Добірка наукової літератури з теми "Insect gut symbiosis"

Stink bugs of the superfamilies Coreoidea and Lygaeoidea establish gut symbioses with environmentally acquired bacteria of the genus Burkholderia sensu lato. In the genus Burkholderia, the stink bug-associated strains form a monophyletic clade, named stink bug-associated beneficial and environmental (SBE) clade (or Caballeronia). Recently, we revealed that members of the family Largidae of the superfamily Pyrrhocoroidea are associated with Burkholderia but not specifically with the SBE Burkholderia; largid bugs harbor symbionts that belong to a clade of plant-associated group of Burkholderia, called plant-associated beneficial and environmental (PBE) clade (or Paraburkholderia). To understand the genomic features of Burkholderia symbionts of stink bugs, we isolated two symbiotic Burkholderia strains from a bordered plant bug Physopellta gutta (Pyrrhocoroidea: Largidae) and determined their complete genomes. The genome sizes of the insect-associated PBE (iPBE) are 9.5 Mb and 11.2 Mb, both of which are larger than the genomes of the SBE Burkholderia symbionts. A whole-genome comparison between two iPBE symbionts and three SBE symbionts highlighted that all previously reported symbiosis factors are shared and that 282 genes are specifically conserved in the five stink bug symbionts, over one-third of which have unknown function. Among the symbiont-specific genes, about 40 genes formed a cluster in all five symbionts; this suggests a “symbiotic island” in the genome of stink bug-associated Burkholderia.

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.

Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola. Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.

ABSTRACTTheRiptortus-Burkholderiasymbiotic system is an experimental model system for studying the molecular mechanisms of an insect-microbe gut symbiosis. When the symbiotic midgut ofRiptortus pedestriswas investigated by light and transmission electron microscopy, the lumens of the midgut crypts that harbor colonizingBurkholderiasymbionts 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 onpurNandpurT, which independently catalyze the same step of bacterial purine biosynthesis. When we disruptedpurNandpurTin theBurkholderiasymbiont, the ΔpurNand ΔpurTmutants grew normally, and only the ΔpurTmutant failed to form biofilms. Notably, the ΔpurTmutant exhibited a significantly lower level of cyclic-di-GMP (c-di-GMP) than the wild type and the ΔpurNmutant, suggesting involvement of the secondary messenger c-di-GMP in the defect of biofilm formation in the ΔpurTmutant, which might operate via impaired purine biosynthesis. The host insects infected with the ΔpurTmutant exhibited a lower infection density, slower growth, and lighter body weight than the host insects infected with the wild type and the ΔpurNmutant. These results show that the function ofpurTof 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.

Insects nurture a panoply of microbial populations that are often obligatory and exist mutually with their hosts. Symbionts not only impact their host fitness but also shape the trajectory of their phenotype. This co-constructed niche successfully evolved long in the past to mark advanced ecological specialization. The resident microbes regulate insect nutrition by controlling their host plant specialization and immunity. It enhances the host fitness and performance by detoxifying toxins secreted by the predators and abstains them. The profound effect of a microbial population on insect physiology and behaviour is exploited to understand the host–microbial system in diverse taxa. Emergent research of insect-associated microbes has revealed their potential to modulate insect brain functions and, ultimately, control their behaviours, including social interactions. The revelation of the gut microbiota–brain axis has now unravelled insects as a cost-effective potential model to study neurodegenerative disorders and behavioural dysfunctions in humans. This article reviewed our knowledge about the insect–microbial system, an exquisite network of interactions operating between insects and microbes, its mechanistic insight that holds intricate multi-organismal systems in harmony, and its future perspectives. The demystification of molecular networks governing insect–microbial symbiosis will reveal the perplexing behaviours of insects that could be utilized in managing insect pests.

AbstractMicroorganisms often live in symbiosis with their hosts, and some are considered mutualists, where all species involved benefit from the interaction. How free-living microorganisms have evolved to become mutualists is unclear. Here we report an experimental system in which non-symbiotic Escherichia coli evolves into an insect mutualist. The stinkbug Plautia stali is typically associated with its essential gut symbiont, Pantoea sp., which colonizes a specialized symbiotic organ. When sterilized newborn nymphs were infected with E. coli rather than Pantoea sp., only a few insects survived, in which E. coli exhibited specific localization to the symbiotic organ and vertical transmission to the offspring. Through transgenerational maintenance with P. stali, several hypermutating E. coli lines independently evolved to support the host’s high adult emergence and improved body colour; these were called ‘mutualistic’ E. coli. These mutants exhibited slower bacterial growth, smaller size, loss of flagellar motility and lack of an extracellular matrix. Transcriptomic and genomic analyses of ‘mutualistic’ E. coli lines revealed independent mutations that disrupted the carbon catabolite repression global transcriptional regulator system. Each mutation reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that single carbon catabolite repression mutations can make E. coli an insect mutualist. These findings provide an experimental system for future work on host–microbe symbioses and may explain why microbial mutualisms are omnipresent in nature.

