during the nonfeeding period, but the identity of this symbiont has not been determined. Here we characterized a novel bacterial symbiont obtained from P. japonensis. Molecular phylogenetic analyses based on 16S rRNA, gyrB, and groEL gene sequences consistently indicated that this symbiont constituted a distinct lineage in the Gammaproteobacteria that has no close relatives but is allied with gut symbionts of acanthosomatid and plataspid stinkbugs, as well as with endocellular symbionts of sharpshooters, tsetse flies, and aphids. The symbiont genes had a remarkably AT-biased nucleotide composition and exhibited significantly accelerated molecular evolution. The symbiont genome was extremely reduced; its size was estimated to be 0.85 Mb. These results suggest that there has been an intimate host-symbiont association over evolutionary time. The symbiont was localized in swollen crypts in a posterior part of the midgut, which was a specialized symbiotic organ. The possibility that the symbiont is involved in uric acid recycling is discussed. The designation "Candidatus Benitsuchiphilus tojoi" is proposed for the symbiont.

DOI： 10.1128/AEM.00616-10

Language：English   Publishing type：Research paper (scientific journal)  

Symbiotic associations with midgut bacteria have been commonly found in diverse phytophagous heteropteran groups, where microbiological characterization of the symbiotic bacteria has been restricted to the stinkbug families Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae. Here we investigated the midgut bacterial symbiont of Cantao ocellatus, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified from the insects of different geographic origins. The bacterium was detected in all 116 insects collected from 9 natural host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, not closely related to gut symbionts of other stinkbugs. Diagnostic PCR and in situ hybridization demonstrated that the bacterium is extracellularly located in the midgut 4th section with crypts. Electron microscopy of the crypts revealed a peculiar histological configuration at the host-symblont interface. Egg sterilization experiments confirmed that the bacterium is vertically transmitted to stinkbug nymphs via egg surface contamination. In addition to the gut symbiont, some individuals of C. ocellatus harbored another bacterial symbiont in their gonads, which was closely related to Sodalis glossinidius, the secondary endosymbiont of tsetse flies. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.

DOI： 10.1128/AEM.00421-10

Language：English   Publishing type：Research paper (scientific journal)  

The West Indian sweet potato weevil, Euscepes postfasciatus, is a notorious pest of the sweet potato, Ipomoea batatas. We examined the potential presence of a bacterial endosymbiont in the pest weevil. The bacterial 16S rRNA gene and groEL gene were detected by PCR from the insects. Cloning, sequencing and molecular phylogenetic analyses of the bacterial genes demonstrated that E. postfasciatus is associated with a γ -proteobacterial endosymbiont of the genus Nardonella. In situ hybridization detected the endosymbiont in the female ovaries, indicating its transovarial transmission through host generations. This study is the first to identify Nardonella from the weevil subfamily Cryptorhynchinae. The potential relevance of the endosymbiont in the biology and management of E. postfasciatus is discussed.

DOI： 10.1303/aez.2010.115

Language：English   Publishing type：Research paper (scientific journal)  

Many insects are dependent on bacterial symbionts that provide essential nutrients (ex. aphid-Buchnera and tsetse-Wiglesworthia associations), wherein the symbionts are harbored in specific cells called bacteriocytes that constitute a symbiotic organ bacteriome. Facultative and parasitic bacterial symbionts like Wolbachia have been regarded as evolutionarily distinct from such obligate nutritional mutualists. However, we discovered that, in the bedbug Cimex lectularius, Wolbachia resides in a bacteriome and appears to be an obligate nutritional mutualist. Two bacterial symbionts, a Wolbachia strain and an unnamed γ-proteobacterium, were identified from different strains of the bedbug. The Wolbachia symbiont was detected fromall of the insects examined whereas the γ-proteobacterium was found in a part of them. The Wolbachia symbiont was specifically localized in the bacteriomes and vertically transmitted via the somatic stemcell niche of germalia to oocytes, infecting the incipient symbiotic organ at an early stage of the embryogenesis. Elimination of the Wolbachia symbiont resulted in retarded growth and sterility of the host insect. These deficiencies were rescued by oral supplementation of B vitamins, confirming the essential nutritional role of the symbiont for the host. The estimated genome size of the Wolbachia symbiont was around 1.3 Mb, which was almost equivalent to the genome sizes of parasitic Wolbachia strains of other insects. These results indicate that bacteriocyte-associated nutritional mutualism can evolve from facultative and prevalent microbial associates like Wolbachia, highlighting a previously unknown aspect of the parasitism-mutualism evolutionary continuum.

