Insect-Microbe Mutualism without Vertical Transmission: a Stinkbug Acquires a Beneficial Gut Symbiont from the Environment Every Generation

The broad-headed bug Riptortus clavatus (Heteroptera: Alydidae) possesses a number of crypts at a posterior midgut region, which house a dense population of a bacterial symbiont belonging to the genus Burkholderia. Although the symbiont is highly prevalent (95 to 100%) in the host populations, the symbiont phylogeny did not reflect the host systematics at all. In order to understand the mechanisms underlying the promiscuous host-symbiont relationship despite the specific and prevalent association, we investigated the transmission mode and the fitness effects of the Burkholderia symbiont in R. clavatus. Inspection of eggs and a series of rearing experiments revealed that the symbiont is not vertically transmitted but is environmentally acquired by nymphal insects. The Burkholderia symbiont was present in the soil of the insect habitat, and a culture strain of the symbiont was successfully isolated from the insect midgut. Rearing experiments by using sterilized soybean bottles demonstrated that the cultured symbiont is able to establish a normal and efficient infection in the host insect, and the symbiont infection significantly improves the host fitness. These results indicated that R. clavatus postnatally acquires symbiont of a beneficial nature from the environment every generation, uncovering a previously unknown pathway through which a highly specific insect-microbe association is maintained. We suggest that the stinkbug-Burkholderia relationship may be regarded as an insect analogue of the well-known symbioses between plants and soil-associated microbes, such as legume-Rhizobium and alder-Frankia relationships, and we discuss the evolutionary relevance of the mutualistic but promiscuous insect-microbe association.     

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.     

Primary Gut Symbiont and Secondary,Sodalis-Allied Symbiont of the Scutellerid Stinkbug Cantao ocellatus

Symbiotic associations with midgut bacteria have been commonly found in diverse phytophagous heteropteran groups, where microbiological characterization of the symbiotic bacteria has been restricted to the stinkbug families Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae. Here we investigated the midgut bacterial symbiont of Cantao ocellatus, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified from the insects of different geographic origins. The bacterium was detected in all 116 insects collected from 9 natural host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, not closely related to gut symbionts of other stinkbugs. Diagnostic PCR and in situ hybridization demonstrated that the bacterium is extracellularly located in the midgut 4th section with crypts. Electron microscopy of the crypts revealed a peculiar histological configuration at the host-symbiont interface. Egg sterilization experiments confirmed that the bacterium is vertically transmitted to stinkbug nymphs via egg surface contamination. In addition to the gut symbiont, some individuals of C. ocellatus harbored another bacterial symbiont in their gonads, which was closely related to Sodalis glossinidius, the secondary endosymbiont of tsetse flies. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.Insects are among the largest animal groups on the earth, embracing 750,000 to several millions of species (37, 52). Diverse insects are symbiotically associated with microorganisms, especially bacteria (5-7). In some insects, symbiotic bacteria are harbored in specialized host cells called bacteriocytes (or mycetocytes), constituting obligate mutualistic associations. For example, Buchnera aphidicola is harbored within bacteriocytes in the abdominal body cavity of almost all aphids and provides essential amino acids that are lacking in the phloem sap diet of the insects (9, 47). Wigglesworthia glossinidia is localized in a midgut-associated bacteriome of tsetse flies and plays pivotal roles in biosynthesis of B vitamins that are deficient in the vertebrate blood diet of the insects (2, 34). These obligate endocellular symbionts are often collectively referred to as “primary symbionts.”In contrast, there are facultative endosymbiotic microorganisms not essential for their host insects, often collectively called “secondary symbionts.” For example, many aphids are known to harbor various facultative symbionts, which belong to distinct lineages in the Gamma- and Alphaproteobacteria (33, 43) and the Mollicutes (10). While the majority of those facultative bacteria are either parasitic or commensalistic for their hosts, some of them affect the host fitness beneficially in particular ecological contexts (29, 32, 36, 44, 51). In addition to the obligate primary symbiont Wigglesworthia, tsetse flies harbor the facultative secondary symbiont Sodalis glossinidius, whose biological function for the hosts is currently elusive (3, 8).Members of the suborder Heteroptera, known as true bugs and consisting of over 38,000 described species, are characterized by their sucking mouthparts, half-membranous forewings, and incomplete metamorphosis (46). In the Heteroptera, symbiotic associations with bacteria are mainly found in phytophagous groups, especially in stinkbugs of the infraorder Pentatomomorpha. These stinkbugs generally possess many sacs or tubular outgrowths, called crypts or ceca, in a posterior region of the midgut, whose lumen is densely populated by a specific bacterial symbiont (7, 16). In some cases, experimental elimination of the symbiotic bacteria resulted in retarded growth and high mortality of the host insects (1, 13, 21, 26, 27, 39), indicating that these gut symbionts play important biological roles. Most of the gut symbionts are vertically transmitted through host generations by such mechanisms as egg surface contamination in the families Pentatomidae and Acanthosomatidae (1, 27, 39, 40, 42), coprophagy in the Cydnidae and Coreidae (22, 45), and capsule transmission in the Plataspidae (20), whereas a case of environmental acquisition has been reported from the Alydidae (26). Thus far, gut symbiotic bacteria of some members of the Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae have been characterized using molecular techniques (21, 23, 25, 27, 38), while phylogenetic and biological aspects of gut symbiotic bacteria have been untouched in many other stinkbug groups.These gut symbiotic bacteria are, despite their extracellular localization, regarded as “primary symbionts” of the stinkbugs. On the other hand, some stinkbugs may, in addition to the gut symbiotic bacteria, also be associated with facultative “secondary symbionts.” For example, Wolbachia infections have been detected from diverse stinkbugs, most of which are probably of parasitic or commensalistic nature (24). Besides Wolbachia, there has been no report on facultative, secondary symbionts from stinkbugs.Members of the family Scutelleridae, often referred to as jewel bugs or shield-backed bugs, are stinkbugs characterized by their greatly enlarged convex scutellum that usually covers the entire abdomen. Some tropical species are also known for their vivid and beautiful body coloration (46). The family contains approximately 80 genera and 450 species, and in Japan, at least 7 genera and 9 species have been recorded (50). In the early 20th century, the presence of symbiotic bacteria was histologically described in midgut crypts of several scutellerid species (16, 31, 42). Since these pioneer works, however, no studies have been conducted on the symbiotic bacteria of scutellerid stinkbugs.Here we investigated the midgut symbiont of Cantao ocellatus, a scutellerid stinkbug widely distributed in Asian countries, including Japan, and known to guard their eggs and newborn nymphs (Fig. ​(Fig.1A)1A) (50). In addition to the gut symbiont, we also identified a Sodalis-allied facultative secondary symbiont from gonads of the insect.Open in a separate windowFIG. 1.(A) Adult female of Cantao ocellatus, guarding hatchlings under her body. (B) Dissected midgut from an adult female of C. ocellatus. 1st, midgut 1st section; 2nd, midgut 2nd section; 3rd, midgut 3rd section; 4th, midgut 4th section with crypts; hg, hindgut. (C) Enlarged image of the midgut 4th section with crypts. Arrowheads indicate three rows of crypts, while a fourth row is hidden behind. Glandular crypts (gc) are developed in adult females specifically, which may be involved in egg surface contamination with the symbiont. (D) An in situ hybridization image of the midgut 4th section, in which red and green signals indicate the gut symbiont and the host nucleus, respectively. Each arrow shows a crypt. (E) An enlarged image of the symbiotic bacteria in the crypts.     

