Yoshitomo Kikuchi
email: kikuchi@aist.go.jp

Tsubasa Ohbayashi

Seonghan Jang

Peter Mergaert

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-labelled symbionts followed by spatiotemporal microscopic observations revealed that after the initial passage of symbionts through the CR, it closes within 12 to 18 hours, 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 non-symbiotic 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.

Introduction

Phenotypic alteration in response to environmental factors is omnipresent in nature, and is thought to be a driving force for generation of evolutionary novelty [START_REF] Gilbert | Ecological developmental biology: integrating epigenetics, medicine, and evolution[END_REF]. In plants and animals that are intimately associated with microbes, the symbiont acquisition is integrated in the hosts' development and sometimes triggers dramatic developmental responses.

Well-known examples are found in the root nodule formation of the legume-Rhizobium symbiosis and the light organ morphogenesis of the squid-Vibrio symbiosis [START_REF] Nyholm | The winnowing: establishing the squid-Vibrio symbiosis[END_REF][START_REF] Kondorosi | A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors[END_REF]. Besides such drastic morphological changes in specific associations, recent studies on mammals and Drosophila gut microbiota have revealed that gut bacteria also play a significant role in proliferation and differentiation of gut epithelial cells and in metabolic and immunity homeostasis [START_REF] Sommer | The gut microbiota-masters of host development and physiology[END_REF][START_REF] Broderick | Microbiota-induced changes in Drosophila melanogaster host gene expression and gut morphology[END_REF]. Similarly, in the plant kingdom, rhizosphere microbiota modify the root and shoot architecture by modulating the phytohormone balance in the plant [START_REF] Vacheron | Plant growth-promoting rhizobacteria and root system functioning[END_REF]. Even non-symbiotic, environmental bacteria can have profound morphological effects on eukaryotes by the production of small molecules that act as essential developmental cues.

The settlement and subsequent metamorphosis of free-swimming larvae of the marine tubeworm Hydroides elegans are trigged by biofilms of the bacterium Pseudoalteromonas luteoviolacea. These biofilms deliver a metamorphosis-triggering effector protein into the larvae via a contractile injection system [START_REF] Shikuma | Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures[END_REF][START_REF] Shikuma | Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling[END_REF][START_REF] Ericson | A contractile injection system stimulates tubeworm metamorphosis by translocating a proteinaceous effector[END_REF]. Another striking example is the triggering of multicellularity from a single founder cell in the choanoflagellate Salpingoaeca rosetta by a sulfonolipid, derived from specific prey bacteria of the Bacteroidetes [START_REF] Alegado | A bacterial sulfonolipid triggers multicellular development in the closest living relatives of animals[END_REF].

The class Insecta consists of over one million described species and is the most diverse animal group in the terrestrial ecosystem. Many insects harbor symbiotic bacteria in specialized cells called bacteriocytes or in gut crypts [START_REF] Buchner | Endosymbiosis of animals with plant microorganism[END_REF][START_REF] Kikuchi | Endosymbiotic bacteria in insects: their diversity and culturability[END_REF]. In these symbiotic organs, bacterial symbionts play metabolic roles for the hosts, such as provision of essential nutrients and/or digestion of indigestible food materials [START_REF] Moran | Genomics and evolution of heritable bacterial symbionts[END_REF][START_REF] Douglas | Lessons from studying insect symbioses[END_REF][START_REF] Utami | Phylogenetic diversity and single-cell genome analysis of "Melainabacteria", a non-photosynthetic cyanobacterial group, in the termite gut[END_REF]. Recent studies have revealed that, in addition, symbionts alter diverse aspects of host phenotypes, including heat tolerance, pathogen resistance, behavior, sex, cuticle hardening, body color, and tolerance to phytotoxins and insecticides [START_REF] Hosokawa | Symbiont acquisition alters behaviour of stinkbug nymphs[END_REF][START_REF] Werren | Wolbachia: master manipulators of invertebrate biology[END_REF][START_REF] Feldhaar | Bacterial symbionts as mediators of ecologically important traits of insect hosts[END_REF][START_REF] Kikuchi | Symbiont-mediated insecticide resistance[END_REF][START_REF] Flórez | Defensive symbioses of animals with prokaryotic and eukaryotic microorganisms[END_REF][START_REF] Tsuchida | Molecular basis and ecological relevance of aphid body colors[END_REF][START_REF] Anbutsu | Small genome symbiont underlies cuticle hardness in beetles[END_REF][START_REF] Itoh | Detoxifying symbiosis: microbe-mediated detoxification of phytotoxins and pesticides in insects[END_REF]. Despite the accumulated knowledge about the diverse symbiosis-mediated phenotypes, symbiont-induced morphogenesis of the symbiotic organ has been poorly described in insects.

The bean bug Riptortus pedestris develops a number of crypts in a posterior region of the midgut, the lumen of which is densely colonized by the betaproteobacterial symbiont, Burkholderia insecticola [START_REF] Kikuchi | Gut symbiotic bacteria of the genus Burkholderia in the broad-headed bugs Riptortus clavatus and Leptocorisa chinensis (Heteroptera: Alydidae)[END_REF][START_REF] Takeshita | Riptortus pedestris and Burkholderia symbiont: an ideal model system for insect-microbe symbiotic associations[END_REF][START_REF] Takeshita | Burkholderia insecticola sp. nov., a gut symbiotic bacterium of the bean bug Riptortus pedestris[END_REF][START_REF] Kaltenpoth | Versatile and dynamic symbioses between insects and Burkholderia bacteria[END_REF]. Infection with B. insecticola improves growth and fecundity of the bean bug [START_REF] Kikuchi | Live imaging of symbiosis: spatiotemporal infection dynamics of a GFP-labelled Burkholderia symbiont in the bean bug Riptortus pedestris[END_REF] probably because the symbiont recycles host metabolic wastes in the midgut crypts [START_REF] Ohbayashi | Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture[END_REF]. Unlike the typical insect-microbe symbioses in which symbionts are vertically transmitted from mother to offspring, the bean bug acquires B. insecticola from environmental soil every generation [START_REF] Kikuchi | Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation[END_REF]. From hatching to adulthood, the bean bug development passes over five instar stages and the symbiont is acquired preferentially at the 2 nd instar but also 3 th instar nymphs are compatible for infection [START_REF] Kikuchi | Specific developmental window for establishment of an insect-microbe gut symbiosis[END_REF].