DOI： 10.1073/pnas.0911476107

Language：English   Publishing type：Research paper (scientific journal)  

Here we investigated the bacterial endosymbionts of weevils of the genus Curculio. From all four species of Curculio weevils examined, a novel group of bacterial gene sequences were consistently identified. Molecular phylogenetic analyses demonstrated that the sequences formed a distinct clade in the Gammaproteobacteria, which was not related to previously known groups of weevil endosymbionts such as Nardonella spp. and Sodalis-allied symbionts. In situ hybridization revealed that the bacterium was intracellularly harbored in a bacteriome associated with larval midgut. In adult females, the bacterium was localized in the germalia at the tip of each overiole, suggesting vertical transmission via ovarial passage. Diagnostic PCR surveys detected high prevalence of the bacterial infection in natural host populations. Electron microscopy identified the reduced cell wall of the bacterial cells, and the bacterial genes exhibited AT-biased nucleotide composition and accelerated molecular evolution, which are suggestive of a long-lasting endosymbiotic association. On the basis of these results, we conclude that the novel endocellular bacteria represent the primary symbiont of Curculio weevils and proposed the designation quot;Condidatus Curculioniphilus buchneri.quot; In addition to quot;Ca. CurcuIioniphilus,quot; we identified Sodalis-allied gammaproteobacterial endosymbionts from the chestnut weevil, Curculio sikkimensis, which exhibited partial infection frequencies in host insect populations and neither AT-biased nucleotide composition nor accelerated molecular evolution. We suggest that such Sodalis-allied secondary symbionts in weevils might provide a potential source for symbiont replacements, as has occurred in an ancestor of Sitophilus grain weevils.

DOI： 10.1128/AEM.02154-09

Language：English   Publishing type：Research paper (scientific journal)  

A symbiotic bacterium of the macaque louse, Pedicinus obtusus, was characterized. The symbiont constituted a gammaproteobacterial lineage distinct from the symbionts of anthropoid primate lice, localized in the midgut epithelium and the ovaries and exhibiting AT-biased genes and accelerated molecular evolution. The designation "Candidatus Puchtella pedicinophila" was proposed for it.

DOI： 10.1128/AEM.00226-09

Language：English   Publishing type：Research paper (scientific journal)  