Fungus-growing termites are eusocial insects that represent one of the most efficient and unique systems for lignocellulose bioconversion, evolved from a sophisticated symbiosis with lignocellulolytic fungi and gut bacterial communities. Despite a plethora of information generated during the last century, some essential information on gut bacterial profiles and their unique contributions to wood digestion in some fungus-growing termites is still inadequate. Hence, using the culture-dependent approach, the present study aims to assess and compare the diversity of lignocellulose-degrading bacterial symbionts within the gut systems of three fungus-growing termites: Ancistrotermes pakistanicus, Odontotermes longignathus, and Macrotermes sp. A total of 32 bacterial species, belonging to 18 genera and 10 different families, were successfully isolated and identified from three fungus-growing termites using Avicel or xylan as the sole source of carbon. Enterobacteriaceae was the most dominant family represented by 68.1% of the total bacteria, followed by Yersiniaceae (10.6%) and Moraxellaceae (9%). Interestingly, five bacterial genera such as Enterobacter, Citrobacter, Acinetobacter, Trabulsiella, and Kluyvera were common among the tested termites, while the other bacteria demonstrated a termite-specific distribution. Further, the lignocellulolytic potential of selected bacterial strains was tested on agricultural waste to evaluate their capability for lignocellulose bioconversion. The highest substrate degradation was achieved with E. chengduensis MA11 which degraded 45.52% of rice straw. All of the potential strains showed endoglucanase, exoglucanase, and xylanase activities depicting a symbiotic role towards the lignocellulose digestion within the termite gut. The above results indicated that fungus-growing termites harbor a diverse array of bacterial symbionts that differ from species to species, which may play an inevitable role to enhance the degradation efficacy in lignocellulose decomposition. The present study further elaborates our knowledge about the termite-bacteria symbiosis for lignocellulose bioconversion which could be helpful to design a future biorefinery.

Termites have long been studied for their symbiotic associations with gut microbes. In the late nineteenth century, this relationship was poorly understood and captured the interest of parasitologists such as Joseph Leidy; this research led to that of twentieth-century biologists and entomologists including Cleveland, Hungate, Trager, and Lüscher. Early insights came via microscopy, organismal, and defaunation studies, which led to descriptions of microbes present, descriptions of the roles of symbionts in lignocellulose digestion, and early insights into energy gas utilization by the host termite. Focus then progressed to culture-dependent microbiology and biochemical studies of host–symbiont complementarity, which revealed specific microhabitat requirements for symbionts and noncellulosic mechanisms of symbiosis (e.g., N2 fixation). Today, knowledge on termite symbiosis has accrued exponentially thanks to omic technologies that reveal symbiont identities, functions, and interdependence, as well as intricacies of host–symbiont complementarity. Moving forward, the merging of classical twentieth-century approaches with evolving omic tools should provide even deeper insights into host–symbiont interplay.

Termites have long been studied for their symbiotic associations with gut microbes. In the late nineteenth century, this relationship was poorly understood and captured the interest of parasitologists such as Joseph Leidy; this research led to that of twentieth-century biologists and entomologists including Cleveland, Hungate, Trager, and Lüscher. Early insights came via microscopy, organismal, and defaunation studies, which led to descriptions of microbes present, descriptions of the roles of symbionts in lignocellulose digestion, and early insights into energy gas utilization by the host termite. Focus then progressed to culture-dependent microbiology and biochemical studies of host–symbiont complementarity, which revealed specific microhabitat requirements for symbionts and noncellulosic mechanisms of symbiosis (e.g., N2 fixation). Today, knowledge on termite symbiosis has accrued exponentially thanks to omic technologies that reveal symbiont identities, functions, and interdependence, as well as intricacies of host–symbiont complementarity. Moving forward, the merging of classical twentieth-century approaches with evolving omic tools should provide even deeper insights into host–symbiont interplay.

