Salmonella enterica serovar Typhimurium (STm) represents a major global health burden. Strains endemic in sub-Saharan Africa cause life-threatening invasive non-typhoidal salmonellosis (iNTS) in vulnerable populations. Studies in the iNTS-like mouse model show that STm induces profound germinal centre (GC) disruption, impairing high-affinity, long-lived antibody and memory B cell formation - affecting nascent and pre-existing GC reactions. Lipopolysaccharide (LPS) and specific STm type 3 secretion effectors drive GC collapse, but the determining bacteria-host interactions are still unclear. Although STm induces an extrafollicular (EF) B cell response generating protective antibodies, their longevity remains unclear. With no licensed human vaccine for iNTS, we propose that vaccine strategies should consider ways to protect GC integrity and include GC parameters as endpoints in preclinical trials.

BACKGROUND AND AIMS:Accumulating evidence suggests the microbiota is a key factor in Disorders of Gut-Brain Interaction (DGBI), by affecting host immune and neural systems. However, the underlying mechanisms remain elusive due to their complexity and clinical heterogeneity of patients with DGBIs. We aimed to identify neuroimmune pathways that are critical in microbiota-gut-brain communication during de novo gut colonization. METHODS:We employed a combination of gnotobiotic and state-of-the-art microbial tools, behavioral analysis, immune and pharmacological approaches. Germ-free wild type, TLR signaling-deficient MyD88-/- Ticam1-/- and lymphocyte-deficient SCID mice were studied before and after colonization with specific pathogen-free microbiota, Altered Schaedler Flora, E. coli or S. typhimurium (permanent or transient colonizers). TLR agonists and antagonists, CCR7 antagonist or immunomodulators were used to study immune pathways. We assessed brain c-Fos, brain-derived neurotrophic factor, and dendritic and glial cells by immunofluorescence, expression of neuroimmune genes by NanoString and performed brain proteomics. RESULTS:Bacterial monocolonization, conventionalization or administration of microbial products to germ-free mice altered mouse behavior similarly, acting through Toll-like receptor or nucleotide-binding oligomerization domain signaling. The process required CD11b+CD11c+CD103+ dendritic cell activation and migration into the brain. The change in behavior did not require the continued presence of bacteria and was associated with activation of multiple neuro-immune networks in the gut and the brain. CONCLUSIONS:Changes in neural plasticity occur rapidly upon initial gut microbial colonization and involve innate immune signaling to the brain, mediated by CD11b+CD11c+CD103+ dendritic cell migration. The results identify a new target with therapeutic potential for DGBIs developing in context of increased gut and blood-brain barrier permeability.

Secondary bile acids, generated through microbial transformation of primary bile acids secreted in bile, play a role in shaping intestinal microbial communities, modulating host immunity, and regulating energy metabolism. In vitro studies have shown that the balance between primary and secondary bile acids strongly affects spore germination, growth, and cellular physiology of Clostridioides difficile , a major nosocomial gut pathogen. In vivo correlations between microbiome composition, bile acid metabolome, and colonization resistance have led to the hypothesis that 7α-dehydroxylating bacteria such as Clostridium scindens protect against C. difficile infection by producing secondary bile acids like deoxycholic acid. However, due to the genetic intractability of known 7α-dehydroxylating species, direct experimental validation of this causal relationship has been challenging. In this study, we leveraged the first available 7α-dehydroxylation-deficient baiH mutant to test the direct role of 7-dehydroxylated bile acid production in C. difficile colonization resistance in vivo. We colonized gnotobiotic mice with isogenic wild-type or baiH strains of the recently described 7α-dehydroxylating species Faecalicatena contorta , including wild-type C. scindens -colonized mice as a positive control. Wild-type F. contorta accumulated 7-dehydroxylated bile acids at levels equivalent to C. scindens , in a strictly baiH -dependent manner. However, despite equivalent bile acid profiles, wild-type F. contorta failed to replicate the C. difficile -restrictive phenotype observed with C. scindens . These findings demonstrate that commensal clostridial 7α-dehydroxylation alone is not sufficient for enhancing colonization resistance to C. difficile . Our results highlight the existence of additional, potentially bile acid-independent mechanisms by which certain commensals mediate protection, with important implications for microbiota-based therapies. Importance 7α-dehydroxylated secondary bile acids, including deoxycholic acid and lithocholic acid, produced by commensal clostridia are widely assumed to inhibit the important nosocomial pathogen Clostridioides difficile , yet their precise role in colonization resistance remains unresolved. Using a defined mouse microbiota and an isogenic Faecalicatena contorta strain pair differing in a single 7α-dehydroxylation gene ( baiH ), we show that restoration of secondary bile acid production is not sufficient to delay C. difficile colonization in vivo. This contrasts with the protective effect of Clostridium scindens , which generates a similar bile acid profile. Our findings uncouple bile acid metabolism from protection and suggest that additional, strain-specific functions – such as nutrient competition or antimicrobial production – play a critical role. Understanding these mechanisms is essential for the rational design of next-generation microbiota-based therapies to prevent or treat recurrent C. difficile infection.