Background: Host-symbiont co-speciation and reductive genome evolution have been commonly observed among obligate endocellular insect symbionts, while such examples have rarely been identified among extracellular ones, the only case reported being from gut symbiotic bacteria of stinkbugs of the family Plataspidae. Considering that gut symbiotic communities are vulnerable to invasion of foreign microbes, gut symbiotic associations have been thought to be evolutionarily not stable. Stinkbugs of the family Acanthosomatidae harbor a bacterial symbiont in the midgut crypts, the lumen of which is completely sealed off from the midgut main tract, thereby retaining the symbiont in the isolated cryptic cavities. We investigated histological, ecological, phylogenetic, and genomic aspects of the unique gut symbiosis of the acanthosomatid stinkbugs. Results: Phylogenetic analyses showed that the acanthosomatid symbionts constitute a distinct clade in the γ-Proteobacteria, whose sister groups are the obligate endocellular symbionts of aphids Buchnera and the obligate gut symbionts of plataspid stinkbugs Ishikawaella. In addition to the midgut crypts, the symbionts were located in a pair of peculiar lubricating organs associated with the female ovipositor, by which the symbionts are vertically transmitted via egg surface contamination. The symbionts were detected not from ovaries but from deposited eggs, and surface sterilization of eggs resulted in symbiont-free hatchlings. The symbiont-free insects suffered retarded growth, high mortality, and abnormal morphology, suggesting important biological roles of the symbiont for the host insects. The symbiont phylogeny was generally concordant with the host phylogeny, indicating host-symbiont co-speciation over evolutionary time despite the extracellular association. Meanwhile, some local host-symbiont phylogenetic discrepancies were found, suggesting occasional horizontal symbiont transfers across the host lineages. The symbionts exhibited AT-biased nucleotide composition, accelerated molecular evolution, and reduced genome size, as has been observed in obligate endocellular insect symbionts. Conclusion: Comprehensive studies of the acanthosomatid bacterial symbiosis provide new insights into the genomic evolution of extracellular symbiotic bacteria: host-symbiont co-speciation and drastic genome reduction can occur not only in endocellular symbiotic associations but also in extracellular ones. We suggest that many more such cases might be discovered in future surveys.

DOI： 10.1186/1741-7007-7-2

Language：English   Publishing type：Research paper (scientific journal)  

In parasitic associations, the evolutionary interest of a symbiont contradicts that of a host, which sometimes causes the phenomena so-called 'parasite manipulation' wherein symbiont infection alters host behaviour to facilitate its vertical/horizontal transmission. In mutualistic associations, meanwhile, symbiont-induced alteration of host behaviour that enhances its transmission has been little described. Here we report such a case in the stinkbug Megacopta punctatissima associated with an obligate gut bacterium. When female stinkbugs lay eggs, small particles called 'symbiont capsules' are deposited underside of the egg mass. Newborn nymphs immediately acquire the symbiont from the capsule, and then aggregate and become quiescent. By manipulating the levels of symbiont supply to newborn nymphs experimentally, we demonstrated that (i) experimental depletion of the symbiont resulted in the occurrence of wandering nymphs, (ii) the less symbiont supply, the more wandering nymphs, and (iii) almost all wandering nymphs were either symbiont-free or symbiont-depleted, whereas the majority of resting nymphs were infected with sufficient titres of the symbiont. These results strongly suggest that the nymphal behaviour is strongly influenced by the success/failure of the symbiont acquisition, thereby ensuring transmission of the essential symbiont and minimizing the energy and time spent for the activity.

DOI： 10.1098/rsbl.2007.0510

Language：English  

Symbiotic microorganisms are universally found in the gut, body cavity, or cells of a wide variety of insects. Some obligate symbionts are of a mutualistic nature and contribute to the fitness of their hosts, whereas other facultative symbionts are rather parasitic and tend to cause negative effects on their hosts (Bourtzis and Miller, 2003, 2006).

DOI： 10.1201/9781420064117

Language：English  

Many animals and plants establish symbiotic associations with benevolent microorganisms. In particular, most of insects that feed exclusively on restricted diets, such as plant sap, vertebrate blood or woody material, usually possess symbiotic microorganisms inside their body. Recent studies have suggested that more than 50% of insect species may be benefited from such endosymbiotic partners. Among these insects, members of the Heteroptera, known as true bugs or stinkbugs, exhibit an extraordinary diversity in their microbial symbiotic system. Such diversity is exemplified by: (i) physiological impact of the symbiotic associations on the host fitness ranging from parasitic to mutualistic; (ii) interdependence of the symbiotic associations ranging from facultative to obligate; (iii) location of the symbiont ranging from extracellular in gut cavity to intracellular in specialized cells; (iv) among the extracellular associations, specialized symbiotic structures such as midgut sacs and tubular outgrowths, whose cavity harbors specific symbiotic bacteria, varying in their number and arrangement; and (v) mechanisms for symbiont transmission ranging from vertical to environmental. Here, the current biological knowledge of the diverse stinkbug-bacteria symbioses is reviewed, which will provide novel insights into and useful model systems for understanding of the host-symbiont interactions and evolution.

