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Microbial symbiosis is widespread among insects. This article reviews our understanding of insect behaviors relevant to commensalistic and mutualistic microbial symbiosis, which has received relatively less attention compared to insect behaviors in parasitic symbiosis. First, we review our knowledge of symbiont transmission behaviors by which the host insects maintain associations with beneficial microorganisms over generations. Some insects that extracellularly harbor mutualistic symbionts exhibit particularly sophisticated behaviors for vertical symbiont transmission. Next, we highlight notable studies on behavioral changes induced by symbiont infection. In the last decade, a number of studies have demonstrated or suggested that mutualistic or commensalistic symbiont infections affect their host behaviors. Finally, future directions regarding these research topics are discussed.

Diverse insects are obligatorily associated with microbial symbionts, wherein the host often develops special symbiotic organs and vertically transmits the symbiont to the next generation. What molecular factors underpin the host-symbiont relationship is of great interest but poorly understood. Here we report a novel protein preferentially produced in a female-specific symbiotic organ of the stinkbug Plautia stali, whose posterior midgut develops numerous crypts to host a Pantoea-allied bacterial mutualist. In adult females, several posteriormost crypts are conspicuously enlarged, presumably specialized for vertical symbiont transmission. We detected conspicuous protein bands specific to the female’s swollen crypts by gel electrophoresis, and identified them as representing a novel mucin-like glycoprotein. Histological inspections confirmed that the mucin protein is localized to the female’s swollen crypts, coexisting with a substantial population of the symbiotic bacteria, and excreted from the swollen crypts to the midgut main tract together with the symbiotic bacteria. Using RNA interference, we successfully suppressed production of the mucin protein in adult females of P. stali. However, although the mucin protein was depleted, the symbiont population persisted in the swollen crypts, and vertical symbiont transmission to the next generation occurred. Possible biological roles and evolutionary trajectory of the symbiosis-related mucin protein are discussed.

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.

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

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

The genome of “Candidatus Regiella insecticola” strain TUt, a facultative bacterial symbiont of the pea aphid Acyrthosiphon pisum, was analyzed. We determined a 2.5-Mb draft genome consisting of 14 contigs; this will contribute to the understanding of the symbiont, which underpins various ecologically adaptive traits of the host insect.

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. Japan Society for the Promotion of Science (JSPS) KAKENHIMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of ScienceGrants-in-Aid for Scientific Research (KAKENHI) [16H04722, 19H03212, JP17H06388]; JST CRESTJapan Science & Technology Agency (JST)Core Research for Evolutional Science and Technology (CREST) [JPMJCR18S7] This work was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 16H04722 and 19H03212 to TK and JP17H06388 to TF, and JST CREST Grant Number JPMJCR18S7 to TK. WOS:000536697300001 2-s2.0-85085620916 PubMed: 32435239

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

Host-microbe symbioses often evolved highly complex developmental processes and colonization mechanisms for establishment of stable associations. It has long been recognized that many insects harbour beneficial bacteria inside specific symbiotic cells (bacteriocytes) or organs (bacteriomes). However, the evolutionary origin and mechanisms underlying bacterial colonization in bacteriocyte/bacteriome formation have been poorly understood. In order to uncover the origin of such evolutionary novelties, we studied the development of symbiotic organs in five stinkbug species representing the superfamily Lygaeoidea in which diverse bacteriocyte/bacteriome systems have evolved. We tracked the symbiont movement within the eggs during the embryonic development and determined crucial stages at which symbiont infection and bacteriocyte formation occur, using whole-mount fluorescence in situ hybridization. In summary, three distinct developmental patterns were observed: two different modes of symbiont transfer from initial symbiont cluster (symbiont ball) to presumptive bacteriocytes in the embryonic abdomen, and direct incorporation of the symbiont ball without translocation of bacterial cells. Across the host taxa, only closely related species seemed to have evolved relatively conserved types of bacteriome development, suggesting repeated evolution of host symbiotic cells and organs from multiple independent origins.

“Candidatus Serratia symbiotica” is a facultative bacterial symbiont of aphids that affects various ecological traits of the host insects. Here, we report the complete genome sequence of “Candidatus Serratia symbiotica” strain IS, consisting of a 2,736,352-bp chromosome and an 82,605-bp plasmid, from the pea aphid Acyrthosiphon pisum.

