Drastic Genome Reduction in an Herbivore's Pectinolytic Symbiont.

Pectin, an integral component of the plant cell wall, is a recalcitrant substrate against enzymatic challenges by most animals. In characterizing the source of a leaf beetle's (Cassida rubiginosa) pectin-degrading phenotype, we demonstrate its dependency on an extracellular bacterium housed in specialized organs connected to the foregut. Despite possessing the smallest genome (0.27 Mb) of any organism not subsisting within a host cell, the symbiont nonetheless retained a functional pectinolytic metabolism targeting the polysaccharide's two most abundant classes: homogalacturonan and rhamnogalacturonan I. Comparative transcriptomics revealed pectinase expression to be enriched in the symbiotic organs, consistent with enzymatic buildup in these structures following immunostaining with pectinase-targeting antibodies. Symbiont elimination results in a drastically reduced host survivorship and a diminished capacity to degrade pectin. Collectively, our findings highlight symbiosis as a strategy for an herbivore to metabolize one of nature's most complex polysaccharides and a universal component of plant tissues.

Host hydrocarbons protect symbiont transmission from a radical host defense.

Symbioses with microbes play a pivotal role in the evolutionary success of insects, and can lead to intimate host-symbiont associations. However, how the host maintains a stable symbiosis with its beneficial partners while keeping antagonistic microbes in check remains incompletely understood. Here, we uncover a mechanism by which a host protects its symbiont from the host's own broad-range antimicrobial defense during transmission. Beewolves, a group of solitary digger wasps (Hymenoptera: Crabronidae), provide their brood cells with symbiotic Streptomyces bacteria that are later transferred to the cocoon and protect the offspring from opportunistic pathogens by producing antibiotics. In the brood cell, however, the symbiont-containing secretion is exposed to a toxic burst of nitric oxide (NO) released by the beewolf egg, which effectively kills antagonistic microorganisms. How the symbiont survives this lethal NO burst remained unknown. Here, we report that upon NO exposure in vitro, the symbionts mount a global stress response, but this is insufficient to ensure survival at brood cell-level NO concentrations. Instead, in vivo bioassays demonstrate that the host's antennal gland secretion (AGS) surrounding the symbionts in the brood cell provides an effective diffusion barrier against NO. This physicochemical protection can be reconstituted in vitro by beewolf hydrocarbon extracts and synthetic hydrocarbons, indicating that the host-derived long-chain alkenes and alkanes in the AGS are responsible for shielding the symbionts from NO. Our results reveal how host adaptations can protect a symbiont from host-generated oxidative and nitrosative stress during transmission, thereby efficiently balancing pathogen defense and mutualism maintenance.

Incipient genome erosion and metabolic streamlining for antibiotic production in a defensive symbiont.

Genome erosion is a frequently observed result of relaxed selection in insect nutritional symbionts, but it has rarely been studied in defensive mutualisms. Solitary beewolf wasps harbor an actinobacterial symbiont of the genus Streptomyces that provides protection to the developing offspring against pathogenic microorganisms. Here, we characterized the genomic architecture and functional gene content of this culturable symbiont using genomics, transcriptomics, and proteomics in combination with in vitro assays. Despite retaining a large linear chromosome (7.3 Mb), the wasp symbiont accumulated frameshift mutations in more than a third of its protein-coding genes, indicative of incipient genome erosion. Although many of the frameshifted genes were still expressed, the encoded proteins were not detected, indicating post-transcriptional regulation. Most pseudogenization events affected accessory genes, regulators, and transporters, but "Streptomyces philanthi" also experienced mutations in central metabolic pathways, resulting in auxotrophies for biotin, proline, and arginine that were confirmed experimentally in axenic culture. In contrast to the strong A+T bias in the genomes of most obligate symbionts, we observed a significant G+C enrichment in regions likely experiencing reduced selection. Differential expression analyses revealed that-compared to in vitro symbiont cultures-"S. philanthi" in beewolf antennae showed overexpression of genes for antibiotic biosynthesis, the uptake of host-provided nutrients and the metabolism of building blocks required for antibiotic production. Our results show unusual traits in the early stage of genome erosion in a defensive symbiont and suggest tight integration of host-symbiont metabolic pathways that effectively grants the host control over the antimicrobial activity of its bacterial partner.