IgA antibodies have an important role in clearing mucosal pathogens. In this study, we have examined the contribution of IgA to the immune control of the gastrointestinal bacterial pathogens Helicobacter pylori and Citrobacter rodentium. Both bacteria trigger a strong local IgA response that results in bacterial IgA coating in mice and in gastritis patients. Class switching to IgA depends on Peyer's patches, T-cells, eosinophils, and eosinophil-derived TGF-(3 in both models. In the case of H. pylori, IgA secretion and bacterial coating also depend on a functional bacterial type IV secretion system, which drives the generation of Th17 cells and the IL-17dependent expression of the polymeric immunoglobulin receptor PIGR. IgA- /- mice are hypercolonized with C. rodentium in all examined tissues, suffer from more severe weight loss and develop more colitis. In contrast, H. pylori is controlled more efficiently in IgA- /- mice than their WT counterparts. The effects of IgA deficiency of the offspring can be compensated by maternal IgA delivered by WT foster mothers. We attribute the improved immune control observed in IgA- /- mice to IgA-mediated protection from complement killing, as H. pylori colonization is restored to wild type levels in a composite strain lacking both IgA and the central complement component C3. IgA antibodies can thus have protective or detrimental activities depending on the infectious agent.

Host-microbiome communication is frequently perturbed in gut pathologies due to microbiome dysbiosis, leading to altered production of bacterial metabolites. Among these, 7α-dehydroxylated bile acids are notably diminished in inflammatory bowel disease patients. Herein, we investigated whether restoration of 7α-dehydroxylated bile acids levels by Clostridium scindens, a human-derived 7α-dehydroxylating bacterium, can reestablish intestinal epithelium homeostasis following colon injury. Gnotobiotic and conventional mice were subjected to chemically-induced experimental colitis following administration of Clostridium scindens. Colonization enhanced the production of 7α-dehydroxylated bile acids and conferred prophylactic and therapeutic protection against colon injury through epithelial regeneration and specification. Computational analysis of human datasets confirmed defects in intestinal cell renewal and differentiation in ulcerative colitis patients while expression of genes involved in those pathways showed a robust positive correlation with 7α-dehydroxylated bile acid levels. Clostridium scindens administration could therefore be a promising biotherapeutic strategy to foster mucosal healing following colon injury by restoring bile acid homeostasis.

Along a food chain, microbiomes occur in each component and often contribute to the functioning or the health of their host or environment. ‘One Health’ emphasizes the connectivity of each component’s health. Chemical stress typically causes dysbiotic microbiomes, but it remains unclear whether chemical stressors consistently affect the microbiomes of food chain components. Here, we challenged food chain components, including water, sediments, soil, plants, and animals, with three chemical stresses consisting of arsenic (toxic trace element), benzoxazinoids (bioactive plant metabolites), and terbuthylazine (herbicide). We analysed 1064 microbiomes to assess their commonalities and differences in their stress responses. We found that chemical stressors overall decreased microbiome diversity in soil, but not in the other microbiomes. In response to stress, all food chain communities strongly shifted in their composition, generally becoming compositionally more similar to each other. In addition, we observed stochastic effects in host-associated communities (plant, animal). Dysbiotic microbiomes were characterized by different sets of bacteria, which responded specifically to the three chemical stressors. Microbial co-occurrence patterns significantly shifted with either decreased (water, sediment, plant, animal) or increased (soil) network sparsity and numbers of keystone taxa following stress treatments. These results suggest major re-distribution of specific taxa in the overall stress- and component-specific responses of microbiomes with the community stability of plant and animal microbiomes being the most affected by chemical stresses.