Since diverse microorganisms abound in the environment, particularly in soils [START_REF] Bahram | Structure and function of the global topsoil microbiome[END_REF], hosts need to sort their specific partner from this microbial community. Furthermore, after symbiont colonization, they have to protect the symbiotic organ against invasion by superinfection to stabilize the mutualistic association. To sort the symbiont, the bean bug develops a specific organ called the "constricted region" (CR), a narrow passage filled with a mucus-like matrix, located in the midgut immediately upstream of the crypt-bearing region [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF]. The CR sorts out contaminating microorganisms while B. insecticola can penetrate into the symbiotic region using an unusual, corkscrew-like flagellar motility [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF][START_REF] Kinosita | Unforeseen swimming and gliding mode of an insect gut symbiont, Burkholderia sp. RPE64, with wrapping of the flagella around its cell body[END_REF][START_REF] Raina | The role of microbial motility and chemotaxis in symbiosis[END_REF]. However, the sorting mechanism is not perfectly tight and some allied non-symbiont Burkholderia species as well as species of its outgroup Pandoraea can nevertheless penetrate the CR [START_REF] Itoh | Host-symbiont specificity determined by microbe-microbe competition in an insect gut[END_REF]. Interestingly, the non-native Burkholderia and Pandoraea species do not fully occupy the lumen of the midgut crypts and co-inoculation analyses revealed that B. insecticola always outcompetes these non-native bacteria inside the crypts, suggesting that the crypt's luminal environment favors only the native symbiont [START_REF] Itoh | Host-symbiont specificity determined by microbe-microbe competition in an insect gut[END_REF]. Although the molecular basis remains unclear, the competition-based winnowing is therefore, thought of as a second-line mechanism ensuring the host-symbiont specificity. In addition to these partner choice mechanisms, here we discovered a third mechanism underpinning the specific gut symbiosis which we call "midgut closure", wherein the CR is closed immediately after colonization of the crypt-bearing midgut region by the symbiont, preventing subsequent infections.

Materials and methods

Insects and bacterial strains

The strain of R. pedestris used in this study was originally collected from a soybean (Glycine max) field in Tsukuba, Ibaraki, Japan, and maintained in the laboratory over ten years. The insects were reared in petri dishes (90 mm in diameter and 20 mm high) at 25°C under a long-day regimen (16 h light, 8 h dark) and fed with soybean seeds and distilled water containing 0.05% ascorbic acid (DWA). For inoculation tests of fluorescent-protein-labelled B. insecticola, a green fluorescent protein (GFP)-labelled strain, RPE225 [START_REF] Kikuchi | Live imaging of symbiosis: spatiotemporal infection dynamics of a GFP-labelled Burkholderia symbiont in the bean bug Riptortus pedestris[END_REF], and a red fluorescent protein (RFP)-labelled strain, KT39 (36), were used. B. insecticola derivatives, including gene-deletion mutants, and non-symbiotic bacteria used in this study are listed in Supplementary Table S1. They were labeled with GFP using the Tn7 minitransposon system, as previously described [START_REF] Kikuchi | Live imaging of symbiosis: spatiotemporal infection dynamics of a GFP-labelled Burkholderia symbiont in the bean bug Riptortus pedestris[END_REF][START_REF] Norris | Stable, site-specific fluorescent tagging constructs optimized for Burkholderia species[END_REF].

Oral inoculation of cultured symbiont cells

The symbiont strains were grown to early log phase in YG medium (containing 30 µg/ml kanamycin for GFP-labelled RPE225 and RFP-labelled KT39) on a gyratory shaker (150 rpm) at 30°C. Bacteria were harvested by centrifugation, re-suspended in DWA, and adjusted to 10 4 cells/µl in DWA. Three types of inoculations were performed: (i) oral inoculation of the GFP strain at the 2 nd instar, (ii) oral inoculation of the GFP strain at the 3 rd instar, and (iii) oral inoculation of the RFP strain at the 2 nd and then the GFP strain inoculated after a defined interval.

(i) Oral inoculation of the GFP strain at 2 nd instar. Immediately after 1 st instar nymphs molted to the 2 nd instar, DWA was removed from the rearing containers so that the nymphs were kept without drinking water overnight. Then 1 µl DWA containing 10 4 cells/µl of the GFP-labelled RPE225 was supplied to the rearing containers, which the 2 nd instar nymphs immediately exploited, leading to the acquisition of Burkholderia symbionts. After drinking, insects were supplied with DWA and the nymphs were further reared in the absence of bacteria for variable times before dissection to analyze their infection status as described below.

(ii) Oral inoculation of the GFP strain at 3 rd instar. Nymphs were kept with soybean seeds and symbiont-free DWA until the 2 nd instar. Immediately after 2 nd instar nymphs molted to the 3 rd instar, the DWA was removed from the rearing containers so that the nymphs were kept without drinking water overnight. Then 1 µl DWA containing 10 4 cells/µl of the GFP-labelled RPE225 was supplied to the rearing containers, which the 3 rd instar nymphs immediately exploited, leading to the acquisition of Burkholderia symbionts. After drinking, insects were supplied with DWA and the nymphs were further reared in the absence of bacteria for variable times before dissection to analyze their infection status as described below.

