Specific Midgut Region Controlling the Symbiont Population in an Insect-Microbe Gut Symbiotic Association

Many insects possess symbiotic bacteria that affect the biology of the host. The level of the symbiont population in the host is a pivotal factor that modulates the biological outcome of the symbiotic association. Hence, the symbiont population should be maintained at a proper level by the host''s control mechanisms. Several mechanisms for controlling intracellular symbionts of insects have been reported, while mechanisms for controlling extracellular gut symbionts of insects are poorly understood. The bean bug Riptortus pedestris harbors a betaproteobacterial extracellular symbiont of the genus Burkholderia in the midgut symbiotic organ designated the M4 region. We found that the M4B region, which is directly connected to the M4 region, also harbors Burkholderia symbiont cells, but the symbionts therein are mostly dead. A series of experiments demonstrated that the M4B region exhibits antimicrobial activity, and the antimicrobial activity is specifically potent against the Burkholderia symbiont but not the cultured Burkholderia and other bacteria. The antimicrobial activity of the M4B region was detected in symbiotic host insects, reaching its highest point at the fifth instar, but not in aposymbiotic host insects, which suggests the possibility of symbiont-mediated induction of the antimicrobial activity. This antimicrobial activity was not associated with upregulation of antimicrobial peptides of the host. Based on these results, we propose that the M4B region is a specialized gut region of R. pedestris that plays a critical role in controlling the population of the Burkholderia gut symbiont. The molecular basis of the antimicrobial activity is of great interest and deserves future study.     

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host–symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host–symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host–symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed.     

Purine Biosynthesis,Biofilm Formation,and Persistence of an Insect-Microbe Gut Symbiosis

The Riptortus-Burkholderia symbiotic system is an experimental model system for studying the molecular mechanisms of an insect-microbe gut symbiosis. When the symbiotic midgut of Riptortus pedestris was investigated by light and transmission electron microscopy, the lumens of the midgut crypts that harbor colonizing Burkholderia symbionts were occupied by an extracellular matrix consisting of polysaccharides. This observation prompted us to search for symbiont genes involved in the induction of biofilm formation and to examine whether the biofilms are necessary for the symbiont to establish a successful symbiotic association with the host. To answer these questions, we focused on purN and purT, which independently catalyze the same step of bacterial purine biosynthesis. When we disrupted purN and purT in the Burkholderia symbiont, the ΔpurN and ΔpurT mutants grew normally, and only the ΔpurT mutant failed to form biofilms. Notably, the ΔpurT mutant exhibited a significantly lower level of cyclic-di-GMP (c-di-GMP) than the wild type and the ΔpurN mutant, suggesting involvement of the secondary messenger c-di-GMP in the defect of biofilm formation in the ΔpurT mutant, which might operate via impaired purine biosynthesis. The host insects infected with the ΔpurT mutant exhibited a lower infection density, slower growth, and lighter body weight than the host insects infected with the wild type and the ΔpurN mutant. These results show that the function of purT of the gut symbiont is important for the persistence of the insect gut symbiont, suggesting the intricate biological relevance of purine biosynthesis, biofilm formation, and symbiosis.     

The specificity of Burkholderia symbionts in the social amoeba farming symbiosis: Prevalence,species, genetic and phenotypic diversity

The establishment of symbioses between eukaryotic hosts and bacterial symbionts in nature is a dynamic process. The formation of such relationships depends on the life history of both partners. Bacterial symbionts of amoebae may have unique evolutionary trajectories to the symbiont lifestyle, because bacteria are typically ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis. In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia bacteria are able to resist amoebal digestion and maintain a persistent relationship that includes carriage throughout the amoeba's social cycle that culminates in spore formation. Some Burkholderia strains allow their host to carry other bacteria, as food. This carried food is released in new environments in a trait called farming. To better understand the diversity and prevalence of Burkholderia symbionts and the traits they impart to their amoebae hosts, we first screened 700 natural isolates of D. discoideum and found 25% infected with Burkholderia. We next used a multilocus phylogenetic analysis and identified two independent transitions by Burkholderia to the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from D. discoideum, as well as strains isolated from other sources, for traits relevant to symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the Burkholderia agricolaris, B. hayleyella, and B. bonniea species were able to form persistent symbiotic associations with D. discoideum. The Burkholderia–Dictyostelium relationship provides a promising arena for further studies of the pathway to symbiosis in a unique system.     