Female-Specific Specialization of a Posterior End Region of the Midgut Symbiotic Organ in Plautia splendens and Allied Stinkbugs

Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae.     

Differential Metabolism of Exopolysaccharides from Probiotic Lactobacilli by the Human Gut Symbiont Bacteroides thetaiotaomicron

Probiotic microorganisms are ingested as food or supplements and impart positive health benefits to consumers. Previous studies have indicated that probiotics transiently reside in the gastrointestinal tract and, in addition to modulating commensal species diversity, increase the expression of genes for carbohydrate metabolism in resident commensal bacterial species. In this study, it is demonstrated that the human gut commensal species Bacteroides thetaiotaomicron efficiently metabolizes fructan exopolysaccharide (EPS) synthesized by probiotic Lactobacillus reuteri strain 121 while only partially degrading reuteran and isomalto/malto-polysaccharide (IMMP) α-glucan EPS polymers. B. thetaiotaomicron metabolized these EPS molecules via the activation of enzymes and transport systems encoded by dedicated polysaccharide utilization loci specific for β-fructans and α-glucans. Reduced metabolism of reuteran and IMMP α-glucan EPS molecules may be due to reduced substrate binding by components of the starch utilization system (sus). This study reveals that microbial EPS substrates activate genes for carbohydrate metabolism in B. thetaiotaomicron and suggests that microbially derived carbohydrates provide a carbohydrate-rich reservoir for B. thetaiotaomicron nutrient acquisition in the gastrointestinal tract.     

Dietary Fructose and Microbiota-Derived Short-Chain Fatty Acids Promote Bacteriophage Production in the Gut Symbiont Lactobacillus reuteri

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Phylogeny of Symbiotic Genes and the Symbiotic Properties of Rhizobia Specific to Astragalus glycyphyllos L.

The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99–100% in the case of nodAC and nifH genes, and 98–99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar “glycyphyllae”, based on nodA and nodC genes sequences.     