(iii) Oral inoculation of the RFP strain, and re-inoculation of the GFP strain after an interval. After 2 nd instar molting, DWA was removed so that nymphs became thirsty, as described above. Then 1 µl DWA containing 10 4 cells/µl of the RFP-labelled strain, KT39, was orally inoculated to the 2 nd instar nymphs. After drinking the bacteria-containing DWA, insects were kept with soybean seeds for 6 h, 12 h, 15 h, 18 h, or 3 days. After the interval, 1 µl DWA containing 10 4 cells/µl of the GFP-labelled RPE225 was supplied to the rearing containers. As for the first infection, insects were deprived of water before the second infection. These nymphs were further reared for two days with symbiont-free DWA, and then dissected to check their infection status of the RFP and GFP strains. In the 3-day-interval group, insects became 3 rd instar during the interval, and the molted nymphs were subjected to the re-inoculation test.

Experimental removal of crypt-colonizing symbionts by antibiotic treatment

To investigate the plasticity of the midgut closure, experimental removal of crypt-colonized B. insecticola by antibiotic treatment followed by re-inoculation of the symbiont was performed. For this experiment, a chrolamphenicol (Cm)-susceptible GFP-labelled strain, KT33, and a Cm-resistant RFP-labelled strain, RPE744, were used (Supplementary Table S1). The GFP-labelled Cm-susceptible KT33 was constructed by the Tn7 minitransposon system [START_REF] Norris | Stable, site-specific fluorescent tagging constructs optimized for Burkholderia species[END_REF], while the RFP-labelled Cm-resistant RPE744 strain carried the stable plasmid pIN29 [START_REF] Vergunst | Burkholderia cenocepacia creates an intramacrophage replication niche in zebrafish embryos, followed by bacterial dissemination and establishment of systemic infection[END_REF]. Immediately after 1 st instar nymphs molted to the 2 nd instar, DWA was removed from the rearing containers and the nymphs were kept without drinking water overnight.

Then DWA containing 10 4 cells/µl of the GFP-labelled Cm-susceptible strain KT33 was supplied to the rearing containers for 24 h. After 24 h, the symbiont-containing DWA was replaced by DWA containing 100 µg/ml chloramphenicol to remove the symbiont. Insects were treated with the antibiotic during the 2 nd instar stage, and after molting to the 3 rd instar, the Cm-resistant RFP-labelled strain RPE744 was inoculated as described above. These nymphs were further reared for two days with symbiont-free DWA, and then dissected to check their infection status with the GFP or RFP strains. To verify the first treatments in the 2 nd instar stage, half of the treated insects (10 insects/20 in total) were sacrificed in the second instar, dissected and their infection status was verified. In addition, two types of control experiments were performed: first, insects were treated with chloramphenicol but without inoculation of the GFP strain in the 2 nd instar stage, and then the RFP-labelled RPE744 strain was inoculated after molting to the 3 rd instar; second, insects were inoculated with the GFP-labelled symbiont at the 2 nd instar stage without chloramphenicol treatment, and the insects were inoculated with the RFP-labelled RPE744 at the 3 rd instar stage.

Methods for "microscopic observations of dissected symbiotic organs", "oral inoculation of colonization-deficient mutants and non-symbiotic bacteria" and "bottleneck estimation" are described in the Supplementary Information.

Results

Closure of the constricted region during symbiont colonization

The midgut of R. pedestris consists of four distinct sections called M1, M2, M3 and M4 (Supplementary Fig. S1). The symbiotic Burkholderia specifically colonizes the crypt-bearing M4 section. In front of M4, a distinct, bulbous part called the M4 bulb (M4B) is developed, wherein symbionts flow from the M4 section and are digested [START_REF] Ohbayashi | Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture[END_REF][START_REF] Kim | Specific midgut region controlling the symbiont population in an insect-microbe gut symbiotic association[END_REF]. The M3 and M4B are connected by the CR. Our previous study using a GFP-labelled Burkholderia symbiont revealed that in the 2 nd instar, colonization of the symbiotic M4B-M4 region of the midgut starts around 6 hours post inoculation (hpi) [START_REF] Kikuchi | Live imaging of symbiosis: spatiotemporal infection dynamics of a GFP-labelled Burkholderia symbiont in the bean bug Riptortus pedestris[END_REF]. Around 24 hpi, the symbiont cells appear in the main tract of the M4 region and start the colonization of the crypts. At 48 hpi most of the crypts are colonized and around 72 hpi, crypts are entirely filled by the symbiont cells. This colonization pattern is then stably maintained during the subsequent nymphal stages.

In order to further detail the dynamics of the infection process, we performed time course observations focusing on the CR and M4B regions of the midgut. As described before, the infection from M3 to M4B through the CR started around 6 hpi and a continuous migration of GFP-labeled bacteria was observed (Fig. 1a andb; Supplementary Fig. S2).

However, around 15 hpi the connection was abruptly interrupted, despite the continuous presence of a dense population of symbiont cells in the M3 (Fig. 1c andd; Supplementary Fig. S2). Subsequently, the M4B was further closed from the side of the CR, and the closure was accomplished around 24 hpi (Fig. 1e-h; Supplementary Fig. S2). Laser Scanning Microscopy (LSM) observations revealed that after symbiont colonization, at 24 hpi, the epithelial cells of M4B are bulged at the luminal side and are arranged like interlocking teeth, closed like a zipper (Fig. 2a andb).

In the M4 crypts, the Burkholderia symbionts started colonization from 12 hpi, and then proliferated in them until they fully occupied the crypts by 48 hpi (Supplementary Fig. S3). Notably, in parallel with the filling of crypts, symbiont cells started to flow backwards from M4 into M4B around 36-42 hpi (Fig. 1i-l; Supplementary Fig. S2). M4B was entirely filled by symbiont cells and became bulbous and maturated around 48 hpi (Fig. 1m-r; Supplementary Fig. S2). However, no overflow was observed from M4B to M3, which at this stage became free of symbionts. LSM observations of tissues stained with phalloidin showed that the CR is kept tightly closed (Fig. 2c andd). The closure and re-opening of M4B were not observed in aposymbiotic insects (Fig. 3a-g). Serial measurements of the CR and M4B (Supplementary Fig. S4) confirmed the marked morphological changes of the CR (Fig. 1s) and M4B (Fig. 1t) after symbiont infection.