Insecticide applications to soil contribute to the development of Burkholderia mediating insecticide resistance in stinkbugs

Some soil Burkholderia strains are capable of degrading the organophosphorus insecticide, fenitrothion, and establish symbiosis with stinkbugs, making the host insects fenitrothion‐resistant. However, the ecology of the symbiotic degrading Burkholderia adapting to fenitrothion in the free‐living environment is unknown. We hypothesized that fenitrothion applications affect the dynamics of fenitrothion‐degrading Burkholderia, thereby controlling the transmission of symbiotic degrading Burkholderia from the soil to stinkbugs. We investigated changes in the density and diversity of culturable Burkholderia (i.e. symbiotic and nonsymbiotic fenitrothion degraders and nondegraders) in fenitrothion‐treated soil using microcosms. During the incubation with five applications of pesticide, the density of the degraders increased from less than the detection limit to around 10⁶/g of soil. The number of dominant species among the degraders declined with the increasing density of degraders; eventually, one species predominated. This process can be explained according to the competitive exclusion principle using V_max and K_m values for fenitrothion metabolism by the degraders. We performed a phylogenetic analysis of representative strains isolated from the microcosms and evaluated their ability to establish symbiosis with the stinkbug Riptortus pedestris. The strains that established symbiosis with R. pedestris were assigned to a cluster including symbionts commonly isolated from stinkbugs. The strains outside the cluster could not necessarily associate with the host. The degraders in the cluster predominated during the initial phase of degrader dynamics in the soil. Therefore, only a few applications of fenitrothion could allow symbiotic degraders to associate with their hosts and may cause the emergence of symbiont‐mediated insecticide resistance.     

Efficiency of partner choice and sanctions in Lotus is not altered by nitrogen fertilization

Eukaryotic hosts must exhibit control mechanisms to select against ineffective bacterial symbionts. Hosts can minimize infection by less-effective symbionts (partner choice) and can divest of uncooperative bacteria after infection (sanctions). Yet, such host-control traits are predicted to be context dependent, especially if they are costly for hosts to express or maintain. Legumes form symbiosis with rhizobia that vary in symbiotic effectiveness (nitrogen fixation) and can enforce partner choice as well as sanctions. In nature, legumes acquire fixed nitrogen from both rhizobia and soils, and nitrogen deposition is rapidly enriching soils globally. If soil nitrogen is abundant, we predict host control to be downregulated, potentially allowing invasion of ineffective symbionts. We experimentally manipulated soil nitrogen to examine context dependence in host control. We co-inoculated Lotus strigosus from nitrogen depauperate soils with pairs of Bradyrhizobium strains that vary in symbiotic effectiveness and fertilized plants with either zero nitrogen or growth maximizing nitrogen. We found efficient partner choice and sanctions regardless of nitrogen fertilization, symbiotic partner combination or growth season. Strikingly, host control was efficient even when L. strigosus gained no significant benefit from rhizobial infection, suggesting that these traits are resilient to short-term changes in extrinsic nitrogen, whether natural or anthropogenic.     

An ecological cost associated with protective symbionts of aphids

Beneficial symbioses are widespread and diverse in the functions they provide to the host ranging from nutrition to protection. However, these partnerships with symbionts can be costly for the host. Such costs, so called “direct costs”, arise from a trade‐off between allocating resources to symbiosis and other functions such as reproduction or growth. Ecological costs may also exist when symbiosis negatively affects the interactions between the host and other organisms in the environment. Although ecological costs can deeply impact the evolution of symbiosis, they have received little attention. The pea aphid Acyrthosiphon pisum benefits a strong protection against its main parasitoids from protective bacterial symbionts. The ecological cost of symbiont‐mediated resistance to parasitism in aphids was here investigated by analyzing aphid behavior in the presence of predatory ladybirds. We showed that aphids harboring protective symbionts expressed less defensive behaviors, thus suffering a higher predation than symbiont‐free aphids. Consequently, our study indicates that this underlined ecological cost may affect both the coevolutionary processes between symbiotic partners and the prevalence of such beneficial bacterial symbionts in host natural populations.     