Roughly 10% to 15% of insect species host heritable symbiotic bacteria known as endosymbionts. The lice parasitizing mammals rely on endosymbionts to provide essential vitamins absent in their blood meals. Here, we describe two bacterial associates from a louse, Proechinophthirus fluctus , which is an obligate ectoparasite of a marine mammal. One of these is a heritable endosymbiont that is not closely related to endosymbionts of other mammalian lice. Rather, it is more closely related to endosymbionts of the genus Sodalis associated with spittlebugs and feather-chewing bird lice. Localization and vertical transmission of this endosymbiont are also more similar to those of bird lice than to those of other mammalian lice. The endosymbiont genome appears to be degrading in symbiosis; however, it is considerably larger than the genomes of other mammalian louse endosymbionts. These patterns suggest the possibility that this Sodalis endosymbiont might be recently acquired, replacing a now-extinct, ancient endosymbiont. From the same lice, we also identified an abundant bacterium belonging to the genus Rickettsia that is closely related to Rickettsia ricketsii , a human pathogen vectored by ticks. No obvious masses of the Rickettsia bacterium were observed in louse tissues, nor did we find any evidence of vertical transmission, so the nature of its association remains unclear. IMPORTANCE Many insects are host to heritable symbiotic bacteria. These heritable bacteria have been identified from numerous species of parasitic lice. It appears that novel symbioses have formed between lice and bacteria many times, with new bacterial symbionts potentially replacing existing ones. However, little was known about the symbionts of lice parasitizing marine mammals. Here, we identified a heritable bacterial symbiont in lice parasitizing northern fur seals. This bacterial symbiont appears to have been recently acquired by the lice. The findings reported here provide insights into how new symbioses form and how this lifestyle is shaping the symbiont genome.

International audience; In the symbiosis of the bean bug Riptortus pedestris with Burkholderia insecticola, the bacteria occupy an exclusive niche in the insect midgut and favor insect development and reproduction. In order to understand how the symbiotic bacteria stably colonize the midgut crypts and which services they provide to the host, we compared the cytology, physiology, and transcriptomics of free-living and midgut-colonizing B. insecticola. The analyses revealed that midgut-colonizing bacteria were smaller in size and had lower DNA content, they had increased stress sensitivity, lost motility, and an altered cell surface. Transcriptomics revealed what kinds of nutrients are provided by the bean bug to the Burkholderia symbiont. Transporters and metabolic pathways of diverse sugars such as rhamnose and ribose, and sulfur compounds like sulfate and taurine were upregulated in the midgut-colonizing symbionts. Moreover, pathways enabling the assimilation of insect nitrogen wastes, i.e. allantoin and urea, were also upregulated. The data further suggested that the midgut-colonizing symbionts produced all essential amino acids and B vitamins, some of which are scarce in the soybean food of the host insect. Together, these findings suggest that the Burkholderia symbiont is fed with specific nutrients and also recycles host metabolic wastes in the insect gut, and in return, the bacterial symbiont provides the host with essential nutrients limited in the insect food, contributing to the rapid growth and enhanced reproduction of the bean bug host.

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.

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.

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

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

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.

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.

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 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.

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 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.

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.

Many insects are ubiquitously associated with multiple endosymbionts, whose infection patterns often exhibit spatial and temporal variations. How such endosymbiont variations are relevant to local adaptation of the host organisms is of ecological interest. Here, we report a comprehensive survey of endosymbionts in natural populations of the chestnut weevil Curculio sikkimensis, whose larvae are notorious pests of cultivated chestnuts and also infest acorns of various wild oaks. From 968 insects representing 55 localities across the Japanese Archipelago and originating from 10 host plant species, we identified six distinct endosymbiont lineages, namely Curculioniphilus, Sodalis, Serratia, Wolbachia, Rickettsia and Spiroplasma, at different infection frequencies (96.7%, 12.8%, 82.3%, 82.5%, 28.2% and 6.8%, respectively) and with different geographical distribution patterns. Multiple endosymbiont infections were very common; 3.18±0.61 (ranging from 1.74 to 5.50) endosymbionts per insect on average in each of the local populations. Five pairs of endosymbionts (Curculioniphilus-Serratia, Curculioniphilus-Wolbachia, Sodalis-Rickettsia, Wolbachia-Rickettsia and Rickettsia-Spiroplasma) co-infected the same host individuals more frequently than expected, while infections with Serratia and Wolbachia were negatively correlated to each other. Infection frequencies of the endosymbionts were significantly correlated with climatic and ecological factors: for example, higher Sodalis, Wolbachia and Rickettsia infections at localities of higher temperature; lower Wolbachia and Rickettsia infections at localities of greater snowfall; and higher Curculioniphilus, Sodalis, Serratia, Wolbachia and Rickettsia infections on acorns than on chestnuts. These patterns are discussed in relation to potential host-endosymbiont co-evolution via local adaptation across geographical populations.

