Insect’s intestinal organ for symbiont sorting PNAS PLUS Tsubasa Ohbayashia, Kazutaka Takeshitaa,b, Wataru Kitagawaa,b, Naruo Nikohc, Ryuichi Kogad, Xian-Ying Mengd, Kanako Tagoe, Tomoyuki Horif, Masahito Hayatsue, Kozo Asanoa, Yoichi Kamagataa,b, Bok Luel Leeg, Takema Fukatsud, and Yoshitomo Kikuchia,b,1 aGraduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan; bBioproduction Research Institute, Hokkaido Center, National Institute of Advanced Industrial Science and Technology, Sapporo 062-8517, Japan; cDepartment of Liberal Arts, The Open University of Japan, Chiba 261-8586, Japan; dBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan; eEnvironmental Biofunction Division, National Institute for Agro-Environmental Sciences, Tsukuba 305-8604, Japan; fEnvironmental Management Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8569, Japan; and gGlobal Research Laboratory, College of Pharmacy, Pusan National University, Pusan 609-735, Korea Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved August 11, 2015 (received for review June 11, 2015) Symbiosis has significantly contributed to organismal adaptation Those controlling mechanisms are of general importance for and diversification. For establishment and maintenance of such host– understanding symbiosis (6, 10). symbiont associations, host organisms must have evolved mecha- Stinkbugs, belonging to the insect order Hemiptera, consist of nisms for selective incorporation, accommodation, and maintenance over 40,000 described species in the world (15). The majority of of their specific microbial partners. Here we report the discovery of a the stinkbugs suck plant sap or tissues, and some of them are previously unrecognized type of animal organ for symbiont sorting. notorious as devastating agricultural pests (16). These plant- In the bean bug Riptortus pedestris, the posterior midgut is morpho- sucking stinkbugs possess a specialized symbiotic organ in their logically differentiated for harboring specific symbiotic bacteria of a alimentary tract: A posterior region of the midgut is morpho- beneficial nature. The sorting organ lies in the middle of the intestine logically differentiated with a number of sacs or tubular out- as a constricted region, which partitions the midgut into an anterior growths, called crypts or ceca, whose inner cavity hosts symbiotic nonsymbiotic region and a posterior symbiotic region. Oral adminis- bacteria (17–21). Usually, a single bacterial species dominates in tration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs the midgut crypts, and elimination of the symbiont causes re- showed that the symbionts pass through the constricted region and tarded growth and increased mortality of the host, which in- colonize the posterior midgut. However, administration of food col- dicates the specific and beneficial nature of the stinkbug gut orings revealed that food fluid enters neither the constricted region symbiosis (20–31). The initial symbiont infection is established by nor the posterior midgut, indicating selective symbiont passage at nymphal feeding, which may be either via vertical transmission the constricted region and functional isolation of the posterior mid- from symbiont-containing maternal secretion supplied upon gut for symbiosis. Coadministration of the GFP-labeled symbiont and oviposition (19–21) or via environmental acquisition from am- red fluorescent protein-labeled Escherichia coli unveiled selective pas- bient microbiota (21–23). What mechanisms underlie the selec- sage of the symbiont and blockage of E. coli at the constricted region, tive establishment of a specific bacterial symbiont in the midgut demonstrating the organ’s ability to discriminate the specific bacterial symbiotic organ despite the oral inoculum contaminated by symbiont from nonsymbiotic bacteria. Transposon mutagenesis and nonsymbiotic microbes has remained largely an enigma, al- screening revealed that symbiont mutants in flagella-related genes though recent studies have started to shed light on the sym- fail to pass through the constricted region, highlighting that both biotic mechanisms underlying the environmental acquisition host’s control and symbiont’s motility are involved in the sorting of specific Burkholderia symbionts in the bean bug Riptortus process. The blocking of food flow at the constricted region is con- pedestris (Hemiptera: Alydidae) (22, 32). Antimicrobial substances served among diverse stinkbug groups, suggesting the evolutionary produced by the midgut epithelia (33, 34) and some symbiont origin of the intestinal organ in their common ancestor. Significance stinkbug | gut symbiosis | partner choice | Burkholderia | flagellar motility In general, animals have a mouth for feeding, an anus for defe- iverse organisms are obligatorily associated with microbial cation, and a gut connecting them for digestion and absorption. Dsymbionts, which significantly contribute to their adaptation However, we discovered that the stinkbug’s gut is functionally and survival (1–3). In such symbiotic associations, the host or- disconnected in the middle by a previously unrecognized organ ganisms often develop specialized cells, tissues, or organs for for symbiont sorting, which blocks food fluid and nonsymbiotic harboring their specific microbial partners [for example, root bacteria but selectively allows passing of a specific bacterial nodules in the legume–Rhizobium symbiosis (4, 5), symbiotic symbiont. Though very tiny and inconspicuous, the organ light organs in the squid–Vibrio symbiosis (6, 7), and bacter- governs the configuration and specificity of stinkbug gut iocytes in the aphid–Buchnera symbiosis (8, 9)]. symbiosis, wherein the posterior gut region is devoid of food These microbial symbionts are either acquired by newborn flow, populated by a specific bacterial symbiont, and trans- hosts from the environment every generation as in the legume– formed into an isolated organ for symbiosis. Mutant analyses Rhizobium and the squid–Vibrio symbioses or transmitted verti- showed that the symbiont’s flagellar motility is needed for cally through host generations as in the aphid–Buchnera symbi- passing the host organ, highlighting intricate host–symbiont osis (10). In the environmentally acquired symbiotic associations, interactions underpinning the symbiont sorting process. it is essential for the host organisms to recognize and incorporate specific symbiotic bacteria while excluding a myriad of nonsym- Author contributions: T.O., T.F., and Y. Kikuchi designed research; T.O., K. Takeshita, W.K., N.N., R.K., X.-Y.M., K. Tago, T.H., M.H., K.A., Y. Kamagata, B.L.L., and Y. Kikuchi biotic environmental microbes (6, 11). In the vertically trans- performed research; T.O. and Y. Kikuchi analyzed data; and T.O., T.F., and Y. Kikuchi mitted symbiotic associations, it is important for the host wrote the paper. organisms to selectively transmit their own symbiotic bacteria The authors declare no conflict of interest. while excluding parasitic/cheating microbial contaminants (12– This article is a PNAS Direct Submission. 14). Hence, it is expected that the host organisms must have 1To whom correspondence should be addressed. Email: [email protected]. EVOLUTION evolved some mechanisms for selective incorporation, accom- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. modation, and maintenance of their specific microbial partners. 1073/pnas.1511454112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1511454112 PNAS | Published online August 31, 2015 | E5179–E5188 Downloaded by guest on September 24, 2021 factors, such as stress-responsive polyester accumulation, cell A wall synthesis, and purine biosynthesis (35–37) might be in- volved in the selective infection of the Burkholderia symbiont to the midgut crypts. Here we address this important symbiotic issue by the dis- covery of a previously unrecognized intestinal organ in the stinkbugs. Though very tiny and inconspicuous, the organ gov- erns the configuration and specificity of the stinkbug gut sym- biosis. Lying in the middle of the midgut, the organ blocks food flow and nonsymbiotic bacteria but selectively allows passing of specific symbiotic bacteria, whereby the stinkbug’s intestine is functionally partitioned into the anterior region specialized for digestion and absorption and the posterior region dedicated to symbiosis. The blocking of food flow by the organ is conserved B across diverse stinkbug families, suggesting the possibility that the organ evolved in their common ancestor and has played substantial roles in their symbiont-mediated adaptation and diversification. Results and Discussion Identification of Constricted Region in Stinkbug Midgut. As in di- verse other stinkbugs (17–21), the midgut of the bean bug C D R. pedestris consists of the following morphologically distinct regions: the voluminous midgut first section (M1), the tubular midgut second section (M2), the ovoid midgut third section (M3), and the midgut fourth section (M4) with numerous crypts densely populated by a specific betaproteobacterial symbiont of the genus Burkholderia, which is orally acquired by nymphal in- sects from the environment every generation (22, 23, 32) (Fig. 1A). A swollen region adorally connected to the M4 is without crypts and called M4 bulb (M4B) (18, 34, 38) (Fig. 1A). Although biological roles