Host–symbiont specificity determined by microbe– microbe competition in an insect gut Hideomi Itoha,1, Seonghan Jangb,1, Kazutaka Takeshitac, Tsubasa Ohbayashid, Naomi Ohnishie,2, Xian-Ying Mengf, Yasuo Mitania, and Yoshitomo Kikuchia,b,g,3 aBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Hokkaido Center, 062-8517 Sapporo, Japan; bGraduate School of Agriculture, Hokkaido University, 060-8589 Sapporo, Japan; cFaculty of Bioresource Sciences, Akita Prefectural University, 010-0195 Akita, Japan; dInstitute for Integrative Biology of the Cell, UMR 9198, CNRS, Commissariat à l’Energie Atomique et aux Énergies Alternatives (CEA), Université Paris-Sud, 91198 Gif-sur-Yvette, France; eResearch Center for Zoonosis Control, Hokkaido University, 001-0020 Sapporo, Japan; fBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba Center, 305-8566 Tsukuba, Japan; and gComputational Bio Big Data Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology, 062-8517 Sapporo, Japan Edited by Joan E. Strassmann, Washington University in St. Louis, St. Louis, MO, and approved September 30, 2019 (received for review July 18, 2019) Despite the omnipresence of specific host–symbiont associations microbe competition on the evolution and stabilization of host– with acquisition of the microbial symbiont from the environment, symbiont specificity is very scarce. little is known about how the specificity of the interaction evolved The bean bug Riptortus pedestris (Heteroptera: Alydidae) is and is maintained. The bean bug Riptortus pedestris acquires a associated with a Burkholderia symbiont that is confined in specific bacterial symbiont of the genus Burkholderia from environ- symbiosis-specific crypts located in the posterior midgut region Burkholderia mental soil and harbors it in midgut crypts. The genus M4 (10). The Burkholderia symbiont is not essential but benefi- consists of over 100 species, showing ecologically diverse lifestyles, cial for growth, reproduction, immunity homeostasis, and pesti- and including serious human pathogens, plant pathogens, and cide resistance of the insect host (11–13). While most insects nodule-forming plant mutualists, as well as insect mutualists. vertically transmit their symbionts, bean bugs acquire their mi- Through infection tests of 34 Burkholderia species and 18 taxo- nomically diverse bacterial species, we demonstrate here that non- crobial partners from the ambient soil in every generation (10, 11). To acquire their specific symbionts from the diverse soil symbiotic Burkholderia and even its outgroup Pandoraea could EVOLUTION stably colonize the gut symbiotic organ and provide beneficial microbiota, stinkbugs appear to utilize a specific organ called the effects to the bean bug when inoculated on aposymbiotic hosts. “constricted region,” a narrow passage filled with a mucus-like However, coinoculation revealed that the native symbiont always matrix, located in the midgut immediately upstream of the crypt- outcompeted the nonnative bacteria inside the gut symbiotic or- bearing region. The constricted region winnows out contami- gan, explaining the predominance of the native Burkholderia sym- nating microorganisms and the Burkholderia symbiont specif- biont in natural bean bug populations. Hence, the abilities for ically penetrates into the symbiotic region (14). In addition to colonization and cooperation, usually thought of as specific traits this specific apparatus for bacterial sorting, the insect expresses of mutualists, are not unique to the native Burkholderia symbiont diverse antimicrobial peptides in the symbiotic organ (13, 15), but, to the contrary, competitiveness inside the gut is a derived which may also play a role in the partner choice. trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont. Significance gut symbiosis | symbiont specificity | stinkbug | Burkholderia | competitiveness How are specific host-symbiont mutualisms stabilized without vertical transmission? This is one of the fundamental questions in evolutionary biology. To ensure specificity, animals and plants number of animals and plants are associated with beneficial have evolved sophisticated sorting mechanisms. Theoretical Amicroorganisms, which provide diverse services to the host studies proposed another mechanism, so-called “competition- species, such as enhanced nutrition and protection from antag- based selection,” where hosts provide a specific environment onists (1, 2). While some symbionts are vertically transmitted to selectively cultivate favorable symbionts and maintain sym- from mother to offspring, many animals and plants acquire in biont quality. Although this mechanism is thought to be powerful