Symbiont infection of 3 rd instar nymphs triggers midgut closure

Our previous study reported that the symbiont acquisition occurs mainly at the 2 nd instar but 3 rd instar nymphs are still competent to acquire the symbiont [START_REF] Kikuchi | Specific developmental window for establishment of an insect-microbe gut symbiosis[END_REF]. To demonstrate the symbiont-dependency of the gut morphological alteration, we kept insects aposymbiotic during the 2 nd instar, and then inoculated symbiont cells after molting and entry into the 3 rd instar (Fig. 3a). While the closure and re-opening of M4B were not observed in the aposymbiotic 2 nd instar stage (Fig. 3b-g), the insects that ingested the symbionts at the 3 rd instar stage showed the closure and re-opening of M4B after symbiont infection in a similar way as during 2 nd instar infection and colonization (Fig. 3h-m). However, the response time in the 3 rd instar stage was delayed compared with the 2 nd instar stage (Fig. 1). These results clearly demonstrated that the drastic morphological alteration of the midgut is triggered by the symbiont.

Midgut closure blocks super-infection of the M4 crypts

To confirm the physical closure of the CR after the symbiont colonization, a re-inoculation test of the symbiont was performed, in which a GFP-labelled symbiont is inoculated to nymphs that already acquired an RFP-labelled symbiont (Supplementary Fig. S5a). Insects accepted GFP-labelled symbionts when the symbionts were re-inoculated after short, 6-12 h, intervals (Supplementary Fig. S5b, Fig. S6). However, insects only rarely acquired the GFP-labelled symbionts when the bacteria were re-inoculated after a 15 h interval (Supplementary Fig. S5b, Fig. S6), which strikingly corresponded to the start of the midgut closure observed by microscopy (Fig. 1), and it never happened when the interval was longer (Supplementary Fig. S5b, Fig. S6).

Accordingly, when the GFP-labelled symbiont was inoculated to 3 rd instar nymphs that already acquired the RFP-labelled symbiont during the 2 nd instar stage (Fig. 4a), insects never acquired the GFP-labelled symbiont (Supplementary Fig. S5b, Fig. S6). LSM observations revealed that, after colonization of the symbiotic region by an RFP-labelled symbiont, no GFP-labelled symbionts were able to enter the CR, although the GFP-labelled symbiont cells were readily detected in the M3 region (Fig. 4b andc). These results confirmed that the morphological closure of the CR after symbiont colonization in M4 crypts and maturation of M4B, effectively prevents subsequent entry of bacteria in the symbiotic organ.

The midgut closure is correlated with a bottleneck of symbiont infection

The rapid closing of the CR, together with its narrow diameter of a few micrometers [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF] could constrict the number of bacteria that can transit from M3 into M4 and thus cause a bottleneck of symbiont infection. Then, how many symbiont cells can enter the symbiotic region before the closing? To determine the bottleneck size, GFP-labelled symbiont cells were co-inoculated with non-labelled wild type symbiont cells in different ratios ranging from one in ten to one in 2x10 5 . The total inoculum per insect was adjusted to 10 5 CFU throughout the experiments. The rationale of the experiment was that if the dilution factor of the GFP-labelled strain is below the bottleneck, the marked strain should readily colonize the crypts while it should not when the dilution factor is higher than the bottleneck. If the dilution factor is around the bottleneck size, some insects should be infected and others not with the GFP-marked strain. Based on the detection rate of GFP-labelled symbiont cells, we concluded that the bottleneck of symbiont infection is 3,950 cells under the used experimental conditions (Supplementary Fig. S7).

Midgut closure is permanent

To investigate the plasticity of the midgut closure, an experimental symbiont removal and re-inoculation test was performed. During the 2 nd instar stage, a GFP-labelled and chloramphenicol sensitive symbiont was allowed to establish in the M4 crypts and was subsequently removed by chloramphenicol treatment. An RFP-labelled and chloramphenicol resistant symbiont was then re-inoculated at the 3 rd instar stage (Fig. 5a).

The chloramphenicol treatment during the 2 nd instar stage efficiently removed the GFP-labelled symbiont cells from the crypts and the insects became aposymbiotic (GFP-Cm in Fig. 5b). The removal was complete since the GFP-labelled symbionts did not reappear after the stop of the chloramphenicol treatment in the 3 rd instar insects (GFP-Cm-RFP in Fig. 5c). Despite this complete elimination of the GFP-labelled symbionts of the first inoculum, no RFP-labelled symbiont was detected in 3 rd instar nymphs when the RFP-labelled symbiont was re-inoculated to the cured insects (GFP-Cm-RFP in Fig. 5c). In control experiments, the re-inoculated RFP-labelled symbiont was accepted when insects were treated with chloramphenicol without inoculation with the GFP-labelled symbiont during the 2 nd instar stage (Cm-RFP in Fig. 5c). On the other hand, the re-inoculated RFP-labelled symbiont was rejected when a GFP-labelled symbiont was inoculated but not cured by chloramphenicol during the 2 nd instar stage (GFP-RFP in Fig. 5c). These results indicate that there is no phenotypic plasticity in the midgut closure (i.e. no re-opening of the closed region) and that the closure is maintained once it is triggered, even in the absence of colonizing symbiont cells in the M4 region.

