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[
International Worm Meeting,
2013]
Like us, C. elegans lives in a microbial world. In its natural habitats of rotting fruits and vegetation, these nematodes proliferate as they dine on an array of microbes. Interactions with microbes span a spectrum from constant confrontation (pathogens) to relative indifference (food) to perhaps even mutual benefit (symbionts). This study identifies these natural microbes and addresses whether microbiome composition influences proliferation of C. elegans in the wild.
To examine this question, we sequenced bacterial 16S (SSU) rDNA amplicons from habitats with wild C. elegans populations collected in France and Spain. Our results show that C. elegans encounters a broad array of bacteria in the wild-especially the divisions (phyla) of Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. An abundance-weighted comparisons of phylogenetic differences (UniFrac) showed distinct clustering by habitat type, as rotting apples clustered separately from other habitats sequenced. Further, rotting apples clustered by large presence of proliferating or small non-proliferating (dauer) populations of worms. C. elegans appear to proliferate in apples with 'simpler' microbiomes (lower diversity, fewer species and Proteobacteria-rich). Specific alpha-proteobacteria were particularly enriched in apples with proliferating worms, while a number of genera were consistently found in apples with non-proliferating worms (e.g., Pseudomonas, several Bacteroidetes, etc.). Population size also correlated with apple rottenness, suggesting bacterial load is key to growth as well.
Similarly, Proteobacteria content does affect C. elegans (N2) growth rate in the lab, as worms grew faster on mixtures (and single isolates) with 80% Proteobacteria versus those with 40% Proteobacteria. Together, these studies define the microbial diet of C. elegans and implicate the natural microbiome as a key determinant of C. elegans' growth in the wild.
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[
International Worm Meeting,
2015]
Like us, C. elegans lives in a microbial world. In its natural habitats of rotting fruits and vegetation, C. elegans encounters a diverse array of microbes, where they serve as diet, microbiota or pathogens. C. elegans is highly tuned and equipped to sense and response to the milieu of microbial products (xenobiotics) that they are bombarded with, as they navigate this microbial landscape. Indeed, several recent studies have demonstrated that animals monitor basic cellular subsystems for microbial targeting (presumably through decreased functionality/efficiency or the like), though the extent to which beneficial microbes may interface with these systems is unknown. Small RNA pathways play a central role in regulating many of the transcriptional and developmental programs that are responsive to microbes, in addition to directly mediating anti-viral immunity. Thus, this study takes a broad look at natural microbes that may specifically engage small RNA pathways to regulate C. elegans physiology.To examine this question, we screened a panel of 565 microbes ('BIGb collection') isolated from C. elegans' natural habitats for microbial-enhancers or -reducers of RNAi (mERI or mRDE, respectively) via co-feeding with several RNAi clones in E. coli (somatic and germline) or alone with a panel of transgenic reporters. In co-feeding experiments, nearly 20% of the microbes reduced RNAi effectiveness (113), while 7.5% enhanced RNAi (42). There was also a strong correlation between the impact of a microbe on C. elegans physiology-beneficial (faster growth, unstressed) or detrimental (slow growth/death, activation of stress reporters)-and its impact on RNAi pathways, with mRDE isolates being more likely to be detrimental and vice versa. Since co-feeding can affect uptake of dsRNA-producing E. coli, we also validated these microbes for impacts on RNAi using a panel of transgene-silencing reporter strains. Together, these data implicate a number of often pathogens (e.g., Pseudomonas, Stenotrophomonas and Enterobacter) as mRDE and beneficial microbes (e.g., Providencia and Bacillus) as mERI. In parallel, we also demonstrate that microbial RNA directly engages a subset of RNAi pathways in regulation of C. elegans growth. Together, these studies expand our understanding of the host systems that are under microbial influence and regulate host health. .
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[
International Worm Meeting,
2011]
Like all of us, C. elegans lives in a microbially dominated world. They naturally proliferate habitats rich in microbes, like rotting fruits and decaying vegetation. Interactions with these as yet unnamed microbes undoubtedly span a spectrum from constant confrontation (pathogens) to relative indifference (food) and perhaps even mutual benefit (symbionts). Interestingly, in contrast to N2, wild C. elegans harbor intestinal microbes. In addition to food, it is tantalizing to speculate that these worms might enlist microbes to improve its fitness (e.g., resistance to pathogens, harmful chemicals, etc.), just as we and many other animals have done. Within a habitat, C. elegans expansion is affected by a host of environmental and intrinsic (genetic) factors. Specifically, similar habitats in close proximity can harbor anywhere from no worms to dauers to actively proliferating populations; our hypothesis is that the mixture of microbes present is a key determinant of C. elegans' success. To examine this question, we performed culture-independent sequencing of microbial small subunit rDNA from habitats with wild C. elegans populations collected during several field seasons and different locations. In addition, we isolated worms to directly examine their more closely associated microbes. Using this dataset, we are able to address: (a) what are the commonly encountered microbes; (b) do groups of microbes correlate with population success; and (c) does C. elegans maintain a microbiota? Initial results from habitats indicate that bacteria belonging to four divisions (phyla), Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria are common in C. elegans' habitats, and occasionally seven more rare divisions are observed. However, there are also great variations in the bacterial diversity and richness among habitats (10-100+ species); we are testing bacterial species found in many habitats for their close association with worms. Notably, cultured microbial mimics of these habitats dramatically alter C. elegans' growth: e.g., proliferation on a Proteobacteria-rich mimic occurs faster than on a Bacteroidetes-rich mimic. Comparisons of responses of wild and lab-raised worms are also in progress. Determination of the microbes in C. elegans natural habitats is a first step in expanding our understanding of how microbes can influence host fitness and resistance to ecological pressures.