Background and Aims Accumulating evidence suggests the microbiota is a key factor in disorders of gut-brain interaction (DGBI), by affecting host immune and neural systems. However, the underlying mechanisms remain elusive due to their complexity and clinical heterogeneity of patients with DGBIs. We aimed to identify neuroimmune pathways that are critical in microbiota-gut-brain communication during de novo gut colonization. Methods We employed a combination of gnotobiotic and state-of-the-art microbial tools, behavioral analysis, immune and pharmacological approaches. Germ-free wild type, MyD88−/− Ticam1−/− and SCID mice were studied before and after colonization with specific pathogen-free microbiota, Altered Schaedler Flora, E. coli or S. typhimurium (permanent or transient colonizers). TLR agonists and antagonists, CCR7 antagonist or immunomodulators were used to study immune pathways. We assessed brain c-Fos, brain-derived neurotrophic factor, and dendritic and glial cells by immunofluorescence, expression of neuroimmune genes by NanoString and performed brain proteomics. Results Bacterial monocolonization, conventionalization or administration of microbial products to germ-free mice altered mouse behavior similarly, acting through Toll-like receptor or nucleotide-binding oligomerization domain signaling. The process required CD11b+CD11c+CD103+ cell activation and migration into the brain. The change in behavior did not require the continued presence of bacteria and was associated with activation of multiple neuro-immune networks in the gut and the brain. Conclusions Changes in neural plasticity occur rapidly upon initial gut microbial colonization and involve innate immune signaling to the brain, mediated by CD11b+CD11c+CD103+ cell migration. The results identify a new target with therapeutic potential for DGBIs developing in context of increased gut and blood-brain barrier permeability. Highlights ### Competing Interest Statement The authors have declared no competing interest. * DGBI : Disorders of Gut-Brain Interaction IBS : Irritable Bowel Syndrome GF : Germ-free ASF : Altered Schaedler Flora SPF : Specific Pathogen Free DC : Dendritic cell TLR : Toll-Like Receptor NOD : Nucleotide-binding Oligomerization Domain CCR7 : Chemokine Receptor 7 BDNF : Brain-Derived Neurotrophic Factor LPS : Lipopolysaccharide SCID : Severe Combined ImmunoDeficiency PBS : Phosphate-Buffered Saline TBS : Tris-Buffered Saline BSA : Bovine Serum Albumin

Arsenobetaine (AB), a major organic arsenic (As) species in seafood, is regarded as safe by current regulatory assessments due to low toxicity and rapid unmodified urinary excretion. This notion has been challenged by reports of AB metabolism by intestinal bacteria in vitro and more recent evidence of in vivo AB metabolism in mice. However, these studies did not establish the causal role of intestinal bacteria in AB transformation in vivo. To address this, we employed gnotobiology and compared the biotransformation of As from naturally AB-rich rodent diet in mice that were either germ-free or colonized with gut microbiota of varying microbial diversity. Our results confirm the in vivo metabolism of AB in the intestine under chronic dietary exposure. The transformation of ingested As was dependent on the presence/absence and complexity of the gut microbiota. Notably, specific toxic As species were absent under germ-free condition. Furthermore, gut microbial colonization was linked to increased As accumulation in the intestinal lumen as well as systemically, along with delayed clearance from the body. These findings emphasize the mammalian gut microbiota as a critical factor in evaluating the safety of AB-accumulating seafoods.