Triggering of midgut closure by colonization-deficient mutants and non-symbiotic bacteria

The rapid midgut closure after infection with the symbiont and its absence in aposymbiotic insects suggest that the passage of bacteria through the CR and/or the colonization of the crypts are required to trigger this developmental response. To test this hypothesis directly, a re-inoculation test of the symbiont was performed, wherein an RFP-labelled B. insecticola was inoculated to nymphs that already acquired a GFP-labelled strain of either colonization-deficient mutants of B. insecticola or non-symbiotic bacteria (Fig. 6a). The following colonization-defective gene-deletion mutants were used: the flagellar mutant fliC::Tn5 which can reach M3 but is completely blocked by the CR [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF] and the purine biosynthesis mutant purL::Tn5 (40), the LPS biosynthesis mutant ΔwaaF (41) and the PHA biosynthesis mutant ΔphaB [START_REF] Kim | Polyester synthesis genes associated with stress resistance are involved in an insect-bacterium symbiosis[END_REF], which can each reach the crypts but cannot proliferate well leading to poor crypt colonization. The insects inoculated with the fliC::Tn5 mutant were colonized by wild-type B. insecticola after re-inoculation indicating no midgut closure by this mutant (Fig. 6c). On the contrary, the purL::Tn5, ΔwaaF, and ΔphaB mutants induced the midgut closure as evidenced by the complete block of infection by the re-inoculated wild type (Fig. 6d-f).

As non-symbiotic bacteria, the following four species were inoculated: Escherichia coli and Cupriavidus taiwanensis which are both blocked at the CR; Burkholderia fungorum and Pandoraea norimbergensis which can both pass through the CR and partially colonize the midgut crypts [START_REF] Itoh | Host-symbiont specificity determined by microbe-microbe competition in an insect gut[END_REF]. Insects inoculated with E. coli or C. taiwanensis did not induce the midgut closure and were colonized by wild-type B. insecticola after re-inoculation (Fig. 6g andh). In contrast, B. fungorum and P. norimbergensis induced the midgut closure and completely blocked the re-inoculated wild type (Fig. 6i andj). Together, these experiments confirm that the midgut closure is triggered only when bacteria have entered the symbiotic organ while their presence in the M3 is not sufficient.

Discussion

Our previous study reported that the bean bug R. pedestris develops in the midgut a mucus-filled sorting organ, the CR. This unusual organ selects the symbiotic Burkholderia from the enormously diverse soil microorganisms that can penetrate the intestine during feeding and drinking [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF]. Through the detailed observations of the symbiont infection process in the 2 nd instar stage, we revealed here additional important features of this selection mechanism. Our observations showed that: (i) the CR as well as the M4B are closed in response to symbiont infection (Fig. 1, Fig. 2; Supplementary Fig. S2); the morphological alteration of the M4B and the CR did not occur in aposymbiotic nymphs, but was reproduced in 3 rd instar nymphs when insects were reared in the absence of bacteria in the 2 nd instar and then inoculated after molting (Fig. 3); (ii) after the closure, the symbiont cells proliferating in the M4 crypts flow back into M4B, which opens again and becomes bulbous and filled with symbiont cells (Fig. 1; Supplementary Fig. S2); (iii) in spite of the M4B opening, the CR is kept closed (Fig. 2), blocking any further infection events (Fig. 4);

(iv) once triggered, the CR is kept closed permanently, even when symbionts are removed from the crypts by antibiotic treatment (Fig. 5); (v) to triggered the closure, the passage of bacteria -symbionts or other species capable to do so -through the CR and their installation in the crypts is required (Fig. 6) and (vi) the midgut closure imposes an infection bottleneck on the symbiont population, limiting the founding population that will populate the symbiotic organ to a maximum of a few thousand cells (Supplementary Fig. S7). Together, these results demonstrate that the establishment of bacteria in the midgut crypts provokes a marked and rapid morphogenic response in the symbiotic organ, which is already largely formed before the establishment of the symbiosis. To our knowledge, this is a rare example in the insects of symbiotic microorganisms that trigger a developmental alteration.

In contrast to the intracellular symbioses that are typically found in insects, the gut symbiotic association of R. pedestris and related stinkbugs is an open system and the M4 symbiotic organ is in direct contact with the digestive tract, located upstream in the intestine (the M1 to M3 midgut regions). Therefore, the symbiotic organ faces a constant risk of invasion by other microorganisms, including cheaters and potential pathogens. From this point of view, the midgut closure found in the bean bug is one of the ultimate mechanisms to limit contamination of the gut symbiotic organ by unwanted microbes. Indeed, the re-inoculation test of the Burkholderia symbiont clearly demonstrated that the closure effectively prevents entrance of bacteria once the symbiont is established in the M4B-M4 region (Fig. 4; Supplementary Fig. S5). Moreover, the midgut closure operates within an intestinal organ that has a very distinctive organization and functioning. The bean bug externally digests the content of seeds of leguminous plants and sucks up liquid food [START_REF] Yasunaga | A Field Guide to Japanese Bugs[END_REF].

By use of food colorings, we showed before that ingested liquid food is completely digested in M3 and absorbed into the hemolymph [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF] and never enters the M4B and M4 region.

Thus the symbiotic organ is isolated from the digestive flow in the intestine and this disconnection is independent of the symbiosis since it is established already in aposymbiotic animals [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF]. Metabolic wastes are removed by the malpighian tubes and excreted via the hindgut, although transcriptome analyses suggest that some metabolic wastes are supplied to the M4 crypts and recycled by the symbiotic Burkholderia [START_REF] Ohbayashi | Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture[END_REF]. We propose that the symbiont-mediated midgut closure, in conjunction with the CR and the unique digestive system, plays a significant role to stabilize the specific host-symbiont interaction in the bean bug.

