The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g. human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans.

Highlights d Development of a model microbiome with high strain-level diversity for C. elegans d Host genetic variation drives distinct microbiome community types in wild C. elegans d Adult gut microbiome composition is driven by insulin signaling networks d Gut microbiome alters host growth and activates insulin signaling pathways

Bathymodiolus mussels dominate animal communities at many hydrothermal vents and cold seeps. Essential to the mussels' ecological and evolutionary success is their association with symbiotic methane- and sulfur-oxidizing gammaproteobacteria, which provide them with nutrition. In addition to these well-known gammaproteobacterial endosymbionts, we found epsilonproteobacterial sequences in metatranscriptomes, metagenomes and 16S rRNA clone libraries as well as by polymerase chain reaction screening of Bathymodiolus species sampled from vents and seeps around the world. These epsilonproteobacterial sequences were closely related, indicating that the association is highly specific. The Bathymodiolus-associated epsilonproteobacterial 16S rRNA sequences were at most 87.6% identical to the closest cultured relative, and 91.2% identical to the closest sequences in public databases. This clade therefore represents a novel family within the Epsilonproteobacteria. Fluorescence in situ hybridization and transmission electron microscopy showed that the bacteria are filamentous epibionts associated with the gill epithelia in two Bathymodiolus species. In animals that host highly specific symbioses with one or a few types of endosymbionts, other less-abundant members of the microbiota can be easily overlooked. Our work highlights how widespread and specific associations with less-abundant microbes can be. Possibly, these microbes play an important role in the survival and health of their animal hosts.

Most autotrophs use the Calvin-Benson-Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts ("Candidatus Thiobarba") of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cycle genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that "Ca. Thiobarba" switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated "Ca. Thiobarba". Direct stable isotope fingerprinting showed that "Ca. Thiobarba" has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment.

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g. human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans .Pseudomonadaceae , and Xanthomonodaceae ) and Bacteroidetes ( Sphingobacteriaceae , Weeksellaceae , Flavobacteriaceae ) (Zhang et al. 2017) .Here we establish a publicly available and well-defined model microbiome for use in C. elegans (CeMbio). This set is composed of 12 bacteria from 9 different families that represent the core microbiome of C. elegans based on analyses and empirical studies of intestinal colonization.These bacterial strains are presented with fully sequenced and annotated genomes, metabolic network reconstructions, and robust protocols for their use in C. elegans studies and beyond. All of the bacteria effectively colonize the C. elegans gut both alone and as a community, which can impact on the growth and development of the host. The CeMbio community has broad application to any aspect of C. elegans biology from aging to pathogenesis, development to neurobiology, and any aspect of physiology where a more natural environment is desired.Ultimately, pairing of this well-defined microbiome and highly-tractable host is envisioned to complement other systems (e.g., (Fraune and Bosch 2010;Brugiroux et al. 2016;Douglas 2019) ) in advancing understanding of the mechanisms of microbiome impact on host health and disease.