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International audience; Many 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.

Figure S1: Phylogenetic placcement of Burkholderia strains used in this study, shown by a maximum likelihood tree. Bold letters indicate strains used for the infection experiment in this study. Stars indicate fenitrothion-degrading strains. B. insecticola RPE64 is mother strain of GFP-labelled strain, RPE225, used in this study. Bootstrap values higher than 70% are depicted at the nodes. Pandoraea norimbergensis LMG 18106 was used as an outgroup.; Figure S2: Fenitrothion-degrading ability of gut-colonizing Burkholderia (a) in the fenitrothion emulsion-containing liquid solution or (b) on minimal agar plate. (a) The optical density of 3-methyl-4-nitrophenol measured by a spectrophotometer at 405nm. (b) The maximum diameter of halo. The statistical significance was analyzed by the t-test with Bonferroni correction. n=10 insects were used in each group. Averages ± SD values are indicated in each bar; Table S1: Burkholderia symbiont strains used in this study.

International audience; How host organisms evolved and maintain specific mutualisms with microorganisms is a fundamental question that is subject to intensive research. In the large majority of insect mutualisms, the host-microbe specificity is maintained by a “partner fidelity” mechanism, mainly through direct symbiont transmission from mother to offspring. Such vertical symbiont transmission is remarkably diverse in insects, including ovarial transmission, milk-gland transmission, coprophagy, egg-smearing, and capsule transmission. In contrast to the insect-microbe symbioses, many animals and plants do not vertically transmit their symbionts but acquire symbionts from ambient environments every generation. Sophisticated “partner choice” mechanisms are at play to establish these mutualisms and maintain them over evolutionary timescales. This symbiont transmission mode, called horizontal transmission or environmental acquisition, is rarely found in insects that maintain specific associations, but recent studies have described this type of symbiosis in a few insect groups. The symbiosis between the bean bug Riptortus pedestris and its gut symbiont Burkholderia insecticola is one of the model systems that is intensively studied to understand how host-symbiont specificity and mutualistic interactions are maintained in insects with horizontal symbiont transmission. Phylogenetic analyses of symbionts in natural insect populations and bacterial inoculation tests in the laboratory revealed a high degree of specificity in this symbiosis while mutant screening of the symbiotic bacterium, genomics and transcriptomics, and histological observations have identified underpinning morphological, genetic and molecular bases. In this chapter, we focus on symbiont transmission modes and mechanisms observed in the amazing diversity of microbial symbioses in insects, highlighting the ways in which they have likely evolved.

International audience; Bacterial 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.

Bacterial 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.