The inoculation tests with colonization-defective mutants (Fig. 6c-f) suggest how the midgut closure is triggered by the symbiont. First, the absence of the midgut closure by the fliC::Tn5 mutant that cannot pass through the CR, indicates that passage through this channel and/or M4 colonization is necessary to trigger the closure but that it cannot be triggered by bacteria that are located at the M3-side of the CR. Second, the results of the colonization-defective mutants purl::Tn5, ΔwaaF, and ΔphaB suggest that a weak colonization of the crypts is sufficient to trigger the midgut closure. Although the molecular mechanism of the midgut closure remains totally unknown, it appears likely that the bean bug recognizes the Burkholderia symbiont either in the CR or in the M4 by epithelial cells in a dose-dependent manner. In the legume-Rhizobium system, a lipochito-oligosaccharide (Nod factor) triggers the nodule formation [START_REF] Zipfel | Plant signalling in symbiosis and immunity[END_REF]. In the case of the bobtail squid, a peptidoglycan fragment, tracheal cytotoxin (TCT), derived from Vibrio fischeri alters morphogenesis of the light organ [START_REF] Koropatnick | Microbial factor-mediated development in a host-bacterial mutualism[END_REF][START_REF] Adin | Mutations in ampG and lytic transglycosylase genes affect the net release of peptidoglycan monomers from Vibrio fischeri[END_REF]. It is notable that in both cases, the host organisms recognize symbiont-associated molecular patterns via immune related receptors to trigger a developmental response [START_REF] Zipfel | Plant signalling in symbiosis and immunity[END_REF][START_REF] Chu | Innate immune recognition of the microbiota promotes host-microbial symbiosis[END_REF]. Gut immunity genes have been reported in R. pedestris [START_REF] Kim | Understanding regulation of the host-mediated gut symbiont population and the symbiont-mediated host immunity in the Riptortus-Burkholderia symbiosis system[END_REF][START_REF] Park | The roles of antimicrobial peptide, rip-thanatin, in the midgut of Riptortus pedestris[END_REF], which may be involved in the here described symbiont-dependent morphogenesis in this insect. The bean bug is an ideal experimental system, where reverse genetic approaches have been established in both the host and in the symbiotic Burkholderia [START_REF] Takeshita | Riptortus pedestris and Burkholderia symbiont: an ideal model system for insect-microbe symbiotic associations[END_REF].

Transcriptomic analyses of the CR during the closure, followed by RNAi screening, and identification of bacterial compounds triggering midgut closure in the absence of live bacteria, could be a strategy leading towards the understanding of the molecular basis underlying the symbiont-triggered morphogenesis in the bean bug.

We recently demonstrated that some non-symbiotic species of the genus Burkholderia and its outgroup Pandoraea can pass through the CR and stably but partially accommodate in the midgut crypts. Moreover, this colonization is beneficial for the host survival and development [START_REF] Itoh | Host-symbiont specificity determined by microbe-microbe competition in an insect gut[END_REF]. Notably, while E. coli and C. taiwanensis showed neither M4 colonization nor induction of the midgut closure (Fig. 6g andh), B. fungorum and P. norimbergensis colonized the midgut crypts and induced the closure (Fig. 6i andj), suggesting that the microbial factors triggering the morphological alteration are conserved in this bacterial lineage. This reveals a potential drawback of the midgut closure mechanism because when induced by non-symbiotic species, it can restrict the access of the native symbiont to the M4 region if the symbiont is not present in the first infection event.

However, these non-symbionts are rarely detected in natural populations of the bean bug [START_REF] Kikuchi | Gut symbiotic bacteria of the genus Burkholderia in the broad-headed bugs Riptortus clavatus and Leptocorisa chinensis (Heteroptera: Alydidae)[END_REF][START_REF] Kikuchi | Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation[END_REF][START_REF] Kikuchi | An ancient but promiscuous host-symbiont association between Burkholderia gut symbionts and their heteropteran hosts[END_REF]. Considering that the symbiotic Burkholderia, non-symbiotic Burkholderia and Pandoraea are very common bacterial groups in soil environments and frequently detected together [START_REF] Itoh | Bacterial population succession and adaptation affected by insecticide application and soil spraying history[END_REF][START_REF] Itoh | Infection dynamics of insecticide-degrading symbionts from soil to insects in response to insecticide spraying[END_REF][START_REF] Stopnisek | Genus-wide acid tolerance accounts for the biogeographical distribution of soil Burkholderia populations[END_REF], it is plausible that, in natural environments, these non-symbiotic species are usually ingested together with symbiotic Burkholderia and are then outcompeted by the native symbiont inside the midgut crypts [START_REF] Itoh | Host-symbiont specificity determined by microbe-microbe competition in an insect gut[END_REF], which ensures the specific association in natural populations of the bean bug.

Bottlenecks imposed on symbiont populations during their transmission or acquisition by the host are thought to strongly affect the population structure and evolution of symbionts [START_REF] Campbell | Changes in endosymbiont complexity drive host-level compensatory adaptations in cicadas[END_REF]. Such bottlenecks have been well described in insect symbioses with vertical transmission. These bottlenecks are large but vary from hundreds or thousands to several millions of symbiont cells transferred from mother to offspring, depending on the insect host and the specific transmission mode [START_REF] Campbell | Changes in endosymbiont complexity drive host-level compensatory adaptations in cicadas[END_REF][START_REF] Mira | Estimating population size and transmission bottlenecks in maternally transmitted endosymbiotic bacteria[END_REF][START_REF] Kaltenpoth | Life cycle and population dynamics of a protective insect symbiont reveal severe bottlenecks during vertical transmission[END_REF][START_REF] Hosokawa | How many symbionts are provided by mothers, acquired by offspring, and needed for successful vertical transmission in an obligate insect-bacterium mutualism?[END_REF]. On the contrary, in the few previously characterized symbioses without vertical transmission, symbionts seem to be selected from the environment in much smaller numbers; experimental studies on the legume-Rhizobium and squid-Vibrio model systems revealed that only a few symbiont cells (mostly only one bacterial cell) initiate the colonization, regardless of symbiont density in the environment [START_REF] Wollenberg | Population structure of Vibrio fischeri within the light organs of Euprymna scolopes squid from two Oahu (Hawaii) populations[END_REF][START_REF] Gage | Analysis of infection thread development using GFP-and DsRed-expressing Sinorhizobium meliloti[END_REF], suggesting a drastic partner choice in the initial stage of the infection. Relative large bottlenecks during vertical transmission seem to be compatible with the fact that the symbiont lineage has been pre-selected efficiently in the previous generations of the host while the very narrow bottlenecks in environmental transmission modes might be required to cope with the extreme richness of microbes in the environment. However, unlike the legume and squid model systems, we determined a symbiont infection bottleneck of about 4000 cells in R. pedestris, indicating a milder sorting, at least in the initial infection stage.