Protection of a defensive symbiont does not constrain the composition of the multifunctional hydrocarbon profile in digger wasps.

Hydrocarbons (HCs) fulfil indispensable functions in insects, protecting against desiccation and serving chemical communication. However, the link between composition and function, and the selection pressures shaping HC profiles remain poorly understood. Beewolf digger wasps (Hymenoptera: Crabronidae) use an antennal gland secretion rich in linear unsaturated HCs to form a hydrophobic barrier around their defensive bacterial symbiont, protecting it from brood cell fumigation by toxic egg-produced nitric oxide (NO). Virtually identical HC compositions mediate desiccation protection and prey preservation from moulding in underground beewolf brood cells. It is unknown whether this composition presents an optimized adaptation to all functions, or a compromise due to conflicting selection pressures. Here, we reconstitute the NO barrier with single and binary combinations of synthetic linear saturated and unsaturated HCs, corresponding to HCs found in beewolves. The results show that pure alkanes as well as 3 : 1 mixtures of alkanes and alkenes resembling the composition of beewolf HCs form efficient protective barriers against NO, indicating that protection can be achieved by different mixtures of HCs. Since in vitro assays with symbiont cultures from different beewolf hosts indicate widespread NO sensitivity, HC-mediated protection from NO is likely important across Philanthini wasps. We conclude that HC-mediated protection of the symbiont from NO does not exert a conflicting selection pressure on the multifunctional HC profile of beewolves.

Colonization dynamics of a defensive insect ectosymbiont.

Beneficial symbionts are horizontally or vertically transmitted to offspring, relying on host- or microbe-mediated mechanisms for colonization. While multiple studies on symbionts transmitted internally or by feeding highlight host adaptations and dynamics of symbiont colonization, less is known for beneficial microbes colonizing host external surfaces, such as the insect cuticle. Here, we investigate the colonization dynamics of a bacterial symbiont that protects eggs and larvae of Lagria villosa beetles against pathogens. After maternal application to the egg surface, symbionts colonize specialized cuticular invaginations on the dorsal surface of larvae. We assessed the colonization time point and investigated the involvement of the host during this process. Symbionts remain on the egg surface before hatching, providing protection. Immediately after hatching, cells from the egg surface colonize the larvae and horizontal acquisition can occur, yet efficiency decreases with increasing larval age. Additionally, passive or host-aided translocation likely supports colonization of the larval symbiotic organs. This may be especially important for the dominant non-motile symbiont strain, while motility of additional strains in the symbiont community might also play a role. Our findings provide insights into the colonization dynamics of cuticle-associated defensive symbionts and suggest alternate or complementary strategies used by different strains for colonization.

Beetle-Bacterial Symbioses: Endless Forms Most Functional.

Beetles are hosts to a remarkable diversity of bacterial symbionts. In this article, we review the role of these partnerships in promoting beetle fitness following a surge of recent studies characterizing symbiont localization and function across the Coleoptera. Symbiont contributions range from the supplementation of essential nutrients and digestive or detoxifying enzymes to the production of bioactive compounds providing defense against natural enemies. Insights on this functional diversity highlight how symbiosis can expand the host's ecological niche, but also constrain its evolutionary potential by promoting specialization. As bacterial localization can differ within and between beetle clades, we discuss how it corresponds to the microbe's beneficial role and outline the molecular and behavioral mechanisms underlying symbiont translocation and transmission by its holometabolous host. In reviewing this literature, we emphasize how the study of symbiosis can inform our understanding of the phenotypic innovations behind the evolutionary success of beetles.