Subcellular architecture and metabolic connection in the planktonic photosymbiosis between Collodaria (radiolarians) and their microalgae

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium). We showed that this symbiosis is very dynamic whereby symbionts interact with different host cells via extracellular vesicles within the colony. 3D electron microscopy revealed that the photosynthetic apparatus of the microalgae was more voluminous in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were stored in the symbiotic microalga in starch granules and purine crystals respectively. Nitrogen was also allocated to the algal nucleolus. In the host, low ¹³C transfer was detected in the Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (c. 40 ppm) and twofold higher in the host, whereas copper concentration increased in symbionts and was detected in the host cell and extracellular vesicles. Sulfur concentration was around two times higher in symbionts (chromatin and pyrenoid) than their host. This study improves our understanding on the functioning of this oceanic photosymbiosis and paves the way for more studies to further assess its biogeochemical significance.     

Loss and resiliency of social amoeba symbiosis under simulated warming

Anthropogenic global change is increasingly raising concerns about collapses of symbiotic interactions worldwide. Therefore, understanding how climate change affects symbioses remains a challenge and demands more study. Here, we look at how simulated warming affects the social ameba Dictyostelium discoideum and its relationship with its facultative bacterial symbionts, Paraburkholderia hayleyella and Paraburkholderia agricolaris. We cured and cross‐infected ameba hosts with different symbionts. We found that warming significantly decreased D. discoideum''s fitness, and we found no sign of local adaptation in two wild populations. Experimental warming had complex effects on these symbioses with responses determined by both symbiont and host. Neither of these facultative symbionts increases its hosts’ thermal tolerance. The nearly obligate symbiont with a reduced genome, P. hayleyella, actually decreases D. discoideum''s thermal tolerance and even causes symbiosis breakdown. Our study shows how facultative symbioses may have complex responses to global change.     

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.     

Bacterial Symbiont Transmission in the Wood-Boring Shipworm Bankia setacea (Bivalvia: Teredinidae)

The Teredinidae (shipworms) are a morphologically diverse group of marine wood-boring bivalves that are responsible each year for millions of dollars of damage to wooden structures in estuarine and marine habitats worldwide. They exist in a symbiosis with cellulolytic nitrogen-fixing bacteria that provide the host with the necessary enzymes for survival on a diet of wood cellulose. These symbiotic bacteria reside in distinct structures lining the interlamellar junctions of the gill. This study investigated the mode by which these nutritionally essential bacterial symbionts are acquired in the teredinid Bankia setacea. Through 16S ribosomal DNA (rDNA) sequencing, the symbiont residing within the B. setacea gill was phylogenetically characterized and shown to be distinct from previously described shipworm symbionts. In situ hybridization using symbiont-specific 16S rRNA-directed probes bound to bacterial ribosome targets located within the host gill coincident with the known location of the gill symbionts. These specific probes were then used as primers in a PCR-based assay which consistently detected bacterial rDNA in host gill (symbiont containing), gonad tissue, and recently spawned eggs, demonstrating the presence of symbiont cells in host ovary and offspring. These results suggest that B. setacea ensures successful inoculation of offspring through a vertical mode of symbiont transmission and thereby enables a broad distribution of larval settlement.     

Insecticide resistance governed by gut symbiosis in a rice pest,Cletus punctiger,under laboratory conditions

Resistance to toxins in insects is generally thought of as their own genetic trait, but recent studies have revealed that gut microorganisms could mediate resistance by detoxifying phytotoxins and man-made insecticides. By laboratory experiments, we here discovered a striking example of gut symbiont-mediated insecticide resistance in a serious rice pest, Cletus punctiger. The rice bug horizontally acquired fenitrothion-degrading Burkholderia through oral infection and housed it in midgut crypts. Fenitrothion-degradation test revealed that the gut-colonizing Burkholderia retains a high degrading activity of the organophosphate compound in the insect gut. This gut symbiosis remarkably increased resistance against fenitrothion treatment in the host rice bug. Considering that many stinkbug pests are associated with soil-derived Burkholderia, our finding strongly supports that a number of stinkbug species could gain resistance against insecticide simply by acquiring insecticide-degrading gut bacteria.     

Epithelial Cell Proliferation Arrest Induced by Lactate and Acetate from Lactobacillus casei and Bifidobacterium breve

In an attempt to identify and characterize how symbiotic bacteria of the gut microbiota affect the molecular and cellular mechanisms of epithelial homeostasis, intestinal epithelial cells were co-cultured with either Lactobacillus or Bifidobacterium as bona fide symbionts to examine potential gene modulations. In addition to genes involved in the innate immune response, genes encoding check-point molecules controlling the cell cycle were among the most modulated in the course of these interactions. In the m-ICcl2 murine cell line, genes encoding cyclin E1 and cyclin D1 were strongly down regulated by L. casei and B. breve respectively. Cell proliferation arrest was accordingly confirmed. Short chain fatty acids (SCFA) were the effectors of this modulation, alone or in conjunction with the acidic pH they generated. These results demonstrate that the production of SCFAs, a characteristic of these symbiotic microorganisms, is potentially an essential regulatory effector of epithelial proliferation in the gut.     