every generation the symbionts from the environment (3). Since in open systems, such as gut symbiosis where contaminants microorganisms are abundant and diverse in the environment, regularly invade, until now little experimental evidence has sup- hosts have evolved elaborate mechanisms for “partner choice” to ported its importance in the evolution of symbioses. Through the ensure the specificity of the associations (4, 5). Well-studied inoculation of an insect host with a large panel of symbiotic and model systems, the legume–Rhizobium and squid–Vibrio symbi- nonsymbiotic bacteria, we demonstrate that microbial competi- oses, have revealed that the partner specificity is achieved by tion is critical to maintain the specificity in an insect gut symbiosis. various host mechanisms, such as signal recognition during ini- tiation and secretion of antimicrobial agents during colonization Author contributions: H.I., S.J., and Y.K. designed research; H.I., S.J., T.O., N.O., X.-Y.M., Y.M., and Y.K. performed research; H.I., S.J., K.T., and Y.K. analyzed data; and H.I., S.J., (6, 7). In addition to such host control mechanisms, theoretical and Y.K. wrote the paper. – modeling studies have suggested the importance of microbe The authors declare no competing interest. microbe competition to ensure the specificity, wherein the host This article is a PNAS Direct Submission. provides a demanding environment to let a favorable microor- Published under the PNAS license. “ ganism grow dominantly (8, 9). The competition-based selec- 1H.I. and S.J. contributed equally to this work. tion” is thought to be more powerful to eliminate contaminants 2Present address: Cancer Precision Medicine Center, Japanese Foundation for Cancer Re- or pathogens and maintain symbiont quality, particularly in open search, 135-850 Koto-ku, Japan. systems, such as the gut, the fungus-farming garden of insects, and 3To whom correspondence may be addressed. Email: [email protected]. the plant rhizosphere, where microbial contamination can regu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. larly occur and potentially disrupt the symbioses. Despite these 1073/pnas.1912397116/-/DCSupplemental. theoretical studies, empirical support for the impact of microbe– First published October 21, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1912397116 PNAS | November 5, 2019 | vol. 116 | no. 45 | 22673–22682 Downloaded by guest on October 2, 2021 To be a bean bug symbiont, therefore, the Burkholderia species Burkholderia cepacia, Burkholderia pseudomallei and Burkholderia should be capable of penetrating the constricted region (initiation), mallei, designated as the “B. cepacia complex and B. pseudomallei” stably colonizing the symbiotic organ (accommodation), and of (BCC&P) clade (22). Some BCC&P species are also reported as course, behaving cooperatively with the host (cooperation). Re- fungal symbionts (23) and beetle symbionts (24). The second clade cent studies have revealed part of the symbiont’s mechanisms in- includes a number of plant growth-promoting rhizobacteria and volved in these processes. The Burkholderia symbiont employs a nodule symbionts of leguminous plants, and is designated as the unique corkscrew flagellar motility to pass through the constricted “plant-associated beneficial and environmental” (PBE) clade (25). region (16); it undergoes cytological and metabolic alterations, This clade, which also includes farming symbionts of the slime mold such as flagella loss and polyhydroxyalkanoate accumulation, to Dictyostelium discoideum (26), was recently even nominated as a cope with the crypt environment (17–19); and it rapidly prolifer- new genus, Paraburkholderia (27). The third clade mainly consists ates to occupy entirely the available space in the lumen of the of environmental species, leaf-nodule symbionts of Rubiaceae plants, symbiotic M4 midgut region, notably by recycling host metabolic and gut symbionts of stinkbugs, and is called the “Burkholderia wastes into essential amino acids and B vitamins in the M4 crypts, ” “ which are thought to be part of the symbiont services provided to glathei clade or the stinkbug-associated beneficial and envi- ” the host (17). ronmental (SBE) clade (10, 28). Also for this clade, a new The bacterial genus Burkholderia (Betaproteobacteria: genus name, Caballeronia, was proposed (29). The outgroup of Burkholderiaceae), comprises over 100 species with taxonomically Burkholderia is the genus Pandoraea (Fig. 1), which are common validated names and is an ecologically very diverse bacterial group soil bacteria, although some of them are opportunistic pathogens (20). Based on genomic phylogeny, the