Abundance and Localization of Symbiotic Bacterial Communities in the Fly Parasitoid Spalangia cameroni.

Multicellular eukaryotes often host multiple microbial symbionts that may cooperate or compete for host resources, such as space and nutrients. Here, we studied the abundances and localization of four bacterial symbionts, Rickettsia, Wolbachia, Sodalis, and Arsenophonus, in the parasitic wasp Spalangia cameroni. Using quantitative PCR (qPCR), we measured the symbionts' titers in wasps that harbor different combinations of these symbionts. We found that the titer of each symbiont decreased as the number of symbiont species in the community increased. Symbionts' titers were higher in females than in males. Rickettsia was the most abundant symbiont in all the communities, followed by Sodalis and Wolbachia. The titers of these three symbionts were positively correlated in some of the colonies. Fluorescence in situ hybridization was in line with the qPCR results: Rickettsia, Wolbachia, and Sodalis were observed in high densities in multiple organs, including brain, muscles, gut, Malpighian tubules, fat body, ovaries, and testes, while Arsenophonus was localized to fewer organs and in lower densities. Sodalis and Arsenophonus were observed in ovarian follicle cells but not within oocytes or laid eggs. This study highlights the connection between symbionts' abundance and localization. We discuss the possible connections between our findings to symbiont transmission success. IMPORTANCE Many insects carry intracellular bacterial symbionts (bacteria that reside within the cells of the insect). When multiple symbiont species cohabit in a host, they may compete or cooperate for space, nutrients, and transmission, and the nature of such interactions would be reflected in the abundance of each symbiont species. Given the widespread occurrence of coinfections with maternally transmitted symbionts in insects, it is important to learn more about how they interact, where they are localized, and how these two aspects affect their co-occurrence within individual insects. Here, we studied the abundance and the localization of four symbionts, Rickettsia, Wolbachia, Sodalis, and Arsenophonus, that cohabit the parasitic wasp Spalangia cameroni. We found that symbionts' titers differed between symbiotic communities. These results were corroborated by microscopy, which shows differential localization patterns. We discuss the findings in the contexts of community ecology, possible symbiont-symbiont interactions, and host control mechanisms that may shape the symbiotic community structure.

Wolbachia causes cytoplasmic incompatibility but not male-killing in a grain pest beetle.

The endosymbiotic Wolbachia is one of the most common intracellular bacteria known in arthropods and nematodes. Its ability for reproductive manipulation can cause unequal inheritance to male and female offspring, allowing the manipulator to spread, but potentially also impact the evolutionary dynamics of infected hosts. Estimated to be present in up to 66% of insect species, little is known about the phenotypic impact of Wolbachia within the order Coleoptera. Here, we describe the reproductive manipulation by the Wolbachia strain wSur harboured by the sawtoothed grain beetle Oryzaephilus surinamensis (Coleoptera, Silvanidae), through a combination of genomics approaches and bioassays. The Wolbachia strain wSur belongs to supergroup B that contains well-described reproductive manipulators of insects and encodes a pair of cytoplasmic incompatibility factor (cif) genes, as well as multiple homologues of the WO-mediated killing (wmk) gene. A phylogenetic comparison with wmk homologues of wMel of Drosophila melanogaster identified 18 wmk copies in wSur, including one that is closely related to the wMel male-killing homologue. However, further analysis of this particular wmk gene revealed an eight-nucleotide deletion leading to a stop-codon and subsequent reading frame shift midsequence, probably rendering it nonfunctional. Concordantly, utilizing a Wolbachia-deprived O. surinamensis population and controlled mating pairs of wSur-infected and noninfected partners, we found no experimental evidence for male-killing. However, a significant ~50% reduction of hatching rates in hybrid crosses of uninfected females with infected males indicates that wSur is causing cytoplasmic incompatibility. Thus, Wolbachia also represents an important determinant of host fitness in Coleoptera.

