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.B vitamins | bacteriome | Cimex lectularius | nutritional mutualism

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

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 hostsymbiont 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.

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.

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 b-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.

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.

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.

The Japanese common broad-headed bugs Riptortus clavatus and Leptocorisa chinensis possess a number of crypts in the posterior region of the midgut, whose lumen contains a copious amount of bacterial cells. We characterized the gut symbiotic bacteria by using molecular phylogenetic analysis, light and electron microscopy, in situ hybridization, and PCR-based detection techniques. Restriction fragment length polymorphism analysis of 16S rRNA gene clones suggested that a single bacterium dominated the microbiota in the crypts of the both bug species. The predominant 16S rRNA gene sequences obtained from different individuals and species of the bugs were not identical but were very similar to each other. Homology searches in the DNA databases revealed that the sequences showed the highest levels of similarity (96% to 99%) to the sequences of Burkholderia spp. belonging to the ␤ subdivision of the class Proteobacteria. In situ hybridization with specific oligonucleotide probes confirmed the localization of the Burkholderia symbiont in the lumen of the midgut crypts. Electron microscopy showed that the lumen of the crypts was filled with rod-shaped bacteria of a single morphotype. Molecular phylogenetic analysis demonstrated that the Burkholderia symbionts of the bugs formed a well-defined monophyletic group, although the group also contained several environmental Burkholderia strains. The phylogenetic relationship of the Burkholderia symbionts did not reflect the relationship of the host bug species at all. The sequences from R. clavatus and the sequences from L. chinensis did not form clades but were intermingled in the phylogeny, suggesting that horizontal transmission of the symbiont might have occasionally occurred between populations and species of the bugs.