BACKGROUND:Bile acids (BAs) are steroid-derived molecules with important roles in digestion, the maintenance of host metabolism, and immunomodulation. Primary BAs are synthesized by the host, while secondary BAs are produced by the gut microbiome through transformation of the former. The regulation of microbial production of secondary BAs is not well understood, particularly the production of 7-dehydroxylated BAs, which are the most potent agonists for host BA receptors. The 7-dehydroxylation of cholic acid (CA) is well established and is linked to the expression of a bile acid-inducible (bai) operon responsible for this process. However, little to no 7-dehydroxylation has been reported for other host-derived BAs (e.g., chenodeoxycholic acid, CDCA or ursodeoxycholic acid, UDCA). RESULTS:Here, we demonstrate that the 7-dehydroxylation of CDCA and UDCA by the human isolate Clostridium scindens is induced when CA is present, suggesting that CA-dependent transcriptional regulation is required for substantial 7-dehydroxylation of these primary BAs. This is supported by the finding that UDCA alone does not promote expression of bai genes. CDCA upregulates expression of the bai genes but the expression is greater when CA is present. In contrast, the murine isolate Extibacter muris exhibits a distinct response; CA did not induce significant 7-dehydroxylation of primary BAs, whereas BA 7-dehydroxylation was promoted upon addition of germ-free mouse cecal content in vitro. However, E. muris was found to 7-dehydroxylate in vivo. CONCLUSIONS:The distinct expression responses amongst strains indicate that bai genes are regulated differently. CA promoted bai operon gene expression and the 7-dehydroxylating activity in C. scindens strains. Conversely, the in vitro activity of E. muris was promoted only after the addition of cecal content and the isolate did not alter bai gene expression in response to CA. The accessory gene baiJ was only upregulated in the C. scindens ATCC 35704 strain, implying mechanistic differences amongst isolates. Interestingly, the human-derived C. scindens strains were also capable of 7-dehydroxylating murine bile acids (muricholic acids) to a limited extent. This study shows novel 7-dehydroxylation activity in vitro resulting from the presence of CA and suggests distinct bai gene expression across bacterial species.

Root exudates contain specialised metabolites that shape the plant's root microbiome. How host-specific microbes cope with these bioactive compounds, and how this ability affects root microbiomes, remains largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound in the maize rhizosphere MBOA (6-methoxybenzoxazolin-2(3H)-one) and formed AMPO (2-amino-7-methoxy-phenoxazin-3-one). AMPO forming bacteria were enriched in the rhizosphere of benzoxazinoid-producing maize and could use MBOA as carbon source. We identified a gene cluster associated with AMPO formation in microbacteria. The first gene in this cluster, bxdA encodes a lactonase that converts MBOA to AMPO in vitro. A deletion mutant of the homologous bxdA genes in the genus Sphingobium, did not form AMPO nor was it able to use MBOA as a carbon source. BxdA was identified in different genera of maize root bacteria. Here we show that plant-specialised metabolites select for metabolisation-competent root bacteria. BxdA represents a benzoxazinoid metabolisation gene whose carriers successfully colonize the maize rhizosphere and thereby shape the plant's chemical environmental footprint.