Multiple microalgal partners in symbiosis with the acantharian Acanthochiasma sp. (Radiolaria)

Acantharia (Radiolaria) are widespread and abundant heterotrophic marine protists, some of which can host endosymbiotic eukaryotic microalgae. Although this photosymbiotic association was first described at the end of the 19th century, the diversity of the symbiotic microalgae remains poorly characterized. Here, we examined the identity of the microalgae associated with the acantharian species Acanthochiasma sp. by sequencing partial 18S and internal transcribed spacer (ITS) ribosomal DNA genes from cultured symbionts and directly from isolated holobiont specimens. Single Acanthochiasma cells contained multiple symbiotic partners, including distantly related dinoflagellates (Heterocapsa sp., Pelagodinium sp., Azadinium sp. and Scrippsiella sp.) as well as a haptophyte (Chrysochromulina sp.). This original association of multiple symbiotic microalgae within a single host cell raises questions about the specificity and functioning of the relationship. These microalgae exhibit the common ecological feature of being abundant and widely distributed in coastal and oceanic waters, some occasionally forming extensive blooms. Some of the microalgal genera found in association with Acanthochiasma (i.e. Pelagodinium and Chrysochromulina) are known to occur in symbiosis with other heterotrophic protists such as Foraminifera and other Radiolaria, whereas Heterocapsa, Scrippsiella and Azadinium have never previously been reported to be involved in putative symbiotic relationships. The unusual association unveiled in this study contributes to our understanding of the ecological and evolutionary significance of photosymbiosis in Acantharia and also provides new insights into the nature of such partnerships in the planktonic realm.     

Host-symbiont co-speciation and reductive genome evolution in gut symbiotic bacteria of acanthosomatid stinkbugs

Background  

Host-symbiont co-speciation and reductive genome evolution have been commonly observed among obligate endocellular insect symbionts, while such examples have rarely been identified among extracellular ones, the only case reported being from gut symbiotic bacteria of stinkbugs of the family Plataspidae. Considering that gut symbiotic communities are vulnerable to invasion of foreign microbes, gut symbiotic associations have been thought to be evolutionarily not stable. Stinkbugs of the family Acanthosomatidae harbor a bacterial symbiont in the midgut crypts, the lumen of which is completely sealed off from the midgut main tract, thereby retaining the symbiont in the isolated cryptic cavities. We investigated histological, ecological, phylogenetic, and genomic aspects of the unique gut symbiosis of the acanthosomatid stinkbugs.     

Bacterial Symbionts of a Devastating Coffee Plant Pest,the Stinkbug Antestiopsis thunbergii (Hemiptera: Pentatomidae)

Stinkbugs of the genus Antestiopsis, so-called antestia bugs or variegated coffee bugs, are notorious pests of coffee plants in Africa. We investigated the symbiotic bacteria associated with Antestiopsis thunbergii, a major coffee plant pest in Rwanda. PCR, cloning, sequencing, and phylogenetic analysis of bacterial genes identified four distinct bacterial lineages associated with A. thunbergii: a gammaproteobacterial gut symbiont and symbionts representing the genera Sodalis, Spiroplasma, and Rickettsia. In situ hybridization showed that the gut symbiont densely occupied the lumen of midgut crypts, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont sparsely and sporadically infected various cells and tissues. Diagnostic PCR survey of 154 A. thunbergii individuals collected at 8 localities in Rwanda revealed high infection frequencies (100% for the gut symbiont, 51.3% for the Sodalis symbiont, 52.6% for the Spiroplasma symbiont, and 24.0% for the Rickettsia symbiont). These results suggest that the gut symbiont is the primary symbiotic associate of obligate nature for A. thunbergii, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont are the secondary symbiotic associates of facultative nature. We observed high coinfection frequencies, i.e., 7.8% of individuals with quadruple infection with all the symbionts, 32.5% with triple infections with the gut symbiont and two of the secondary symbionts, and 39.6% with double infections with the gut symbiont and any of the three secondary symbionts, which were statistically not different from the expected coinfection frequencies and probably reflected random associations. The knowledge of symbiotic microbiota in A. thunbergii will provide useful background information for controlling this devastating coffee plant pest.     

