Burkholderia insecticola sp. nov., a gut symbiotic bacterium of the bean bug Riptortus pedestris

A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64(T), was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei Glade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314(T) (100%), Burkholderia turbans LMG 29316(T) (99.52%) and Burkholderia ptereochthonis LMG 29326(T) (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome BLAST Distance Phylogeny confirmed B. peredens LMG 29314(T), B. ptereochthonis LMG 29326(T) and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA-DNA hybridization experiments unambiguously demonstrated that strain RPE64(T) represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C-16:0, C-18:1 omega 7c and C-17:0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64(T) from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64(T) is classified as representing a novel species of the genus Burkholderia, for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64(T) (=NCIMB 15023(T)=JCM 31142(T))

Burkholderia insecticola triggers midgut closure in the bean bug Riptortus pedestris to prevent secondary bacterial infections of midgut crypts

In addition to abiotic triggers, biotic factors such as microbial symbionts can alter development of multicellular organisms. Symbiont-mediated morphogenesis is well-investigated in plants and marine invertebrates but rarely in insects despite the enormous diversity of insect-microbe symbioses. The bean bug Riptortus pedestris is associated with Burkholderia insecticola which are acquired from the environmental soil and housed in midgut crypts. To sort symbionts from soil microbiota, the bean bug develops a specific organ called the "constricted region" (CR), a narrow and symbiont-selective channel, located in the midgut immediately upstream of the crypt-bearing region. In this study, inoculation of fluorescent protein-labeled symbionts followed by spatiotemporal microscopic observations revealed that after the initial passage of symbionts through the CR, it closes within 12-18 h, blocking any potential subsequent infection events. The "midgut closure" developmental response was irreversible, even after symbiont removal from the crypts by antibiotics. It never occurred in aposymbiotic insects, nor in insects infected with nonsymbiotic bacteria or B. insecticola mutants unable to cross the CR. However, species of the genus Burkholderia and its outgroup Pandoraea that can pass the CR and partially colonize the midgut crypts induce the morphological alteration, suggesting that the molecular trigger signaling the midgut closure is conserved in this bacterial lineage. We propose that this drastic and quick alteration of the midgut morphology in response to symbiont infection is a mechanism for stabilizing the insect-microbe gut symbiosis and contributes to host-symbiont specificity in a symbiosis without vertical transmission

Burkholderia Gut Symbionts Associated with European and Japanese Populations of the Dock Bug Coreus marginatus (Coreoidea: Coreidae)

International audienceInsects of the heteropteran superfamilies Coreoidea and Lygaeoidea are consistently associated with symbionts of a specific group of the genus Burkholderia, called the "stinkbug-associated beneficial and environmental (SBE)" group. The symbiosis is maintained by the environmental transmission of symbionts. We investigated European and Japanese populations of the dock bug Coreus marginatus (Coreoidea: Coreidae). High nymphal mortality in reared aposymbiotic insects suggested an obligate host-symbiont association in this species. Molecular phylogenetic analyses based on 16S rRNA gene sequences revealed that all 173 individuals investigated were colonized by Burkholderia, which were further assigned to different subgroups of the SBE in a region-dependent pattern

A Peptidoglycan Amidase Mutant of Burkholderia insecticola Adapts an L-form-like Shape in the Gut Symbiotic Organ of the Bean Bug Riptortus pedestris