Nutritional symbionts enhance structural defence against predation and fungal infection in a grain pest beetle.

Many insects benefit from bacterial symbionts that provide essential nutrients and thereby extend the hosts' adaptive potential and their ability to cope with challenging environments. However, the implications of nutritional symbioses for the hosts' defence against natural enemies remain largely unstudied. Here, we investigated whether the cuticle-enhancing nutritional symbiosis of the saw-toothed grain beetle Oryzaephilus surinamensis confers protection against predation and fungal infection. We exposed age-defined symbiotic and symbiont-depleted (aposymbiotic) beetles to two antagonists that must actively penetrate the cuticle for a successful attack: wolf spiders (Lycosidae) and the fungal entomopathogen Beauveria bassiana. While young beetles suffered from high predation and fungal infection rates regardless of symbiont presence, symbiotic beetles were able to escape this period of vulnerability and reach high survival probabilities significantly faster than aposymbiotic beetles. To understand the mechanistic basis of these differences, we conducted a time-series analysis of cuticle development in symbiotic and aposymbiotic beetles by measuring cuticular melanisation and thickness. The results reveal that the symbionts accelerate their host's cuticle formation and thereby enable it to quickly reach a cuticle quality threshold that confers structural protection against predation and fungal infection. Considering the widespread occurrence of cuticle enhancement via symbiont-mediated tyrosine supplementation in beetles and other insects, our findings demonstrate how nutritional symbioses can have important ecological implications reaching beyond the immediate nutrient-provisioning benefits.

Selective advantages favour high genomic AT-contents in intracellular elements.

Extrachromosomal genetic elements such as bacterial endosymbionts and plasmids generally exhibit AT-contents that are increased relative to their hosts' DNA. The AT-bias of endosymbiotic genomes is commonly explained by neutral evolutionary processes such as a mutational bias towards increased A+T. Here we show experimentally that an increased AT-content of host-dependent elements can be selectively favoured. Manipulating the nucleotide composition of bacterial cells by introducing A+T-rich or G+C-rich plasmids, we demonstrate that cells containing GC-rich plasmids are less fit than cells containing AT-rich plasmids. Moreover, the cost of GC-rich elements could be compensated by providing precursors of G+C, but not of A+T, thus linking the observed fitness effects to the cytoplasmic availability of nucleotides. Accordingly, introducing AT-rich and GC-rich plasmids into other bacterial species with different genomic GC-contents revealed that the costs of G+C-rich plasmids decreased with an increasing GC-content of their host's genomic DNA. Taken together, our work identifies selection as a strong evolutionary force that drives the genomes of intracellular genetic elements toward higher A+T contents.

Demonstrating the role of symbionts in mediating detoxification in herbivores.

Plant toxins constitute an effective defense against herbivorous animals. However, many herbivores have evolved adaptations to cope with dietary toxins through detoxification, excretion, sequestration, target site insensitivity and/or via behavioral avoidance. While these adaptations are often directly encoded in herbivore genomes, evidence is accumulating that microbial symbionts can reduce the dose of plant toxins by metabolizing or sequestering them prior to absorption by the herbivore. Here, we describe a few well-studied examples to assess such symbiont-mediated detoxification and showcase different approaches that have been used for their analyses. These include: (i) a host phenotypic route in which the symbiotic association is manipulated to reveal host fitness costs upon toxin exposure in the presence/absence of detoxifying symbionts, including function restoration after symbiont re-infection, (ii) a molecular microbiological approach that focuses on the identification and characterization of microbial genes involved in plant toxin metabolism, and (iii) an analytical chemical route that aims to characterize the conversion of the toxin to less harmful metabolites in vivo and link conversion to the activities of a detoxifying symbiont. The advantages and challenges of each approach are discussed, and it is argued that a multi-pronged strategy combining phenotypic, molecular, and chemical evidence is needed to unambiguously demonstrate microbial contributions to plant toxin reduction and the importance of these processes for host fitness. Given the interdisciplinary nature of the topic, we aim to provide a guideline to researchers interested in symbiont-mediated detoxification and hope to encourage future studies that contribute to a more comprehensive and mechanistic understanding of detoxification in herbivores and their symbionts.