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.

We explored the origin of all-female broods resulting from male death in a Hokkaido population of Lymantria dispar through genetic crosses based on the earlier experiments done by Goldschmidt and by testing for the presence of endosymbionts that are known to cause male killing in some insect species. The mitochondrial DNA haplotypes of the all-female broods in Hokkaido were different from those of normal Hokkaido females and were the same as those widely distributed in Asia, including Tokyo (TK). Goldschmidt obtained all-female broods through backcrossing, that is, F1 females obtained by a cross between TK females (L. dispar japonica) and Hokkaido males (L. dispar praeterea) mated with Hokkaido males. He also obtained all-male broods by mating Hokkaido females with TK males. Goldschmidt inferred that female- and male-determining factors were weakest in the Hokkaido subspecies and stronger in the Honshu (TK) subspecies. According to his theory, the females of all-female broods mated with Honshu males should produce normal sex-ratio broods, whereas weaker Hokkaido sexes would be expected to disappear in F1 or F2 generations after crossing with the Honshu subspecies. We confirmed both of Goldschmidt's results: in the case of all-female broods mated with Honshu males, normal sex-ratio broods were produced, but we obtained only all-female broods in the Goldschmidt backcross and obtained an all-male brood in the F1 generation of a Hokkaido female crossed with a TK male. We found no endosymbionts in all-female broods by 4,′6-diamidino-2-phenylindole (DAPI) staining. Therefore, the all-female broods observed in L. dispar are caused by some incompatibilities between Honshu and Hokkaido subspecies.

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.

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.

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 from all 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 stem cell 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.

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.

We investigated the infection dynamics of endosymbiotic bacteria in the developmental course of the mealybugs Planococcus kraunhiae and Pseudococcus comstocki . Molecular phylogenetic analyses identified a betaproteobacterium and a gammaproteobacterium from each of the mealybug species. The former bacterium was related to the β-endosymbionts of other mealybugs, i.e., “ Candidatus Tremblaya princeps,” and formed a compact clade in the Betaproteobacteria . Meanwhile, the latter bacterium was related to the γ-endosymbionts of other mealybugs but belonged to distinct clades in the Gammaproteobacteria . Whole-mount in situ hybridization confirmed the peculiar nested formation in the endosymbiotic system of the mealybugs: the β-endosymbiont cells were present in the cytoplasm of the bacteriocytes, and the γ-endosymbiont cells were located in the β-endosymbiont cells. In nymphal and female development, a large oval bacteriome consisting of a number of bacteriocytes was present in the abdomen, wherein the endosymbionts were harbored. In male development, strikingly, the bacteriome progressively degenerated in prepupae and pupae and became almost unrecognizable in adult males. In the degeneration process, the γ-endosymbionts disappeared more rapidly than the β-endosymbionts did. Quantitative PCR analyses revealed that (i) the population dynamics of the endosymbionts in female development reflected the reproductive activity of the insects, (ii) the population dynamics of the endosymbionts were strikingly different between female development and male development, (iii) the endosymbiont populations drastically decreased in male development, and (iv) the γ-endosymbiont populations decreased more rapidly than the β-endosymbiont populations in male development. Possible mechanisms underlying the uncoupled regulation of the β- and γ-endosymbiont populations are discussed in relation to the establishment and evolution of this unique prokaryote-prokaryote endosymbiotic system.

Bacteria of the genus Wolbachia are intracellular symbionts that are found in a broad range of insects and cause a variety of reproductive alterations, including male killing, parthenogenesis, feminization and cytoplasmic incompatibility. Although Wolbachia has not been cultured outside its host cells, it can be co-cultured with insect cell lines in vitro, and this technique would facilitate studies on Wolbachia. In this study, we inoculated five Wolbachia strains into two insect cell lines to examine the ability of the cell lines to harbor different infections. The Wolbachia strains used were wKue, derived from Ephestia kuehniella, wCauA and wCauB, from Cadra cautella, and wMel and wMelPop, from Drosophila melanogaster. The cell lines used were Sf9 of Spodoptera frugiperda (Lepidoptera) and AeAl2 of Aedes albopictus (Diptera). Immunofluorescence staining with an antibody against Wolbachia surface protein showed that wCauA was successfully maintained in both AeAl2 and Sf9 cells and that wCauB and wKue were maintained in AeAl2 cells. The infection levels, determined by real-time quantitative PCR targeting the Wolbachia 16S rRNA gene, varied among different infections.