Thus, the bean bug seems to adopt a different strategy than legumes and the squid and enables a range of bacteria, including some non-symbiont species, to pass the gate of the symbiotic organ (i.e. the CR). However, it applies then a severe selection inside the crypts of the symbiotic organ where non-symbiotic bacteria are very efficiently outcompeted by symbiont species [START_REF] Itoh | Host-symbiont specificity determined by microbe-microbe competition in an insect gut[END_REF].

The CR is widely conserved among diverse stinkbug species that harbor gut symbiotic bacteria in midgut crypts [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF], including the Bukholderia-associated coreoid and lygaoeid species and members of the superfamily Pentatomoidea that vertically transmit gamma-proteobacterial symbionts. As the risk of contamination is a common issue in these gut symbiotic systems, it is plausible that the midgut closure after symbiont colonization is prevalent among the stink bug species. In the case of members of the Plataspidae and Urostylididae families belonging to the Pentatomoidea superfamily, the M3 and M4B are only joint by a thin connective tissue in adult insects [START_REF] Ohbayashi | Insect's intestinal organ for symbiont sorting[END_REF][START_REF] Schneider | Beiträge zur Kenntnis der symbiontischen Einrichtungen der Heteropteren[END_REF][START_REF] Fukatsu | Capsule-transmitted gut symbiotic bacterium of the Japanese common plataspid stinkbug, Megacopta punctatissima[END_REF][START_REF] Kaiwa | Symbiont-supplemented maternal investment underpinning host's ecological adaptation[END_REF], suggesting that the CR is indeed closed and completely disconnected from the M3 after symbiont infection in these species. However, in case of the brown-winged green bug Plautia stali, the CR becomes looser after the 5 th instar stage is reached and a food coloring can pass through from M3 to M4 [START_REF] Oishi | Morphogenesis and development of midgut symbiotic organ of the stinkbug Plautia stali (Hemiptera: Pentatomidae)[END_REF]. Thus, a morphological diversity of the CR exists among stinkbug species and may reflect a functional difference of this organ. This potential divergence requires comprehensive comparative analyses between different stinkbug species.

Recent microbiome analyses have revealed that a number of animals possess diverse but highly specialized microbiota in their gut, which are maintained by various physiochemical, molecular, and structural mechanisms. In human gut microbiota, mucus layers containing specific polysaccharides and antimicrobial peptides (AMPs) control the microbiota and facilitate specific colonization of Bacteroides and Acinatobacter in colon crypts [START_REF] Donaldson | Gut biogeography of the bacterial microbiota[END_REF]. In termites and honey bees, the hypoxia condition of the gut lumen remarkably contributes to shape their specific microbiota [START_REF] Utami | Phylogenetic diversity and single-cell genome analysis of "Melainabacteria", a non-photosynthetic cyanobacterial group, in the termite gut[END_REF][START_REF] Brune | The termite gut microflora as an oxygen sink: microelectrode determination of oxygen and pH gradients in guts of lower and higher termites[END_REF][START_REF] Zheng | Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling[END_REF]. In Drosophila, immune-related AMPs and reactive oxygen species limit the growth of pathogenic bacteria and play a significant role to establish its indigenous gut microbiota [START_REF] Buchon | Gut homeostasis in a microbial world: insights from Drosophila melanogaster[END_REF][START_REF] Lee | Gut microbiota-generated metabolites in animal health and disease[END_REF]. A more extreme case is found in the Sonoran Desert turtle ant, wherein a proventriculus valve located between the foregut and midgut filters out bacteria and keeps an exclusively specific microbiota in the midgut [START_REF] Lanan | A bacterial filter protects and structures the gut microbiome of an insect[END_REF]. Among these diverse mechanisms, the midgut closure reported here is an additional and unique mechanism to maintain the population of a specific bacterial partner and prevent chronic contamination with unwanted microbes. Our findings expand the understanding of how animals sustain specific gut symbiosis. In the Cm-RFP control experiment, 2 nd instar insects are kept aposymbiotic and fed with chloramphenicol (Cm) in the drinking water; after molting, these insects are infected with the RFP strain. In the GFP-RFP control experiment, 2 nd instar insects are infected with the GFP strain present in the drinking water and after molting, the 3 rd instars are exposed to the RFP strain present in the drinking water. In the GFP-Cm-RFP experiment, 2 nd instar insects are infected with the GFP strain present in the drinking water; after 24 h, this drinking water is replaced by chloramphenicol-containing drinking water; the 3 rd instars are then exposed to the RFP strain. In all infection schemes, the infection status was analyzed by dissection and microscopy in 3 dpi 2 nd instar insects and in 3 dpi

Fig. 1 .

 1 Fig. 1. Morphological alteration of the constricted region and M4B during symbiont

Fig. 2 .

 2 Fig. 2. Closing of the constricted region. (a) At 24 h after inoculation, the CR and the

Fig. 3 .

 3 Fig. 3. The closure of the constricted region and M4B during symbiont colonization in

Fig. 4 .

 4 Fig. 4. The midgut closure prevents subsequent infections. (a) Experimental set-up of

Fig. 5 .

 5 Fig. 5. The midgut closure is irreversible. (a) Infection schemes for three series of experiments. In the Cm-RFP control experiment, 2 nd instar insects are kept aposymbiotic

  3 rd instar insects. (b) Infection status in 2 nd instar insects. (c) Infection status in 3 rd instar insects. In (b) and (c), red and green fluorescence, DIC and merged images are shown. Note the "fluorescence" panels are merged images of red and green.

Fig. 6 .