Language：English   Publishing type：Research paper (scientific journal)  

Vertical symbiont transmission is among the most pivotal processes for maintenance of symbiotic associations. However, it is poorly understood whether and how the levels of resource allocation and investment upon vertical transmission are regulated. The stinkbug Megacopta punctatissima is obligatorily associated with the gut symbiotic bacterium 'Candidatus Ishikawaella capsulata', whose transmission is mediated by a unique mechanism called 'symbiont capsule'. We investigated the population dynamics of the symbiont during vertical transmission in the host-symbiont mutualism. The stinkbug mothers produced one capsule for around 3.6 eggs irrespective of clutch size, suggesting a strict maternal control over symbiont supply for the offspring. However, experimental manipulation of egg/capsule ratios revealed that one capsule is sufficient for symbiont transmission to six nymphs. Quantitative polymerase chain reaction analyses demonstrated that a capsule contains 1.2 × 10⁸ symbionts, a newborn nymph possesses 2 × 10 ⁷ symbionts from a capsule, and thus one capsule certainly contains a sufficient amount of symbiont cells for six nymphs. These results indicated that the stinkbug mothers produce 1.7 times more symbiont capsules than needed. The newborn nymphs consistently harboured around 2 × 10⁷ symbionts, also suggesting a nymphal control over symbiont transmission. The threshold symbiont titre minimally needed for successful vertical transmission was estimated to be 1.9 × 10⁶ symbionts, which is only 1/10 of the actual symbiont titre detected in a newborn nymph. These results illuminate several ecological factors that may be relevant to parental and offspring controls over symbiotic resource allocation through host insect generations.

DOI： 10.1111/j.1365-294X.2007.03592.x

Language：English   Publishing type：Research paper (scientific journal)  

The origin of specific insect genotypes that enable efficient use of agricultural plants is an important subject not only in applied fields like pest control and management but also in basic disciplines like evolutionary biology. Conventionally, it has been presupposed that such pest-related ecological traits are attributed to genes encoded in the insect genomes. Here, however, we report that pest status of an insect is principally determined by symbiont genotype rather than by insect genotype. A pest stinkbug species, Megacopta punctatissima, performed well on crop legumes, while a closely related non-pest species, Megacopta cribraria, suffered low egg hatch rate on the plants. When their obligate gut symbiotic bacteria were experimentally exchanged between the species, their performance on the crop legumes was, strikingly, completely reversed: the pest species suffered low egg hatch rate, whereas the non-pest species restored normal egg hatch rate and showed good performance. The low egg hatch rates were attributed to nymphal mortality before or upon hatching, which were associated with the symbiont from the non-pest stinkbug irrespective of the host insect species. Our finding sheds new light on the evolutionary origin of insect pests, potentially leading to novel approaches to pest control and management.

DOI： 10.1098/rspb.2007.0620

Language：English   Publishing type：Research paper (scientific journal)  

The broad-headed bug Riptortus clavatus (Heteroptera: Alydidae) possesses a number of crypts at a posterior midgut region, which house a dense population of a bacterial symbiont belonging to the genus Burkholderia. Although the symbiont is highly prevalent (95 to 100%) in the host populations, the symbiont phylogeny did not reflect the host systematics at all. In order to understand the mechanisms underlying the promiscuous host-symbiont relationship despite the specific and prevalent association, we investigated the transmission mode and the fitness effects of the Burkholderia symbiont in R. clavatus. Inspection of eggs and a series of rearing experiments revealed that the symbiont is not vertically transmitted but is environmentally acquired by nymphal insects. The Burkholderia symbiont was present in the soil of the insect habitat, and a culture strain of the symbiont was successfully isolated from the insect midgut Rearing experiments by using sterilized soybean bottles demonstrated that the cultured symbiont is able to establish a normal and efficient infection in the host insect, and the symbiont infection significantly improves the host fitness. These results indicated that R. clavatus postnatally acquires symbiont of a beneficial nature from the environment every generation, uncovering a previously unknown pathway through which a highly specific insect-microbe association is maintained. We suggest that the stinkbug-Burkholderia relationship may be regarded as an insect analogue of the well-known symbioses between plants and soil-associated microbes, such as legume-Rhizobium and alder-Frankia relationships, and we discuss the evolutionary relevance of the mutualistic but promiscuous insect-microbe association.