Insects are generally associated with gut bacterial communities that benefit the hosts with respect to diet digestion, limiting resource supplementation, pathogen defense, and ecological niche expansion. Heteroptera (true bugs) represent one of the largest and most diverse insect lineages and comprise species consuming different diets and inhabiting various ecological niches, even including underwater. However, the bacterial symbiotic associations have been characterized for those basically restricted to herbivorous stink bugs of the infraorder Pentatomomorpha. The gut microbiota associated with the megadiverse heteropteran lineages and the implications of ecological and diet variance remain largely unknown. Here, we conducted a bacterial 16S rRNA amplicon sequencing of the gut microbiota across 30 species of true bugs representative of different ecological niches and diets. It was revealed that Proteobacteria and Firmicute were the predominant bacterial phyla. Environmental habitats and diets synergistically contributed to the diversity of the gut bacterial community of true bugs. True bugs living in aquatic environments harbored multiple bacterial taxa that were not present in their terrestrial counterparts. Carnivorous true bugs possessed distinct gut microbiota compared to phytophagous species. Particularly, assassin bugs of the family Reduviidae possessed a characterized gut microbiota predominantly composed of one Enterococcus with different Proteobacteria, implying a specific association between the gut bacteria and host. Overall, our findings highlight the importance of the comprehensive surveillance of gut microbiota association with true bugs for understanding the molecular mechanisms underpinning insect–bacteria symbiosis.

Insects are one of the most fascinating taxa on Earth: their diversity, diffusion, colonization of different niches are unparalleled in the animal kingdom. Besides, they have a remarkable impact on human life: they are parasites for people, animals and crops, vectors of diseases, pollinators, and even breeding animals (e.g. honeybees, silkworms). This extraordinary evolutionary success and diversification is partially due to the symbiotic relationships that insects have with a wide range of bacteria. These symbionts can be divided into primary, secondary symbionts and gut bacteria. Primary symbionts are found in very specialized cells (the bacteriocytes), strictly maternally transmitted and not cultivable. They are essential for their host, and vice-versa: they can actually be considered part of a single organism called “holobiont”. Secondary symbionts are not necessary for the host survival, although often beneficial, and they can inhabit various organs and tissues. In this category fall also reproductive parasites, as Wolbachia, which spreads in the population by maternal transmission, manipulating the reproduction of the host to favour the birth of infected daughters. Finally, gut bacteria are a more vague category, comprising organisms that live in the insect intestine because they are ingested with the diet, but also symbionts that establish a close relationship with the host, being essential for its survival and development. The roles of all these microorganisms are, to different extents, important for the insect physiology. Primary symbionts are generally essential to complement unbalanced diets and secondary ones contribute to the host fitness, while reproduction parasites deeply affect the reproduction mode of their hosts. Even commensals have been demonstrated to influence the development, mating choice and immune responses in Drosophila flies. For these reasons, the understanding of the biology of an insect can not do without the characterisation of its microbiota. In the second chapter of my PhD thesis, a review on the microbial ecology techniques applied to the study of insect microbial communities gives an overview on the methods that can be applied to this purpose. On one hand, molecular analyses based on the 16S gene sequencing, such as 16S rRNA barcoding (pyrotag) and Denaturing Gradient Gel Electrophoresis (DGGE) are the most powerful methods to get a complete picture of the microbial community composition and structure. Microscopic localisation of symbionts can be also achieved by Fluorescent In Situ Hybridisation. On the other hand, the isolation of bacteria allows to deeply characterize the cultivable fraction, verifying through direct in vitro tests the activities of the strains. Taking advantage of a strain collection isolated from the target insect, the symbiotic relationship can be investigated through in vivo experiments. The more common ones involve i) the labeling of the strains with fluorescent proteins and the recolonization of the insects, to evaluate