16S rRNA amplicon sequencing is a fundamental tool for characterizing prokaryotic microbial communities. While short-read 16S rRNA sequencing is a proven standard for examining complex microbiomes, it cannot achieve taxonomic classification beyond genus level. Third-generation sequencing technologies, particularly nanopore sequencing, have allowed for full-length 16S rRNA gene sequencing enabling enhanced taxonomic resolution to species and strain levels.Precise species-to-strain level classification is crucial in investigating low-complexity microbiota. This research presents an efficient pipeline using full-length 16S rRNA amplicon nanopore sequencing, spanning library prep to computational analysis for low-complexity microbiota composition analysis. We applied this pipeline to a defined intestinal bacterial community in gnotobiotic mice to evaluate different methods.Our findings revealed that the proprietary barcoded universal primers 27F-1492R from Oxford Nanopore Technologies(ONT) 16S amplicon sequencing kit did not efficiently amplify the 16S rRNA gene of Bifidobacterium species. Addressing this constraint, we designed degenerate primers and employed ONT’s native barcoding kit for library preparation. We developed a customized wet lab and bioinformatics pipeline for processing and classifying amplicon reads at the species level.Validation of the protocol using a mock community DNA sample with known composition confirmed a reduced analytical bias. Additionally, our method surpassed Illumina short-read V3-V4 amplicon sequencing, achieving accurate species-level classification compared to Illumina’s genus-level accuracy.This pipeline is tailored for analyzing the composition of low-complexity microbiota from natural ecosystems and synthetic/gnotobiotic communities. It is cost- and time-effective and therefore accessible for small-scale studies that would otherwise be hindered by the typically long turnaround times of NGS services.Importance 16S rRNA amplicon sequencing is conventionally used to identify microbes and determine their composition in microbial communities. Deep amplicon sequencing of complex microbiomes is well established using short-read sequencing targeting variable regions of the 16S rRNA gene. Short reads enable the classification of bacteria until the genus level in the taxa hierarchy, whereas long reads provide better chances of identifying bacteria to species and even strain levels. This study introduces a streamlined approach for analyzing simple microbial communities using full-length 16S rRNA amplicon nanopore long read sequencing. This approach surpasses Illumina sequencing in species accuracy, is cost-effective and time-efficient. Tailored for low-complexity microbiota, it facilitates studies in natural or synthetic communities, especially beneficial for smaller-scale projects with limited resources.

Abstract Background Accumulating evidence suggests that gut microbiota affects brain development and its function. It is well known that compared with conventional mice (SPF), germ-free (GF) mice display higher exploratory behavior, which normalizes after bacterial colonization. However, little is known about the underlying mechanisms and first critical steps initiating microbiota-gut-brain communication, which lead to establishment of normal behavior. Purpose To investigate the role of immune system in the establishment of normal behavior after bacterial colonization. Method We assessed behavior in GF mice before and after colonization with SPF microbiota, Altered Schaedler Flora (ASF) or the single bacterial strain E. coli JM83, and compared them to SPF mice, using the light-dark preference and tail suspension tests. Levels of brain-derived neurotrophic factor (BDNF) and c-Fos expression were measured by immunofluorescence in the hippocampus and amygdala. Colonic and brain gene expression were assessed using a NanoString technology. The immunodeficient MyD88-/- Ticam1- and SCID mice were used to study the role of the innate and adaptive immune systems. To demonstrate the role of the dendritic cells (DCs), we measured behavior before and after mono-colonization with E. coli JM83 in GF mice treated with cosalane and fingolimod, that inhibit DCs activation and migration, respectively. Brain levels of CD11b, CD11c and CD103 as DCs markers was assessed by immunofluorescence. Result(s) Compared to SPF mice, GF mice showed higher exploratory and less depressive-like behavior. The ex-germ-free mice colonized with ASF microbiota, or mono-colonized with E. coli JM83 showed similar normalization of behavior as those colonized with SPF microbiota. Mono-colonization with E. coli reduced both BDNF and c-fos levels in the hippocampus and amygdala. While colonization of GF SCID mice induced same change in behavior as in wild-type mice, GF MyD88-/-Ticam1-/- mice did not alter their behavior. Mono-colonization affected multiple genes in the colon and the brain, associated with innate immunity and neural plasticity. Treatment with both cosalane and fingolimod prevented behavioral changes after colonization, which was paralleled by absence of CD11b+CD103+CD11c+ cells in the brain, otherwise found in high numbers in control mono-colonized mice and absent in germ-free mice. Conclusion(s) The innate immune system, through activation and migration of intestinal dendritic cells into the brain, initiates the neuro-immune signaling within the gut-brain axis and leads to normalization of behavior after bacterial colonization. Our findings may impact several psychiatric conditions, in which altered innate immune signaling has been implicated. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; Balsam Family Foundation Disclosure of Interest None Declared

SUMMARY Communities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N + 1/N−1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N−1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N−1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies.