International audienceBacterial cell shapes may be altered by the cell cycle, nutrient availability, environmental stress, and interactions with other organisms. The bean bug Riptortus pedestris possesses a symbiotic bacterium, Burkholderia insecticola, in its midgut crypts. This symbiont is a typical rod-shaped bacterium under in vitro culture conditions, but changes to a spherical shape inside the gut symbiotic organ of the host insect, suggesting the induction of morphological alterations in B. insecticola by host factors. The present study revealed that a deletion mutant of a peptidoglycan amidase gene (amiC), showing a filamentous chain form in vitro, adapted a swollen L-form-like cell shape in midgut crypts. Spatiotemporal observations of the ΔamiC mutant in midgut crypts revealed the induction of swollen cells, particularly prior to the molting of insects. To elucidate the mechanisms underlying in vivo-specific morphological alterations, the symbiont was cultured under 13 different conditions and its cell shape was examined. Swollen cells, similar to symbiont cells in midgut crypts, were induced when the mutant was treated with fosfomycin, an inhibitor of peptidoglycan precursor biosynthesis. Collectively, these results strongly suggest that the Burkholderia symbiont in midgut crypts is under the control of the host insect via a cell wall-attacking agent

Dual oxidase enables insect gut symbiosis by mediating respiratory network formation

International audienceMost animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris , Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola . Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects

Host-symbiont specificity determined by microbe-microbe competition in an insect gut

Despite the omnipresence of specific host-symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont

Intercontinental diversity of <i>Caballeronia</i> gut symbionts in the conifer pest bug <i>Leptoglossus occidentalis</i>

International audienceMany stinkbugs in the superfamily Coreoidea (Hemiptera: Heteroptera) develop crypts in the posterior midgut, harboringCaballeronia (Burkholderia) symbionts. These symbionts form a monophyletic group in Burkholderia sensu lato, called the“stinkbug-associated beneficial and environmental (SBE)” group, recently reclassified as the new genus Caballeronia. SBEsymbionts are separated into the subclades SBE-α and SBE-β. Previous studies suggested a regional effect on the symbiontinfection pattern; Japanese and American bug species are more likely to be associated with SBE-α, while European bugspecies are almost exclusively associated with SBE-β. However, since only a few insect species have been investigated, itremains unclear whether region-specific infection is general. We herein investigated Caballeronia gut symbionts in diverseJapanese, European, and North American populations of a cosmopolitan species, the Western conifer seed bug Leptoglossusoccidentalis (Coreoidea: Coreidae). A molecular phylogenetic analysis of the 16S rRNA gene demonstrated that SBE-βwas the most dominant in all populations. Notably, SBE-α was rarely detected in any region, while a third clade, the“Coreoidea clade” occupied one fourth of the tested populations. Although aposymbiotic bugs showed high mortality, SBEα- and SBE-β-inoculated insects both showed high survival rates; however, a competition assay demonstrated that SBE-βoutcompeted SBE-α in the midgut crypts of L. occidentalis. These results strongly suggest that symbiont specificity inthe Leptoglossus-Caballeronia symbiotic association is influenced by the host rather than geography, while the geographicdistribution of symbionts may be more important in other bugs

Transposon sequencing reveals the essential gene set and genes enabling gut symbiosis in the insect symbiont Caballeronia insecticola

Abstract Caballeronia insecticola is a bacterium belonging to the Burkholderia genus sensu lato , able to colonize multiple environments like soils and the gut of the bean bug Riptortus pedestris . To identify the essential genome of a bacterium is a first step in the understanding of its lifestyles. We constructed a saturated Himar1 mariner transposon library and revealed by transposon-sequencing (Tn-seq) that 498 protein-coding genes constitute the essential genome of C. insecticola for growth in free-living conditions. By comparing essential gene sets of C. insecticola and seven related Burkholderia s.l. strains, only 120 common genes were identified indicating that a large part of the essential genome is strain-specific. In order to reproduce specific nutritional conditions that are present in the gut of R. pedestris , we grew the mutant library in minimal media supplemented with candidate gut nutrients and identified several condition-dependent fitness-defect genes by Tn-seq. To validate the robustness of the approach, insertion mutants in six fitness genes were constructed and their growth-deficiency in media supplemented with the corresponding nutrient was confirmed. The mutants were further tested for their efficiency in R. pedestris gut colonization, confirming that gluconeogenic carbon sources, taurine and inositol, are nutrients consumed by the symbiont in the gut

Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture

International audienceIn 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