their localisation and colonisation ability, ii) the assessment of the detrimental effects of symbionts deprivation on the hosts, and iii) the comparison of insects monoassociated with different strains to check the effects on host fitness. To further analyse the interaction between bacteria and their hosts from a genetic point of view, advanced techniques, such as Signature Tagged Mutagenesis or In Vivo Expression Technology, can be performed. Many of these techniques have been applied in the case studies here presented, in which the microbial communities associated to three insect pests have been characterised. In the third chapter is presented a study on the spotted-wing fly Drosophila suzukii. Unlike its relative D. melanogaster, which feeds on rotten fruit, this fly feeds and lays eggs on healthy fruits. The most damaged crops are members of the Drupaceae family (e.g. cherries) and berries (strawberries, raspberries, blueberries). The bacterial community associated to this pest have been characterised with a focus on acetic acid bacteria (AAB), important symbionts of many sugar-feeding insects. According to our findings, D. suzukii harbours a diverse community of AAB, detected both in the isolate collection and in culture-independent screenings (pyrotag, DGGE). They are primarily localised in the gut, attached to the peritrophic matrix, as showed by FISH micrographs. The ability of three AAB species (Gluconobacter oxydans, Acetobacter tropicalis and Acetobacter indonesiensis) to colonise the gut has been proved by recolonization experiments of the insect using GFP-marked strains. In the fourth chapter, the bacterial community of the wood-feeding beetle Rhynchophorus ferrugineus has been analysed. Commonly named Red Palm Weevil (RPW), this insect is an important pest for palm trees. The plants are damaged mainly by the larvae, which dig tunnels in the trunks until pupation. Bacteria associated to the red palm weevil have been studied primarily by molecular means (pyrotag). Our results outline that the bacteria hosted by R. ferrugineus are mainly acquired from the environment while feeding. Indeed, a sharp difference has been registered between field-caught and bred specimens. While field caught RPW harbour more bacterial taxa which are in common with their feeding plants, the animals fed on apple in the laboratory show a higher prevalence of lactic acid and acetic acid bacteria, which presumably grow on the rotten fruit. The latter result is further confirmed by the bacterial isolations performed on apple-fed specimens. Besides, the DNA sequence of a primary symbiont, Candidatus Nardonella, has been detected. This bacterium has been shown to inhabit a wide range of insects of the same family of the RPW, Curculionidae. The fifth chapter is about the gut bacterial community of Psacothea hilaris hilaris. Native of Japan and east China, this longicorn beetle (family: Cerambicidae) arrived in Italy as a consequence of the wood trade, and settled as a stable population in a small area in Como province. Its larvae dig tunnels in the trunks of the trees of the Moraceae family, while the adults feed on leaves. The most damaged by its feeding habits are mulberry and fig trees. This beetle hosts a variegate gut microbiota, that, as shown by DGGE, greatly changes according to the diet and to the gut tract examined. The cultivable fraction of this microbiota has been tested for several activities that proved the capability of the community as a whole to exploit the food sources in the insect gut (primarily, sugars from plant cell walls) and to assist their host in carbon and nitrogen absorption. Thus, even if acquired from the environment, these bacteria seem to be adapted to a symbiotic lifestyle. From the comparison among these three studies, some conclusions can be drawn. All three case studies outline the importance of the diet in shaping the insect microbial community. In detail, wild insects always show higher diversity and individual variability in their associated microbiota. Reared insects appear, on the contrary, dominated by the species that can rapidly grow on laboratory diets, such as Lactobacillales and Enterobacteriales. Secondly, these studies depict a more accurate image of the commensal bacteria, which are not merely acquired by chance through feeding, but are capable to actively colonize insect guts, and to efficiently exploit this niche to multiply and spread in the environment. Finally, the research data point out that the origin and the function of many of the organisms detected in insects are yet poorly understood. For this reason, these studies can be considered a basis to for future research, aimed to a more in-depth understanding of the roles of these bacteria and their interactions with the hosts.