Biosynthesis of Sinapigladioside, an Antifungal Isothiocyanate from Burkholderia Symbionts.

Sinapigladioside is a rare isothiocyanate-bearing natural product from beetle-associated bacteria (Burkholderia gladioli) that might protect beetle offspring against entomopathogenic fungi. The biosynthetic origin of sinapigladioside has been elusive, and little is known about bacterial isothiocyanate biosynthesis in general. On the basis of stable-isotope labeling, bioinformatics, and mutagenesis, we identified the sinapigladioside biosynthesis gene cluster in the symbiont and found that an isonitrile synthase plays a key role in the biosynthetic pathway. Genome mining and network analyses indicate that related gene clusters are distributed across various bacterial phyla including producers of both nitriles and isothiocyanates. Our findings support a model for bacterial isothiocyanate biosynthesis by sulfur transfer into isonitrile precursors.

Evolutionary stability of antibiotic protection in a defensive symbiosis.

The increasing resistance of human pathogens severely limits the efficacy of antibiotics in medicine, yet many animals, including solitary beewolf wasps, successfully engage in defensive alliances with antibiotic-producing bacteria for millions of years. Here, we report on the in situ production of 49 derivatives belonging to three antibiotic compound classes (45 piericidin derivatives, 3 streptochlorin derivatives, and nigericin) by the symbionts of 25 beewolf host species and subspecies, spanning 68 million years of evolution. Despite a high degree of qualitative stability in the antibiotic mixture, we found consistent quantitative differences between species and across geographic localities, presumably reflecting adaptations to combat local pathogen communities. Antimicrobial bioassays with the three main components and in silico predictions based on the structure and specificity in polyketide synthase domains of the piericidin biosynthesis gene cluster yield insights into the mechanistic basis and ecoevolutionary implications of producing a complex mixture of antimicrobial compounds in a natural setting.

Microbiome-assisted carrion preservation aids larval development in a burying beetle.

The ability to feed on a wide range of diets has enabled insects to diversify and colonize specialized niches. Carrion, for example, is highly susceptible to microbial decomposers, but is kept palatable several days after an animal's death by carrion-feeding insects. Here we show that the burying beetle Nicrophorus vespilloides preserves carrion by preventing the microbial succession associated with carrion decomposition, thus ensuring a high-quality resource for their developing larvae. Beetle-tended carcasses showed no signs of degradation and hosted a microbial community containing the beetles' gut microbiota, including the yeast Yarrowia In contrast, untended carcasses showed visual and olfactory signs of putrefaction, and their microbial community consisted of endogenous and soil-originating microbial decomposers. This regulation of the carcass' bacterial and fungal community and transcriptomic profile was associated with lower concentrations of putrescine and cadaverine (toxic polyamines associated with carcass putrefaction) and altered levels of proteases, lipases, and free amino acids. Beetle-tended carcasses develop a biofilm-like matrix housing the yeast, which, when experimentally removed, leads to reduced larval growth. Thus, tended carcasses hosted a mutualistic microbial community that promotes optimal larval development, likely through symbiont-mediated extraintestinal digestion and detoxification of carrion nutrients. The adaptive preservation of carrion coordinated by the beetles and their symbionts demonstrates a specialized resource-management strategy through which insects modify their habitats to enhance fitness.

Versatile and Dynamic Symbioses Between Insects and Burkholderia Bacteria.