A Ribose-Scavenging System Confers Colonization Fitness on the Human Gut Symbiont Bacteroides thetaiotaomicron in a Diet-Specific Manner

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Evolutionary Origin and Host Range of Plagiotoma lumbrici (Ciliophora,Hypotrichia), an Obligate Gut Symbiont of Lumbricid Earthworms

Four common earthworm species, the anecic Lumbricus terrestris, the endogeic Octolasion tyrteum as well as the epigeic Eisenia fetida and Dendrobaena veneta, were examined for the presence of the microbial gut symbiont Plagiotoma lumbrici. The evolutionary origin of this endobiotic microbe was reconstructed, using the 18S rRNA gene, the ITS1‐5.8S‐ITS2 region, and the first two domains of the 28S rRNA gene. Plagiotoma lumbrici was exclusively detected in the anecic Lumbricus terrestris. Multigene analyses and the ITS2 secondary structure robustly determined the phylogenetic home of Plagiotoma lumbrici populations within the oxytrichid Dorsomarginalia (Spirotrichea: Hypotrichia) as a sister taxon of the free‐living Hemiurosomoida longa. This indicates that earthworms obtained their gut endosymbiont by ingesting soil/leaf litter containing oxytrichine ciliates that became adapted to the intestinal tract of earthworms. Interestingly, according to the literature data, Plagiotoma lumbrici was detected in multiple anecic and some epigeic but never in endogeic earthworms. These observations suggest that Plagiotoma lumbrici might be adapted to certain gut conditions and the lifestyle of anecic Lumbricidae, such as Lumbricus, Aporrectodea, and Scherotheca, as well as of some co‐occurring epigeic Lumbricus species.     

Capsule-Transmitted Gut Symbiotic Bacterium of the Japanese Common Plataspid Stinkbug, Megacopta punctatissima

The Japanese common plataspid stinkbug, Megacopta punctatissima, deposits small brown particles, or symbiont capsules, on the underside of the egg mass for the purpose of transmission of symbiotic bacteria to the offspring. We investigated the microbiological aspects of the bacteria contained in the capsule, such as microbial diversity, phylogenetic placement, localization in vivo, and fitness effects on the host insect. Restriction fragment length polymorphism analysis of 16S ribosomal DNA clones revealed that a single bacterial species dominates the microbiota in the capsule. The bacterium was not detected in the eggs but in the capsules, which unequivocally demonstrated that the bacterium is transmitted to the offspring of the insect orally rather than transovarially, through probing of the capsule content. Molecular phylogenetic analysis showed that the bacterium belongs to the γ-subdivision of the Proteobacteria. In adult insects the bacterium was localized in the posterior section of the midgut. Deprivation of the bacterium from the nymphs resulted in retarded development, arrested growth, abnormal body coloration, and other symptoms, suggesting that the bacterium is essential for normal development and growth of the host insect.     

Phenotypic Variance Predicts Symbiont Population Densities in Corals: A Modeling Approach

Background

We test whether the phenotypic variance of symbionts (Symbiodinium) in corals is closely related with the capacity of corals to acclimatize to increasing seawater temperatures. Moreover, we assess whether more specialist symbionts will increase within coral hosts under ocean warming. The present study is only applicable to those corals that naturally have the capacity to support more than one type of Symbiodinium within the lifetime of a colony; for example, Montastraea annularis and Montastraea faveolata.

Methodology/Principal Findings

The population dynamics of competing Symbiodinium symbiont populations were projected through time in coral hosts using a novel, discrete time optimal–resource model. Models were run for two Atlantic Ocean localities. Four symbiont populations, with different environmental optima and phenotypic variances, were modeled to grow, divide, and compete in the corals under seasonal fluctuations in solar insolation and seawater temperature. Elevated seawater temperatures were input into the model 1.5°C above the seasonal summer average, and the symbiont population response was observed for each location. The models showed dynamic fluctuations in Symbiodinium populations densities within corals. Population density predictions for Lee Stocking Island, the Bahamas, where temperatures were relatively homogenous throughout the year, showed a dominance of both type 2, with high phenotypic variance, and type 1, a high-temperature and high-insolation specialist. Whereas the densities of Symbiodinium types 3 and 4, a high-temperature, low-insolation specialist, and a high-temperature, low-insolation generalist, remained consistently low. Predictions for Key Largo, Florida, where environmental conditions were more seasonally variable, showed the coexistence of generalists (types 2 and 4) and low densities of specialists (types 1 and 3). When elevated temperatures were input into the model, population densities in corals at Lee Stocking Island showed an emergence of high-temperature specialists. However, even under high temperatures, corals in the Florida Keys were dominated by generalists.