La stabilité évolutive des relations hôte-microbe est cruciale pour la symbiose. La transmission verticale des symbiotes microbiens des parents à la progéniture est bien établie, mais l'acquisition environnementale par transmission horizontale de symbiotes nécessite des adaptations spécifiques. Les insectes de l'infra-ordre Pentatomomorpha disposent d'un mécanisme efficace pour l'acquisition de leur symbiote à partir du sol. Ces insectes possèdent une architecture intestinale distinctive contenant une région postérieure, appelée M4, composée de centaines de cryptes, constituant une niche spécifique pour abriter des symbiotes intestinaux bénéfiques. Les Coreoidea sélectionnent spécifiquement des bactéries Caballeronia. Ma thèse explore la spécificité de cette association et les mécanismes bactériens sous-jacents. Trois espèces de Coreoidea (Riptortus pedestris, Leptoglossus occidentalis, Coreus marginatus) montrent une préférence pour des sous-clades spécifiques de Caballeronia, influencée par la compétition interspécifique. La région M4 est dominée par une seule espèce bactérienne, suggérant une forte pression de sélection. La spécificité de la souche est alignée avec un avantage en termes de fitness reproductif. Des criblages génétiques ont révélé des fonctions cruciales pour la colonisation des cryptes, notamment le chimiotactisme, la résistance aux peptides antimicrobiens et de la capacité à utiliser des sources de carbone néoglucogéniques, la taurine et l'inositol, suggérant que l'hôte fournit ce type de métabolites comme nutriments aux symbiotes. Ces découvertes démontrent que malgré une grande diversité microbienne environnementale, les insectes sélectionnent des symbiotes spécifiques grâce à des mécanismes multifactoriels
The evolutionary stability of host-microbe relationships is crucial for symbiosis. Vertical transmission of microbial symbionts from parents to offspring is well established, but environmental acquisition through horizontal transmission of symbionts requires specific adaptations. Insects of the infraorder Pentatomomorpha have an effective mechanism for acquiring their symbionts from the soil. These insects possess a distinctive intestinal architecture with a posterior region called M4, composed of hundreds of crypts that provide a specific niche for harboring beneficial gut symbionts. Coreoidea specifically select Caballeronia bacteria. My thesis explores the specificity of this association and the underlying bacterial mechanisms. Three species of Coreoidea (Riptortus pedestris, Leptoglossus occidentalis, Coreus marginatus) show a preference for specific subclades of Caballeronia, influenced by interspecific competition. The M4 region is dominated by a single bacterial species, suggesting strong selective pressure. Strain specificity is aligned with a reproductive fitness advantage. Genetic screenings revealed crucial functions for crypt colonization, including chemotaxis, resistance to antimicrobial peptides, and the ability to utilize neoglucogenic carbon sources such as taurine and inositol, suggesting that the host provides these metabolites as nutrients to the symbionts. These findings demonstrate that despite high environmental microbial diversity, insects select specific symbionts through multifactorial mechanisms