Symbiotic associations with microorganisms represent major sources of ecological and evolutionary innovations in insects. Multiple insect taxa engage in symbioses with bacteria of the genus Burkholderia, a diverse group that is widespread across different environments and whose members can be mutualistic or pathogenic to plants, fungi, and animals. Burkholderia symbionts provide nutritional benefits and resistance against insecticides to stinkbugs, defend Lagria beetle eggs against pathogenic fungi, and may be involved in nitrogen metabolism in ants. In contrast to many other insect symbioses, the known associations with Burkholderia are characterized by environmental symbiont acquisition or mixed-mode transmission, resulting in interesting ecological and evolutionary dynamics of symbiont strain composition. Insect-Burkholderia symbioses present valuable model systems from which to derive insights into general principles governing symbiotic interactions because they are often experimentally and genetically tractable and span a large fraction of the diversity of functions, localizations, and transmission routes represented in insect symbioses.

De novo biosynthesis of simple aromatic compounds by an arthropod (Archegozetes longisetosus).

The ability to synthesize simple aromatic compounds is well known from bacteria, fungi and plants, which all share an exclusive biosynthetic route-the shikimic acid pathway. Some of these organisms further evolved the polyketide pathway to form core benzenoids via a head-to-tail condensation of polyketide precursors. Arthropods supposedly lack the ability to synthesize aromatics and instead rely on aromatic amino acids acquired from food, or from symbiotic microorganisms. The few studies purportedly showing de novo biosynthesis via the polyketide synthase (PKS) pathway failed to exclude endosymbiotic bacteria, so their results are inconclusive. We investigated the biosynthesis of aromatic compounds in defence secretions of the oribatid mite Archegozetes longisetosus. Exposing the mites to a diet containing high concentrations of antibiotics removed potential microbial partners but did not affect the production of defensive benzenoids. To gain insights into benzenoid biosynthesis, we fed mites with stable-isotope labelled precursors and monitored incorporation with mass spectrometry. Glucose, malonic acid and acetate, but not phenylalanine, were incorporated into the benzenoids, further evidencing autogenous biosynthesis. Whole-transcriptome sequencing with hidden Markov model profile search of protein domain families and subsequent phylogenetic analysis revealed a putative PKS domain similar to an actinobacterial PKS, possibly indicating a horizontal gene transfer.

Effects, interactions, and localization of Rickettsia and Wolbachia in the house fly parasitoid, Spalangia endius.

Many insect species harbor facultative microbial symbionts that affect their biology in diverse ways. Here, we studied the effects, interactions, and localization of two bacterial symbionts-Wolbachia and Rickettsia-in the parasitoid Spalangia endius. We crossed between four S. endius colonies-Wolbachia only (W), Rickettsia only (R), both (WR), and none (aposymbiotic, APS) (16 possible crosses) and found that Wolbachia induces incomplete cytoplasmic incompatibility (CI), both when the males are W or WR. Rickettsia did not cause reproductive manipulations and did not rescue the Wolbachia-induced CI. However, when R females were crossed with W or WR males, significantly less offspring were produced compared with that of control crosses. In non-CI crosses, the presence of Wolbachia in males caused a significant reduction in offspring numbers. Females' developmental time was significantly prolonged in the R colony, with adults starting to emerge one day later than the other colonies. Other fitness parameters did not differ significantly between the colonies. Using fluorescence in situ hybridization microscopy in females, we found that Wolbachia is localized alongside Rickettsia inside oocytes, follicle cells, and nurse cells in the ovaries. However, Rickettsia is distributed also in muscle cells all over the body, in ganglia, and even in the brain.

Insect-Associated Bacteria Assemble the Antifungal Butenolide Gladiofungin by Non-Canonical Polyketide Chain Termination.

Genome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously-overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol-derived C3 building block. The key role of an A-factor synthase (AfsA)-like offloading domain was corroborated by CRISPR-Cas-mediated gene editing, which facilitated precise excision within a PKS domain.