The current study focuses on a symbiotic bacterium found in the slender pigeon louse, Columbicola columbae (Insecta: Phthiraptera). Molecular phylogenetic analyses indicated that the symbiont belongs to the gamma subdivision of the class Proteobacteria and is allied to Sodalis glossinidius , the secondary symbiont of tsetse flies ( Glossina spp.) and also to the primary symbiont of grain weevils ( Sitophilus spp.). Relative-rate tests revealed that the symbiont of C. columbae exhibits accelerated molecular evolution in comparison with the tsetse fly symbiont and the weevil symbiont. Whole-mount in situ hybridization was used to localize the symbiont and determine infection dynamics during host development. In first- and second-instar nymphs, the symbionts were localized in the cytoplasm of oval bacteriocytes that formed small aggregates on both sides of the body cavity. In third-instar nymphs, the bacteriocytes migrated to the central body and were finally located in the anterior region of the lateral oviducts, forming conspicuous tissue formations called ovarial ampullae. In adult females, the symbionts were transmitted from the ovarial ampullae to developing oocytes in the ovarioles. In adult males, the bacteriocytes often disappeared without migration. A diagnostic PCR survey of insects collected from Japan, the United States, Australia, and Argentina detected 96.5% (109/113) infection, with a few uninfected male insects. This study provides the first microbial characterization of a bacteriocyte-associated symbiont from a chewing louse. Possible biological roles of the symbiont are discussed in relation to the host nutritional physiology associated with the feather-feeding lifestyle.

When the butterfly Eurema hecabe is infected with two different strains ( w HecCI2 and w HecFem2) of the bacterial endosymbiont Wolbachia , genetic males are transformed into functional females, resulting in production of all-female broods. In an attempt to understand how and when the Wolbachia endosymbiont feminizes genetically male insects, larval insects were fed an antibiotic-containing diet beginning at different developmental stages until pupation. When the adult insects emerged, strikingly, many of them exhibited sexually intermediate traits in their wings, reproductive organs, and genitalia. The expression of intersexual phenotypes was strong in the insects treated from first instar, moderate in the insects treated from third instar, and weak in the insects treated from fourth instar. The insects treated from early larval instar grew and pupated normally but frequently failed to emerge and died in the pupal case. The dead insects in the pupal case contained lower densities of the feminizing Wolbachia endosymbiont than the successfully emerged insects, although none of them were completely cured of the symbiont infection. These results suggest the following: (i) the antibiotic treatment suppressed the population of feminizing Wolbachia endosymbionts; (ii) the suppression probably resulted in attenuated feminizing activity of the symbiont, leading to expression of intersexual host traits; (iii) many of the insects suffered pupal mortality, possibly due to either intersexual defects or Wolbachia -mediated addiction; and hence (iv) the feminizing Wolbachia endosymbiont continuously acts on the host insects during larval development for expression of female phenotypes under a male genotype. Our finding may prompt reconsideration of the notion that Wolbachia -induced reproductive manipulations are already complete before the early embryonic stage and provide insights into the mechanism underlying the symbiont-induced reversal of insect sex.

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.

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host–symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ’s ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host’s control and symbiont’s motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria . The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation “ Candidatus Riesia pediculicola” for the louse symbionts.

In Japanese populations of the pea aphid Acyrthosiphon pisum, four types of facultative endosymbiotic bacteria, Serratia symbiotica, Regiella insecticola, Rickettsia sp. and Spiroplasma sp., have been identified in addition to the essential endosymbiont Buchnera aphidicola. On the vetch Vicia sativa and other leguminous plants, Aphis craccivora, Megoura crassicauda and A. pisum frequently co-occur and form mixed colonies. In this study, the endosymbiotic microbiotae in A. craccivora and M. crassicauda collected from different localities in Japan were investigated in comparison with the endosymbiotic microbiotae in sympatric samples of A. pisum. While the A. pisum samples frequently contained one or more of the four symbionts, the A. craccivora samples were infected with only Serratia and the M. crassicauda samples contained none of the facultative symbionts. When the four symbionts were artificially transferred from A. pisum to M. crassicauda by hemolymph injection, Regiella and Spiroplasma established heritable infections in the novel host while Serratia and Rickettsia did not. The Regiella infection in M. crassicauda was stably maintained with 100% fidelity for over 50 generations, whereas the Spiroplasma infection was unstable. On the basis of these results, we discussed the possible mechanisms whereby the different infection patterns with the facultative symbionts are maintained in the sympatric aphid communities.