DOI： 10.1128/AEM.00067-07

Language：English   Publishing type：Research paper (scientific journal)  

We investigated the influence of the presence of large males on the mating tactics of small males of the stink bug Megacopta punctatissima (Montandon) (Hemiptera: Plataspidae) by using laboratory experiments. In the presence of large males, the mating acceptance rate of females (the proportion of copulating males to the number of courting males) and the mating success (the proportion of copulating males to the total number of males) of large males were significantly higher than those of small males. Therefore, sexual selection favors larger male body size at mating. Although only 15.4% of small males courted females in the presence of large males, 57.5% of small males courted females in the absence of large males. Consequently, the mating success of small males was conspicuously higher in the absence of large males than in the presence of large males. We suggest that small males adopt an alternative mating strategy in which their courtship behavior is the same as that of large males, but their decision to court females depends on the presence or absence of large rival males.

DOI： 10.1603/0013-8746(2006)99[974:AMSOSM]2.0.CO;2

Language：English   Publishing type：Research paper (scientific journal)  

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.

DOI： 10.1128/AEM.00803-06

Language：English   Publishing type：Research paper (scientific journal)  

Host-symbiont cospeciation and reductive genome evolution have been identified in obligate endocellular insect symbionts, but no such example has been identified from extracellular ones. Here we first report such a case in stinkbugs of the family Plataspidae, wherein a specific gut bacterium is vertically transmitted via "symbiont capsule." In all of the plataspid species, females produced symbiont capsules upon oviposition and their gut exhibited specialized traits for capsule production. Phylogenetic analysis showed that the plataspid symbionts constituted a distinct group in the γ-Proteobacteria, whose sister group was the aphid obligate endocellular symbionts Buchnera. Removal of the symbionts resulted in retarded growth, mortality, and sterility of the insects. The host phylogeny perfectly agreed with the symbiont phylogeny, indicating strict host-symbiont cospeciation despite the extracellular association. The symbionts exhibited AT-biased nucleotide composition, accelerated molecular evolution, and reduced genome size, as has been observed in obligate endocellular insect symbionts. These findings suggest that not the endocellular conditions themselves but the population genetic attributes of the vertically transmitted symbionts are probably responsible for the peculiar genetic traits of these insect symbionts. We proposed the designation "Candidatus Ishikawaella capsulata" for the plataspid symbionts. The plataspid stinkbugs, wherein the host-symbiont associations can be easily manipulated, provide a novel system that enables experimental approaches to previously untouched aspects of the insect-microbe mutualism. Furthermore, comparative analyses of the sister groups, the endocellular Buchnera and the extracellular Ishikawaella, would lead to insights into how the different symbiotic lifestyles have affected their genomic evolution.

DOI： 10.1371/journal.pbio.0040337

Language：English   Publishing type：Research paper (scientific journal)  

In stinkbugs of the family Plataspidae, adult females deposit small brownish particles, containing specific symbiotic bacteria inside, on the underside of their egg mass. Newborn nymphs ingest the content of the unique structure, called "symbiont capsule", whereby vertical transmission of the symbiont occurs. We investigated the fine structure and the formation process of the symbiont capsule in the Japanese common plataspid stinkbug, Megacopta punctatissima, by using light and electron microscopy. It was demonstrated that (i) the capsule consists of three structural components, namely "symbionts", "matrix" and "envelope"; (ii) the posterior midgut of adult females is characterized by several specific sections with peculiar anatomical traits, including "thin crypt-bearing midgut (TCM) section", "swollen crypt-bearing midgut (SCM) section" and "brownish enlarged midgut (BEM) end section"; (iii) the different capsule components, symbionts, matrix and envelope, are produced and/or supplied by the specialized midgut sections, TCM, SCM and BEM, respectively; and (iv) the capsule components are stored in BEM and excreted during oviposition to produce the symbiont capsules. These results strongly suggested that the host insect incurs a substantial cost for the symbiont transmission. Ecological and evolutionary implications of the highly developed, female-specific system for symbiont transmission were discussed.