Established Cotton Stainer Gut Bacterial Mutualists Evade Regulation by Host Antimicrobial Peptides.

Symbioses with microorganisms are ubiquitous in nature and confer important ecological traits to animal hosts but also require control mechanisms to ensure homeostasis of the symbiotic interactions. In addition to protecting hosts against pathogens, animal immune systems recognize, respond to, and regulate mutualists. The gut bacterial symbionts of the cotton stainer bug, Dysdercus fasciatus, elicit an immune response characterized by the upregulation of c-type lysozyme and the antimicrobial peptide pyrrhocoricin in bugs with their native gut microbiota compared to that in dysbiotic insects. In this study, we investigated the impact of the elicited antimicrobial immune response on the established cotton stainer gut bacterial symbiont populations. To this end, we used RNA interference (RNAi) to knock down immunity-related genes hypothesized to regulate the symbionts, and we subsequently measured the effect of this silencing on host fitness and on the abundance of the major gut bacterial symbionts. Despite successful downregulation of target genes by both ingestion and injection of double-stranded RNA (dsRNA), the silencing of immunity-related genes had no effect on either host fitness or the qualitative and quantitative composition of established gut bacterial symbionts, indicating that the host immune responses are not actively involved in the regulation of the nutritional and defensive gut bacterial mutualists. These results suggest that close associations of bacterial symbionts with their hosts can result in the evolution of mechanisms ensuring that symbionts remain insensitive to host immunological responses, which may be important for the evolutionary stability of animal-microbe symbiotic associations.IMPORTANCE Animal immune systems are central for the protection of hosts against enemies by preventing or eliminating successful infections. However, in the presence of beneficial bacterial mutualists, the immune system must strike a balance of not killing the beneficial symbionts while at the same time preventing enemy attacks. Here, using the cotton stainer bug, we reveal that its long-term associated bacterial symbionts are insensitive to the host's immune effectors, suggesting adaptation to the host's defenses, thereby strengthening the stability of the symbiotic relationship. The ability of the symbionts to elicit host immune responses but remain insensitive themselves may be a mechanism by which the symbionts prime hosts to fight future pathogenic infections.

Angiosperm to Gymnosperm host-plant switch entails shifts in microbiota of the Welwitschia bug, Probergrothius angolensis (Distant, 1902).

The adaptation of herbivorous insects to new host plants is key to their evolutionary success in diverse environments. Many insects are associated with mutualistic gut bacteria that contribute to the host's nutrition and can thereby facilitate dietary switching in polyphagous insects. However, how gut microbial communities differ between populations of the same species that feed on different host plants remains poorly understood. Most species of Pyrrhocoridae (Hemiptera: Heteroptera) are specialist seed-feeders on plants in the family Malvaceae, although populations of one species, Probergrothius angolensis, have switched to the very distantly related Welwitschia mirabilis plant in the Namib Desert. We first compared the development and survival of laboratory populations of Pr. angolensis with two other pyrrhocorids on seeds of Welwitschia and found only Pr. angolensis was capable of successfully completing its development. We then collected Pr. angolensis in Namibia from Malvaceae and Welwitschia host plants, respectively, to assess their bacterial and fungal community profiles using high-throughput amplicon sequencing. Comparison with long-term laboratory-reared insects indicated stable associations of Pr. angolensis with core bacteria (Commensalibacter, Enterococcus, Bartonella and Klebsiella), but not with fungi or yeasts. Phylogenetic analyses of core bacteria revealed relationships to other insect-associated bacteria, but also found new taxa indicating potential host-specialized nutritional roles. Importantly, the microbial community profiles of bugs feeding on Welwitschia versus Malvaceae revealed stark and consistent differences in the relative abundance of core bacterial taxa that correlate with the host-plant switch; we were able to reproduce this result through feeding experiments. Thus, a dynamic gut microbiota may provide a means for insect adaptation to new host plants in new environments when food plants are extremely divergent.