Conclusions/Significance

Predictions at higher seawater temperatures showed endogenous shuffling and an emergence of the high-temperature Symbiodinium specialists, even though their phenotypic variance was low. The model shows that sustaining these “hidden” specialists becomes advantageous under thermal stress conditions, and shuffling symbionts may increase the corals'' capacity to acclimatize but not adapt to climatechange–induced ocean warming.     

Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis.     

Genome-Wide Association Studies of the Human Gut Microbiota

The bacterial composition of the human fecal microbiome is influenced by many lifestyle factors, notably diet. It is less clear, however, what role host genetics plays in dictating the composition of bacteria living in the gut. In this study, we examined the association of ~200K host genotypes with the relative abundance of fecal bacterial taxa in a founder population, the Hutterites, during two seasons (n = 91 summer, n = 93 winter, n = 57 individuals collected in both). These individuals live and eat communally, minimizing variation due to environmental exposures, including diet, which could potentially mask small genetic effects. Using a GWAS approach that takes into account the relatedness between subjects, we identified at least 8 bacterial taxa whose abundances were associated with single nucleotide polymorphisms in the host genome in each season (at genome-wide FDR of 20%). For example, we identified an association between a taxon known to affect obesity (genus Akkermansia) and a variant near PLD1, a gene previously associated with body mass index. Moreover, we replicate a previously reported association from a quantitative trait locus (QTL) mapping study of fecal microbiome abundance in mice (genus Lactococcus, rs3747113, P = 3.13 x 10⁻⁷). Finally, based on the significance distribution of the associated microbiome QTLs in our study with respect to chromatin accessibility profiles, we identified tissues in which host genetic variation may be acting to influence bacterial abundance in the gut.     

Specific Eco-evolutionary Contexts in the Mouse Gut Reveal Escherichia coli Metabolic Versatility

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A Cell-Surface GH9 Endo-Glucanase Coordinates with Surface Glycan-Binding Proteins to Mediate Xyloglucan Uptake in the Gut Symbiont Bacteroides ovatus

Dietary fiber is an important food source for members of the human gut microbiome. Members of the dominant Bacteroidetes phylum capture diverse polysaccharides via the action of multiple cell surface proteins encoded within polysaccharide utilization loci (PUL). The independent activities of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) for the harvest of various glycans have been studied in detail, but how these proteins work together to coordinate uptake is poorly understood. Here, we combine genetic and biochemical approaches to discern the interplay between the BoGH9 endoglucanase and the xyloglucan-binding proteins SGBP-A and SGBP-B from the Bacteroides ovatus xyloglucan utilization locus (XyGUL). The expression of BoGH9, a weakly active xyloglucanase in isolation, is required in a strain that expresses a non-binding version of SGBP-A (SGBP-A*). The crystal structure of the BoGH9 enzyme suggests the molecular basis for its robust activity on mixed-linkage β-glucan compared to xyloglucan. However, catalytically inactive site-directed mutants of BoGH9 fail to complement the deletion of the active BoGH9 in a SGBP-A* strain. We also find that SGBP-B is needed in an SGBP-A* background to support growth on xyloglucan, but that the non-binding SGBP-B* protein acts in a dominant negative manner to inhibit growth on xyloglucan. We postulate a model whereby the SGBP-A, SGBP-B, and BoGH9 work together at the cell surface, likely within a discrete complex, and that xyloglucan binding by SGBP-B and BoGH9 may facilitate the orientation of the xyloglucan for transfer across the outer membrane.     

Association of the Hepatocyte Growth Factor Gene with Keratoconus in an Australian Population

Purpose

A previous study has indicated suggestive association of the hepatocyte growth factor (HGF) gene with Keratoconus. We wished to assess this association in an independent Caucasian cohort as well as assess its association with corneal curvature.

Participants

Keratoconus patients were recruited from private and public clinics in Melbourne, Australia. Non-keratoconic individuals were identified from the Genes in Myopia (GEM) study from Australia. A total of 830 individuals were used for the analysis including 157 keratoconic and 673 non keratoconic subjects.

Methods

Tag single nucleotide polymorphisms (tSNPs) were chosen to encompass the hepatocyte growth factor gene as well as 2 kb upstream of the start codon through to 2 kb downstream of the stop codon. Logistic and linear regression including age and gender as covariates were applied in statistical analysis with subsequent Bonferroni correction.

Results

Ten tSNPs were genotyped. Following statistical analysis and multiple testing correction, a statistically significant association was found for the tSNP rs2286194 {p = 1.1×10-³ Odds Ratio 0.52, 95% CI - 0.35, 0.77} for keratoconus. No association was found between the 10 tSNPs and corneal curvature.

Conclusions

These findings provide additional evidence of significant association of the HGF gene with Keratoconus. This association does not appear to act through the corneal curvature route.