Les bactéries du sol sont adaptées pour survivre dans ce milieu et pour faire face à divers organismes y vivant, comme d'autres bactéries, champignons, plantes et insectes. Pour mieux connaitre ces adaptations et comprendre si ces adaptations se chevauchent ou sont spécifiques à chacun de ces modes de vie, j'ai utilisé l'approche dites « Transposon sequencing » (Tn-seq) pour identifier les gènes essentiels et conditionnellement essentiels dans deux bactéries du sol bien connues, Caballeronia insecticola et Sinorhizobium meliloti. J'ai utilisé des cribles Tn-seq réalisés dans les milieux naturels des microbes (in situ), combinés à des expériences in vitro. La sélection des conditions in vitro a été guidée par des analyses transcriptomiques, des études physiologiques, la génétique, la génomique et des analyses biochimiques, ainsi que par les expériences Tn-seq in situ. Ces conditions in vitro consistent en plusieurs facteurs de stress (comme des peptides antimicrobiens ou AMP) ou de conditions nutritionnelles (panel de sources de carbone, d'azote et de soufre) et physiologiques (motilité et chimiotaxie) que les microbes rencontrent dans leurs milieux naturels. Ces conditions in vitro simplifiées décomposent les conditions naturelles en composants uniques et facilitent ainsi l'interprétation des cribles Tn-seq in situ. C. insecticola est une bactérie polyvalente qui établit des interactions spécifiques avec des insectes, des plantes, des champignons et d'autres bactéries. J'ai analysé quatre modes de vie différents de C. insecticola avec l'approche Tn-seq : le sol, la rhizosphère des plants de soja, l'organe symbiotique intestinal de l'insecte Riptortus pedestris et la surface des hyphes des champignons Cunninghamella. Pour les interactions bactéries-bactéries, je me suis concentré sur la compétition entre la souche de rhizobium S. meliloti et la souche productrice de toxines Rhizobium sp. Pop5, car cette interaction est bien caractérisée et repose sur la production du AMP phazolicine par la souche Pop5.Au total, 34 cribles chez C. insecticola et 4 cribles chez S. meliloti ont été réalisés et analysés, ce qui a permis de découvrir des phénotypes pour 1 162 gènes de C. insecticola et 264 gènes de S. meliloti. Chez C. insecticola, le génome essentiel, c'est-à-dire l'ensemble des gènes qui ne peuvent être supprimés et qui sont donc indispensables à la vie bactérienne, a été défini. Il est constitué de 498 gènes, avec des gènes codant des fonctions cellulaires attendues, comme la transcription, la traduction, la production d'énergie, la biosynthèse de l'enveloppe cellulaire et le cycle cellulaire, ou des gènes moins attendus comme ceux impliqués dans la modification spécifique de la partie lipidique A du lipopolysaccharide avec des groupes 4-amino-4-désoxy-L arabinose. Les résultats des différents cribles ont été vérifiés en utilisant des mutants d'insertion ou de délétion de C. insecticola et S. meliloti et en caractérisant leur phénotype dans les conditions in situ et in vitro appropriées. Au total, 23 mutants de C. insecticola et 8 mutants de S. meliloti ont été phénotypés. Dans chaque cas, le phénotypage de ces mutants a confirmé les données Tn-seq, illustrant la robustesse et le potentiel de la méthode.Parmi les fonctions bactériennes cruciales dans tous les milieux naturels, tant chez C. insecticola que chez S. meliloti, figure l'enveloppe bactérienne, ce qui suggère qu'elle constitue un bouclier contre les agressions environnementales, comme les AMPs fréquemment produits par d'autres organismes. La motilité bactérienne et la chimiotaxie chez C. insecticola sont cruciales dans l'interaction avec les insectes et dans le sol, lorsque les bactéries circulent sur les hyphes fongiques. Enfin, chaque milieu impose des contraintes métaboliques spécifiques aux bactéries. Ces travaux ont mis en évidence des adaptations à la fois généralistes et spécifiques à l'environnement chez les bactéries du sol
Soil bacteria are adapted to survive in their abiotic soil environment as well as to cope with different organisms, including other bacteria, fungi, plants and insects with which they share that environment. With the objective to contribute to the understanding of these adaptations and to answer the question if adaptations are overlapping or unique for each of these lifestyles, I used the transposon-sequencing (Tn-seq) approach to identify essential and conditionally fitness genes in two well-studied soil bacteria, Caballeronia insecticola and Sinorhizobium meliloti. The experimental strategy consisted in the use of Tn-seq screens performed in the natural, in situ environments of the microbes combined with multiple in vitro experiments in synthetic environments. The selection of these in vitro conditions was informed by available transcriptome analyses, physiological studies, genetics, genomics and biochemical analyses as well as the in situ Tn-seq experiments themselves. The selected in vitro conditions were a variety of stressors (e.g. antimicrobial peptides or AMPs) or nutritional (e.g. a panel of carbon, nitrogen and sulphur sources) and physiological (e.g. motility and chemotaxis) conditions that the microbes encounter in their natural environments. These simplified synthetic conditions decompose the complexity of natural conditions in single components and facilitate thereby the interpretation of the in situ Tn-seq screens.C. insecticola is a versatile bacterium establishing specific interactions with insects, plants, fungi and other bacteria. I analyzed four different lifestyles of C. insecticola with the Tn-seq approach: soil, the rhizosphere of soybean plants, the gut symbiotic organ of the insect Riptortus pedestris and the surface of the hyphae of Cunninghamella fungi. For bacteria-bacteria interactions, I focused on the competition of the rhizobium strain S. meliloti with the toxin producing strain Rhizobium sp. Pop5 because this interaction is well characterized and based on the production of the AMP phazolicin by the strain Pop5.In total, 34 screens in C. insecticola and 4 screens in S. meliloti were performed and analysed, resulting in the discovery of phenotypes for 1162 C. insecticola genes and 264 S. meliloti genes. In C. insecticola, the essential genome, i.e. the set of genes that cannot be removed and that are therefore indispensable to support bacterial life, was precisely defined. I found that it is constituted of 498 genes, including the genes encoding the expected cellular functions, like transcription, translation, energy production, cell envelope biosynthesis and cell cycle, but also less expected genes like those involved in the specific modification of the lipid A moiety of lipopolysaccharide with 4-amino-4-deoxy-L arabinose groups. Results of the different Tn-seq screens were verified by independent experiments, using insertion or deletion mutants of C. insecticola and S. meliloti in selected genes and characterization of the phenotype of these mutants in the relevant environmental and in vitro conditions. In total, 23 mutants in C. insecticola and 8 mutants in S. meliloti were phenotyped. In each case, the phenotyping of these mutants confirmed the Tn-seq data, illustrating the robustness and potential of the method.Among the crucial bacterial functions in all natural environments, in both C. insecticola and S. meliloti, is the bacterial envelope, suggesting that it constitutes a shield, fending of environmental stresses, in particular AMPs frequently produced by other organisms. Bacterial motility and chemotaxis in C. insecticola are particularly important in the interaction with insects but also in the soil, when bacteria hitchhike on fungal hyphae. Finally, each environment imposes specific metabolic constraints on the bacteria. Together, this work highlighted both generalist and environment-specific adaptations in soil bacteria