OmpA-Mediated Biofilm Formation Is Essential for the Commensal Bacterium Sodalis glossinidius To Colonize the Tsetse Fly Gut

Many bacteria successfully colonize animals by forming protective biofilms. Molecular processes that underlie the formation and function of biofilms in pathogenic bacteria are well characterized. In contrast, the relationship between biofilms and host colonization by symbiotic bacteria is less well understood. Tsetse flies (Glossina spp.) house 3 maternally transmitted symbionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined that Sodalis forms biofilms in the tsetse gut and that this process is influenced by the Sodalis outer membrane protein A (OmpA). Mutant Sodalis strains that do not produce OmpA (Sodalis ΔOmpA mutants) fail to form biofilms in vitro and are unable to colonize the tsetse gut unless endogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms, Sodalis ΔOmpA mutant cells are exposed to and eliminated by tsetse''s innate immune system, suggesting that biofilms help Sodalis evade the host immune system. Tsetse is the sole vector of pathogenic African trypanosomes, which also reside in the fly gut. Acquiring a better understanding of the dynamics that promote Sodalis colonization of the tsetse gut may enhance the development of novel disease control strategies.     

A single-cell view of ammonium assimilation in coral–dinoflagellate symbiosis

Assimilation of inorganic nitrogen from nutrient-poor tropical seas is an essential challenge for the endosymbiosis between reef-building corals and dinoflagellates. Despite the clear evidence that reef-building corals can use ammonium as inorganic nitrogen source, the dynamics and precise roles of host and symbionts in this fundamental process remain unclear. Here, we combine high spatial resolution ion microprobe imaging (NanoSIMS) and pulse-chase isotopic labeling in order to track the dynamics of ammonium incorporation within the intact symbiosis between the reef-building coral Acropora aspera and its dinoflagellate symbionts. We demonstrate that both dinoflagellate and animal cells have the capacity to rapidly fix nitrogen from seawater enriched in ammonium (in less than one hour). Further, by establishing the relative strengths of the capability to assimilate nitrogen for each cell compartment, we infer that dinoflagellate symbionts can fix 14 to 23 times more nitrogen than their coral host cells in response to a sudden pulse of ammonium-enriched seawater. Given the importance of nitrogen in cell maintenance, growth and functioning, the capability to fix ammonium from seawater into the symbiotic system may be a key component of coral nutrition. Interestingly, this metabolic response appears to be triggered rapidly by episodic nitrogen availability. The methods and results presented in this study open up for the exploration of dynamics and spatial patterns associated with metabolic activities and nutritional interactions in a multitude of organisms that live in symbiotic relationships.     

Evidence of Environmental and Vertical Transmission of Burkholderia Symbionts in the Oriental Chinch Bug,Cavelerius saccharivorus (Heteroptera: Blissidae)

The vertical transmission of symbiotic microorganisms is omnipresent in insects, while the evolutionary process remains totally unclear. The oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae), is a serious sugarcane pest, in which symbiotic bacteria densely populate the lumen of the numerous tubule-like midgut crypts that the chinch bug develops. Cloning and sequence analyses of the 16S rRNA genes revealed that the crypts were dominated by a specific group of bacteria belonging to the genus Burkholderia of the Betaproteobacteria. The Burkholderia sequences were distributed into three distinct clades: the Burkholderia cepacia complex (BCC), the plant-associated beneficial and environmental (PBE) group, and the stinkbug-associated beneficial and environmental group (SBE). Diagnostic PCR revealed that only one of the three groups of Burkholderia was present in ∼89% of the chinch bug field populations tested, while infections with multiple Burkholderia groups within one insect were observed in only ∼10%. Deep sequencing of the 16S rRNA gene confirmed that the Burkholderia bacteria specifically colonized the crypts and were dominated by one of three Burkholderia groups. The lack of phylogenetic congruence between the symbiont and the host population strongly suggested host-symbiont promiscuity, which is probably caused by environmental acquisition of the symbionts by some hosts. Meanwhile, inspections of eggs and hatchlings by diagnostic PCR and egg surface sterilization demonstrated that almost 30% of the hatchlings vertically acquire symbiotic Burkholderia via symbiont-contaminated egg surfaces. The mixed strategy of symbiont transmission found in the oriental chinch bug might be an intermediate stage in evolution from environmental acquisition to strict vertical transmission in insects.