Globally, enteropathogenic bacteria are a major cause of morbidity and mortality.1-3 Campylobacter, Salmonella, Shiga-toxin-producing Escherichia coli, and Listeria are among the top five most commonly reported zoonotic pathogens in the European Union.4 However, not all individuals naturally exposed to enteropathogens go on to develop disease. This protection is attributable to colonization resistance (CR) conferred by the gut microbiota, as well as an array of physical, chemical, and immunological barriers that limit infection. Despite their importance for human health, a detailed understanding of gastrointestinal barriers to infection is lacking, and further research is required to investigate the mechanisms that underpin inter-individual differences in resistance to gastrointestinal infection. Here, we discuss the current mouse models available to study infections by non-typhoidal Salmonella strains, Citrobacter rodentium (as a model for enteropathogenic and enterohemorrhagic E. coli), Listeria monocytogenes, and Campylobacter jejuni. Clostridioides difficile is included as another important cause of enteric disease in which resistance is dependent upon CR. We outline which parameters of human infection are recapitulated in these mouse models, including the impact of CR, disease pathology, disease progression, and mucosal immune response. This will showcase common virulence strategies, highlight mechanistic differences, and help researchers from microbiology, infectiology, microbiome research, and mucosal immunology to select the optimal mouse model.

BACKGROUND & AIMS:Although chronic diarrhea and constipation are common, the treatment is symptomatic because their pathophysiology is poorly understood. Accumulating evidence suggests that the microbiota modulates gut function, but the underlying mechanisms are unknown. We therefore investigated the pathways by which microbiota modulates gastrointestinal motility in different sections of the alimentary tract. METHODS:Gastric emptying, intestinal transit, muscle contractility, acetylcholine release, gene expression, and vasoactive intestinal polypeptide (VIP) immunoreactivity were assessed in wild-type and Myd88-/-Trif-/- mice in germ-free, gnotobiotic, and specific pathogen-free conditions. Effects of transient colonization and antimicrobials as well as immune cell blockade were investigated. VIP levels were assessed in human full-thickness biopsies by Western blot. RESULTS:Germ-free mice had similar gastric emptying but slower intestinal transit compared with specific pathogen-free mice or mice monocolonized with Lactobacillus rhamnosus or Escherichia coli, the latter having stronger effects. Although muscle contractility was unaffected, its neural control was modulated by microbiota by up-regulating jejunal VIP, which co-localized with and controlled cholinergic nerve function. This process was responsive to changes in the microbial composition and load and mediated through toll-like receptor signaling, with enteric glia cells playing a key role. Jejunal VIP was lower in patients with chronic intestinal pseudo-obstruction compared with control subjects. CONCLUSIONS:Microbial control of gastrointestinal motility is both region- and bacteria-specific; it reacts to environmental changes and is mediated by innate immunity-neural system interactions. By regulating cholinergic nerves, small intestinal VIP plays a key role in this process, thus providing a new therapeutic target for patients with motility disorders.

Bacterial pathogens employ a plethora of virulence factors for host invasion, and their use is tightly regulated to maximize infection efficiency and manage resources in a nutrient-limited environment. Here we show that during Escherichia coli stationary phase the 3 ' UTR-derived small non-coding RNA FimR2 regulates fimbrial and flagellar biosynthesis at the post-transcriptional level, leading to biofilm formation as the dominant mode of survival under conditions of nutrient depletion. FimR2 interacts with the translational regulator CsrA, antagonizing its functions and firmly tightening control over motility and biofilm formation. Generated through RNase E cleavage, FimR2 regulates stationary phase biology by fine-tuning target mRNA levels independently of the chaperones Hfq and ProQ. The Salmonella enterica orthologue of FimR2 induces effector protein secretion by the type III secretion system and stimulates infection, thus linking the sRNA to virulence. This work reveals the importance of bacterial sRNAs in modulating various aspects of bacterial physiology including stationary phase and virulence.

The microbes indigenous to helminth species are a major obstacle to deciphering host-parasite interactions. Repurposing a system of reversible bacterial colonization, we have generated germ-free Heligomosomoides polygyrus bakeri (Hpb) larvae that maintain the sterility of axenic mice upon infection. This protocol provides a valuable tool for controlled studies of helminth-microbiota-immune interactions.

The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.