Insects while feeding, encounter a wide array of hydrocarbon polymers in their diet and the digestive tracts of various insects contain microbial symbionts that aid in the degradation of these polymers. Thus the idea of insects as synthetic polymer bio-degraders was established. Soon various insect, like mealworms, flour beetles, weevils, wax moths etc. particularly from the Coleopteran and Lepidopteran orders, were identified to have remarkable abilities to consume and degrade a wide range of synthetic polymers like polyethylene, polyurethane, polypropylene, polystyrene and polyvinyl chloride into lower molecular weight, simple, and nontoxic molecules which are eventually excreted as fecula. In this review we aim at congregating the diversity of polymer degrading insect fauna and understanding the underlying mechanism in which the insect’s digestive enzymes works in synergy with the gut microbiota to digest complex synthetic polymers.

Scientists have been knocking the wood to ascertain the symbiotic relationships of tiny living creatures, that is, microorganisms with other beings such as plants, animals, insects, and humans. The concept of “symbiosis” got its existence in 1879, which means “living together.” Microorganisms show a great deal of diverse interactions such as commensalism (moochers), mutualism (both benefitted), and parasitism (one benefitted and other unharmed) with other living beings and mutualism being the most common of all, thus forming a range of antagonistic to cooperative symbiotic relationships. These tiny creatures interact with plants by forming lichens (fungi and algae), mycorrhizae (plants and roots of higher plants), root noodles (Rhizobium) and acting as keyworkers in plant’s rhizosphere promoting growth and development. Microbial community also extends itself to kingdom Animalia establishing relationships with phylum Mammalia including humans, animals, and the most abundant species of phylum Arthropoda, that is, insects such as termites, which have colonization of bacteria in gut to digest wood cellulose. Scientists have discovered that most studied organisms—mussels found in deep-sea hydrothermal vents too live in a mutualistic association whereby bacteria get protection and mussels get nutrition as bacteria use chemicals from hydrothermal fluid producing organic compounds.

The Mediterranean fruit fly Ceratitis capitata (medfly) is a major pest throughout the world and one of the most destructive. Several strategies for controlling this pest have been proposed, including the sterile insect technique (SIT). The SIT’s effectiveness against the medfly is well documented. Sterile medflies, on the other hand, can perform poorly. Reduced mating compatibility and mating competitiveness in the field may be caused by genetic and symbiotic differences between natural and laboratory medfly populations. Probiotic gut symbionts have been shown to facilitate control strategies and improve male medfly fitness. They are equally effective in the live and inactivated forms when administered to medfly adults or larvae. They have been shown to modulate a large set of inducible effector molecules including antimicrobial peptides (AMP) and stress-responsive proteins. The selection procedures of probiotics for their use in the medfly rearing process are reviewed, and other pathways for selection are proposed based on recent in silico studies. This chapter summarizes the most relevant evidence from scientific literature regarding potential applications of probiotics in medfly as an innovative tool for biocontrol, while also shedding light on the spectrum of symbiotic relationships in medfly that may serve as a powerful symbiotic integrative control approach.

Abstract The study of endosymbioses continues to accentuate the extraordinary impact that microorganisms have had on the ecology and evolution of invertebrates, especially insects. One only has to learn of the studies of Buchneria and Wolbachia and their interactions with a wide variety of arthropods to wonder at the dramatic effect of endosymbionts on their hosts (van Meer et al. 1999; Dale et al. 2001; Funk et al. 2001). The study of eukaryotic endosymbionts, however, has lagged somewhat behind that of the prokaryotes. Nevertheless, fungi are increasingly being recognized as important endosymbionts of insects. It is difficult to predict which insects might be associated with fungi because even close relatives feeding on similar nutrient resources may vary in whether they are associated with bacterial or fungal symbionts, or for that matter with any symbiont (Buchner 1965; Martin 1987).