 6 Fig. 6. Midgut closure requires passage through the constricted region and crypt colonization. (a) Infection scheme: 2 nd instar insects were infected with different GFP-labelled strains and after molting, 3 rd instars were infected with the wild type RFP

Fig. S3 .Fig. S4 .

 S3S4 Fig. S2. Morphological alteration of the constricted region and M4B during symbiont colonization in 2 nd instar nymphs of R. pedestris. (a-i) A GFP-labelled Burkholderia symbiont was orally administered, and the infection dynamics and morphogenesis of the M4B region were observed. Merged fluorescence and DIC images are shown. M4B is closed during symbiont infection (c-f) and reopened by backflow of symbiont cells during the colonization (g-i). Abbreviations: M3, midgut 3 rd section; M4, midgut 4 th section; CR, constricted region; M4B, M4 bulb. Bars: 0.1 mm.

Acknowledgements

We thank H. Ooi (AIST) for technical assistance. Part of the fluorescence microscopy was performed at the Imagerie-Gif facilities (http://www.i2bc.paris-saclay.

fr/spip.php?rubrique184). This study was supported by the JSPS-CNRS Bilateral Open Partnership Joint Research Project to YK and PM, the Ministry of Education, Culture,

Sports, Science and Technology (MEXT) KAKENHI to YK (18KK0211), and the JSPS Research Fellowship for Young Scientists to TO (20170267 and 19J01106) and SJ (201911493).

Author contributions

YK, TO and PM designed the study. YK, TO and SJ performed inoculation tests, antibiotic treatments, and microscopy observations. TO and PM measured morphological change of gut parts and bottleneck of symbiont infection. YK and PM wrote the manuscript.

Competing interests

The authors declare that they have no competing interests.

SUPPLEMENTARY INFORMATION

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

Yoshitomo Kikuchi, Tsubasa Ohbayashi, Seonghan Jang and Peter Mergaert

Supplementary Materials and Methods

Microscopic observations of dissected symbiotic organs

The insects administered with the GFP-labeled or RFP labelled strains were dissected in phosphate-buffered saline (PBS: 137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4 [pH7.4]) using fine forceps under a dissection microscope (S8APO, Leica; MZ FZ III, Leica). Pictures of the dissected tissues were taken by a digital camera (EC3, Leica) and the width of the CR and M4B were measured using ImageJ software (Schneider et al. 2012). To investigate the infection process, freshly dissected midguts were stained with 4',6-diamidino-2-phenylindole (DAPI) and observed under an epifluorescence microscope (DMI4000B, Leica). For analysis of phalladoidin-stained tissues, the dissected midguts were fixed with 4% paraformaldehyde for 10 min at room temperature, washed twice in PBS, incubated in PBS containing 0.1% Triron X-100 for 5 min, stained with 0.5 µM of SYTOX Green and 5 units/ml of Alexa Fluor555 phalloidin (Thermofisher) in PBS for 20 min, washed twice in PBS, and mounted on silane-coated glass slides and observed with a confocal laser scanning microscope (TCS SP8, Leica).

Oral inoculation of colonization-deficient mutants and non-symbiotic bacteria

Immediately after 1 st instar nymphs molted to the 2 nd instar, DWA was removed from the rearing containers so that the nymphs were kept without drinking water overnight and became thirsty. Then DWA containing 10 4 cells/µl of each GFP-labelled strain was supplied to the rearing containers for 24 h. After 24 h, the symbiont-containing DWA was replaced by symbiont-free DWA, and the nymphs were further reared in the absence of bacteria during the instar. After the 2 nd instar nymphs molted to 3 rd instar, DWA containing 10 4 cells/µl of the RFP-labelled KT39 was supplied to the rearing containers for 24 h. These nymphs were further reared for two days with symbiont-free DWA, and then dissected to check their infection status of the GFP and RFP strains.

Bottleneck estimation

To estimate how many symbiont cells can infect the symbiotic region before the midgut closure, co-inoculation of the GFP-labelled strain RPE225 (Rf r , Km r ) and the nonlabelled wild type strain RPE75 (Rf r ) was performed. The non-labelled symbiont and GFP-labelled symbiont were mixed in different ratios (GFP-labelled symbiont:nonlabelled symbiont = 1:10, 1:10 2 , 1:10 3 , 1:1.65x10 3 , 1:5.0x10 3 , 1:10 4 , 1:1.5x10 4 , 1: 4.6x10 4 or 1:2.2x10 5 ). Symbiont cells were diluted in DWA to a cell density of 10 5 CFU/µl, and 2 nd instar nymphs were fed with 1 µl of the solution as above. Six to sixteen insects were inoculated per dilution. Three days after inoculation, the symbiotic organ (M4+M4B) was dissected and its content was plated on a YG plate containing kanamycin 30 µg/ml to check whether GFP-labelled symbionts entered the symbiotic organ. Insects were counted as positive for infection with the GFP-labelled symbiont if at least one colony of the GFPlabelled symbiont was detected.

a These strains were used in the experimental removal of crypt-colonizing symbionts by antibiotic treatment (Fig. 5). b GFP/RFP was labelled by the Tn7 minitransposon system. c RFP was labelled with the RFP-encoding stable plasmid pIN29. In the oral administration experiments, second-instar nymphs were fed with GFP-labelled and non-labelled symbiont strains, mixed in different ratios (1:10; 1:10 2 ; 1:10 3 ; 1:1.65x10 3 ; 1:5.0x10 3 ; 1:10 4 ; 1:1.5x10 4 ; 1: 4.6x10 4 and 1:2.2x10 5 ), and the percentage of host individuals colonized by the GFP-labelled symbiont was subsequently determined by plating on selective medium. The dilution ratio at which 50% of the bean bugs carry the GFP-labelled symbiont, indicated by the dotted lines, was determined by using the drc package in R software; the equation probit is (y) = 2.2913+99.08/(1+exp(1.18*(x-3950)).