DOI： 10.1016/j.femsec.2005.06.002

Language：English   Publishing type：Research paper (scientific journal)  

The Japanese common plataspid stinkbug, Megacopta punctatissima, deposits small brown particles, or symbiont capsules, on the underside of the egg mass for the purpose of transmission of symbiotic bacteria to the offspring. We investigated the microbiological aspects of the bacteria contained in the capsule, such as microbial diversity, phylogenetic placement, localization in vivo, and fitness effects on the host insect. Restriction fragment length polymorphism analysis of 16S ribosomal DNA clones revealed that a single bacterial species dominates the microbiota in the capsule. The bacterium was not detected in the eggs but in the capsules, which unequivocally demonstrated that the bacterium is transmitted to the offspring of the insect orally rather than transovarially, through probing of the capsule content. Molecular phylogenetic analysis showed that the bacterium belongs to the γ-subdivision of the Proteobacteria. In adult insects the bacterium was localized in the posterior section of the midgut. Deprivation of the bacterium from the nymphs resulted in retarded development, arrested growth, abnormal body coloration, and other symptoms, suggesting that the bacterium is essential for normal development and growth of the host insect.

DOI： 10.1128/AEM.68.1.389-396.2002

Language：English   Publishing type：Research paper (scientific journal)  

Copulation duration and daily reproductive activity pattern of the stink bug Megacopta punctatissima Montandon were investigated in the laboratory and the field. Mean copulation duration was >10 h regardless of sex ratio and few copulations continued for more than 24 h in the laboratory. In the field, mating aggregations and copulations were found from 1400 to 1000 hours on the next day, but bugs tended not to copulate from 1000 to 1400 hours. This daily reproductive activity pattern seemed to restrict the copulation duration to about 24 h at the longest. Sperm transfer from a male to a female spermatheca was complete within 2-4 h. Thus, it appears that copulations over 4 h serve as postcopulatory mate guarding. However, copulations did not always continue until pviposition because female oviposited after about a 7-d interval. Our findings suggest the possibility that prolonged copulations that do not continue until oviposition may also function as postcopulatory mate guarding within daily reproductive time. Alternative hypotheses, such as copulatory courship, for prolonged copulation of M. punctatissisma are also discussed.

DOI： 10.1603/0013-8746(2001)094[0750:SOPCUT]2.0.CO;2

Language：English   Publishing type：Research paper (scientific journal)  

In the stink bug, Megacopta punctatissima, which forms mating aggregations, male mating behaviors and the copulatory success of males were examined in experimental populations with 4 different sex ratios. In the male-biased sex ratio, more males tended to stay in "copulatory aggregations" including at least a mating pair rather than in "non-copulatory aggregations" including no mating pair, despite a scarcity of non-copulating females in such aggregations. However, when copulating females were included, there were more females in the copulatory aggregations than those in the non-copulatory aggregations. Therefore, the results appear to suggest that males also regarded copulating females as potential mates. Males which had immigrated to an aggregation showed a higher copulatory success than resident males in the aggregation at a male-biased sex ratio. Moreover, males showed a large variation in mating success, and those which had a higher mating success tended to stay in aggregations of female-biased sex ratios. These results suggest that females may choose their mates in mating aggregations, and that the choice may influence male mating success more acutely in a male-biased sex ratio.