Symbiotic bacteria have been shown to influence host reproduction and defense against biotic and abiotic stressors, and this relates to possible development of a symbiont-based control strategy. This project was based on the hypothesis that symbionts have a significant impact on Culicoides fitness and vector competence for animal viruses. The original objectives in our proposal were: 1. Molecular identification and localization of the newly-discovered symbiotic bacteria within C. imicola and C. schultzei in Israel and C. sonorensis in California. 2. Determination of the prevalence of symbiotic bacteria within different vector Culicoides populations. 3. Documentation of specific symbiont effects on vector reproduction and defense: 3a) test for cytoplasmic incompatibility in Cardinium-infected species; 3b) experimentally evaluate the role of the symbiont on infection or parasitism by key Culicoides natural enemies (iridescent virus and mermithid nematode). 4. Testing the role(s) of the symbionts in possible protection against infection of vector Culicoides by BTV. According to preliminary findings and difficulties in performing experimental procedures performed in other insect symbiosis systems where insect host cultures are easily maintained, we modified the last two objectives as follows: Obj. 3, we tested how symbionts affected general fitness of Israeli Culicoides species, and thoroughly described and evaluated the correlation between American Culicoides and their bacterial communities in the field. We also tried alternative methods to test symbiont-Culicoides interactions and launched studies to characterize low-temperature stress tolerances of the main US vector, which may be related to symbionts. Obj. 4, we tested the correlation between EHDV (instead of BTV) aquisition and Cardinium infection. Culicoides-bornearboviral diseases are emerging or re-emerging worldwide, causing direct and indirect economic losses as well as reduction in animal welfare. One novel strategy to reduce insects’ vectorial capacity is by manipulating specific symbionts to affect vector fitness or performance of the disease agent within. Little was known on the bacterial tenants occupying various Culicoides species, and thus, this project was initiated with the above aims. During this project, we were able to describe the symbiont Cardinium and whole bacterial communities in Israeli and American Culicoides species respectively. We showed that Cardinium infection prevalence is determined by land surface temperature, and this may be important to the larval stage. We also showed no patent significant effect of Cardinium on adult fitness parameters. We showed that the bacterial community in C. sonorensis varies significantly with the host’s developmental stage, but it varies little across multiple wastewater pond environments. This may indicate some specific biological interactions and allowed us to describe a “core microbiome” for C. sonorensis. The final set of analyses that include habitat sample is currently done, in order to separate the more intimately-associated bacteria from those inhabiting the gut contents or cuticle surface (which also could be important). We were also able to carefully study other biological aspects of Culicoides and were able to discriminate two species in C. schultzei group in Israel, and to investigate low temperature tolerances of C. sonorensis that may be related to symbionts. Scientific implications include the establishment of bacterial identification and interactions in Culicoides (our work is cited in other bacteria-Culicoides studies), the development molecular identification of C. schultzei group, and the detailed description of the microbiome of the immature and matched adult stages of C. sonorensis. Agricultural implications include understanding of intrinsic factors that govern Culicoides biology and population regulation, which may be relevant for vector control or reduction in pathogen transmission. Being able to precisely identify Culicoides species is central to understanding Culicoides borne disease epidemiology.

Whiteflies are sap-sucking insects that harbor obligatory symbiotic bacteria to fulfill their dietary needs, as well as a facultative microbial community with diverse bacterial species. The sweetpotato whitefly Bemisia tabaci (Gennadius) is a severe agricultural pest in many parts of the world. This speciesconsists of several biotypes that have been distinguished largely on the basis of biochemical or molecular diagnostics, but whose biological significance is still unclear. The original objectives of the project were (i) to identify the specific complement of prokaryotic endosymbionts associated with select, well-studied, biologically and phylogeographically representative biotypes of B. tabaci, and (ii) to attempt to 'cure’ select biotypes of certain symbionts to permit assessment of the affect of curing on whitefly fitness, gene flow, host plant preference, and virus transmission competency.To identify the diversity of bacterial community associated with a suite of phylogeographically-diverseB. tabaci, a total of 107 populations were screened using general Bacteria primers for the 16S rRNA encoding gene in a PCR. Sequence comparisons with the available databases revealed the presence of bacteria classified in the: Proteobacteria (66%), Firmicutes (25.70%), Actinobacteria (3.7%), Chlamydiae (2.75%) and Bacteroidetes (<1%). Among previously identified bacteria, such as the primary symbiont Portiera aleyrodidarum, and the secondary symbionts Hamiltonella, Cardinium and Wolbachia, a Rickettsia sp. was detected for the first time in this insect family. The distribution, transmission, and localization of the Rickettsia were studied using PCR and fluorescence in situ hybridization (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened as well as some populations screened in the Arizona laboratory, but not in all individuals within each population. FISH analysis of B. tabaci eggs, nymphs and adults, revealed a unique concentration of Rickettsia around the gut and follicle cells as well as its random distribution in the haemolymph, but absence from the primary symbiont housing cells, the bacteriocytes. Rickettsia vertical transmission on the one hand and its partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.To test for the possible involvement of Wolbachia and Cardiniumin the reproductive isolation of different B. tabacibiotypes, reciprocal crosses were preformed among populations of the Cardinium-infected, Wolbachia-infected and uninfected populations. The crosses results demonstrated that phylogeographically divergent B. tabaci are reproductively competent and that cytoplasmic incompatibility inducer-bacteria (Wolbachia and Cardinium) both interfered with, and/or rescued CI induced by one another, effectively facilitating bidirectional female offspring production in the latter scenario.This knowledge has implications to multitrophic interactions, gene flow, speciation, fitness, natural enemy interactions, and possibly, host preference and virus transmission. Although extensive and creative attempts undertaken in both laboratories to cure whiteflies of non-primary symbionts have failed, our finding of naturally uninfected individuals have permitted the establishment of Rickettsia-, Wolbachia- and Cardinium-freeB. tabaci lines, which are been employed to address various biological questions, including determining the role of these bacteria in whitefly host biology.