DOI： 10.1303/aez.2000.93

Event date： 2025.3

Language：Japanese  

Venue：幕張メッセ（千葉市）   Country：Japan  

Event date： 2025.3

Language：Japanese  

Venue：幕張メッセ（千葉市）   Country：Japan  

Event date： 2025.3

Language：Japanese  

Venue：幕張メッセ（千葉市）   Country：Japan  

Event date： 2025.3

Language：Japanese  

Venue：幕張メッセ（千葉市）   Country：Japan  

Event date： 2024.3

Language：Japanese  

Venue：仙台国際センター（仙台市）   Country：Japan  

Event date： 2024.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン（横浜市）   Country：Japan  

Event date： 2023.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：熊本大学（熊本市）   Country：Japan  

Event date： 2023.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：Japan  

Event date： 2023.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：枚方   Country：Japan  

Event date： 2023.2

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：つくば   Country：Japan  

Event date： 2022.6

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

ツノカメムシ科ではメス親が上に覆いかぶさる形で卵塊を保護する。この保護行動の適応的意義に関する定量的な研究はElasmucha属のみを対象としており、他属においては保護行動の断片的な報告しかない。最近の分子系統解析によって、ツノカメムシ科におけるメス親による子の保護は属ごとに独立に進化したことが明らかにされている。したがってElasmucha属以外の保護行動の適応的意義には再検討の余地がある。本研究ではSastragala属のエサキモンキツノカメムシにおける卵塊保護の適応的意義の解明および卵の捕食者の特定をおこなった。野外集団において卵塊からメス親を除去したところ、メス親を除去しない場合に比べて卵の生存率が有意に低くなった。また、メス親除去に加えて徘徊性捕食者を排除した実験では卵の生存率が有意に高くなった。メス親を除去した卵塊では卵の消失および損傷が確認され、その一因としてアリ類およびハナカメムシ類による捕食が観察された。また、今回の調査では卵寄生蜂による寄生はまったく観察されなかった。実験室内での行動観察では、メス親の体の傾けや羽ばたきがアリ類に対する防衛に有効であることが明らかとなった。

Event date： 2022.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2022.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2020.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2020.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名城大学   Country：Japan  

Event date： 2020.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名城大学   Country：Japan  

Event date： 2020.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名城大学   Country：Japan  

Event date： 2019.3 - 2019.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：筑波大学   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：筑波大学   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京大学   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京大学   Country：Japan  

Event date： 2018.5

Language：Japanese  

Country：Japan  

Event date： 2018.5

Language：Japanese  

Country：Japan  

Event date： 2017.3 - 2018.5

Language：Japanese  

Country：Japan  

Event date： 2015.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：山形   Country：Japan  

多くの昆虫類において親は子の生存率を高めるためにさまざまな方法で子の世話をする。カメムシ類では卵の保護、栄養卵の供給、必須共生細菌を含む分泌物の供給、子への給餌などが知られている。本講演ではノコギリカメムシにおいて新たに発見した非常にユニークなメス親の行動について報告する。本種の後脚脛節には性的二型が見られ、メスではその一部が幅広く発達していた。また、この拡幅部位には性成熟にともなって菌類の菌糸束が見られるようになった。産卵時の行動を観察したところ、メス親は卵を産んだ後に後脚脛節上の菌を反対側の後脚のふ節でかきとって卵に付けた。この行動は左右交互に約2分間にわたって続き、卵を一個産下するたびに必ず繰り返された。菌を接種された卵の表面からは数日のうちにおびただしい数の菌糸が伸長して最終的には卵塊全体を被うまでに成長した。一方、メスの後脚脛節を切除することによって菌の接種を妨げると卵から菌糸が伸長することはなかった。分子系統解析の結果から、共生菌はバッカクキン科に属する子嚢菌であることが明らかになった。このメス親の行動が子の世話として機能している可能性について議論する。

Type：Competition, symposium, etc. 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：1

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：1

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：2

Type：Competition, symposium, etc. 

Number of participants：109