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[
International Worm Meeting,
2009]
Animals have evolved in a microbial world. Microbes exert influence on a broad range of physiologic processes, from shear survival to development to regulation of energy balance. C. elegans is highly tuned to microbial cues as potential food. While E. coli is fed often to lab strains, such associations are not likely to occur in the wild. Indeed, culture-based assessments of natural C. elegans habitats, such as compost and rotting fruits, indicate the presence of a broad range of bacterial phylotypes (M.-A. Felix, unpublished). To more broadly examine the spectrum of microbes encountered by wild C. elegans populations, we performed culture-independent sequencing of bacterial 16S rDNA from eight habitats harboring wild C. elegans populations. The data indicates that these animals most commonly encounter bacteria belonging to four phylogenetic divisions (phyla), Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. Our goal is to use this natural association to explore conserved endocrine responses to microbes in C. elegans. To this end, we selected a panel of ten bacteria that were identified in at least three independent habitats and assayed various measures of energy balance. Growth rates, brood size and feeding rates of C. elegans are coarse assessments of the food quality and potential modulation of energy store partitioning. We found that C. elegans (N2) exhibited a spectrum of growth rates on these wild microbes compared to E. coli. These differences cannot solely be attributed to bacterial cell size or wall thickness, as two closely related gram-positive Firmicutes have opposite impacts on growth rates, suggesting additional factors are involved. Decreased brood sizes were also observed in animals grown on three diverse bacteria. Feeding rates were diminished on four microbes, with a Bacteroidetes being the most dramatic. Two of these microbes exhibit decreased Nile Red staining, though biochemical analyses of lipid content are ongoing. To allow for better classification of these growth responses by food quality-based measures, we are employing additional assays of C. elegans'' activity and behavior. These results suggest that exposure to commonly encountered wild microbes distinctly alters C. elegans energy metabolism, which may represent co-evolved endocrine responses to microbes.
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Carr, Chris, Rowedder, Holli, Plunkett, Guy, Melo, Justine, Durfee, Tim, Nusbaum, Chad, Glasner, Jeremy, Russ, Carsten, Ruvkun, Gary, Sykes, Sean, Samuel, Buck S., Young, Sarah
[
International Worm Meeting,
2011]
Like other metazoans, C. elegans fitness (success) within its microbe-rich habitats depends on a tight balance of energy acquisition and expenditure. Thus, it is also highly tuned to microbial cues that allow it to separate potential food or friend from foe. Accordingly, some microbial signals have been postulated to influence fat storage in parallel to endogenous endocrine cues. Several studies also show that the E. coli-adapted N2-Bristol strain is especially sensitive to 'minor' differences in E. coli strains: faster growth rates, increased progeny delivery rates, and less fat retention is seen when worms consume HB101 compared to OP50. Perhaps due to this fitness benefit, worms also exhibit increased satiety and a behavioral preference for HB101. Thus, we have sought to identify the E. coli gene products that modulate C. elegans fitness. To this end, we have sequenced E. coli genomes routinely used in C. elegans cultivation: HB101 (2 isolates), OP50 (2 isolates) and HT115. Despite little variation among strain isolates, 350 and 412 genes are 'unique' to OP50 and HB101, respectively. Many are organized into clusters, and represent a range of gene functions: e.g., carbohydrate utilization (96), cell wall/LPS modification (42), amino acid metabolism (21), regulation (41), the Cascade system (6) and fatty acid metabolism (5). Phenotype microarrays were also used to confirm the metabolic defects. In order to systematically test the impact of these microbial gene products on C. elegans' fitness, we assembled nearly 200 single gene mutants with defined function in a 'neutral' and consistent genetic background (E. coli K12). We then used a number of assays to test a mutant's impact on N2 growth, broods, body size and fat storage. Our analyses indicate that both genes in core metabolism and transport/biosynthesis of conserved mediators of host interaction-autoinducers, biogenic amines, short-chain fatty acids and LPS-influence N2 fitness. Studies of these small molecules as sensory or nutritive cues to C. elegans directly or via regulation of E. coli metabolism are ongoing. However, results so far indicate that the microbial milieu of signals may be just as important of a determinant of C. elegans' fitness as the nutritional potential for supporting growth of a population within a given habitat.
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2010]
Like all of us, C. elegans lives in a microbially dominated world. It naturally colonizes habitats rich in microbes, such as rotting fruits and vegetation. Interactions with these as yet unnamed microbes undoubtedly span a spectrum from constant confrontation (pathogens) to relative indifference (food) and perhaps even mutual benefit (symbionts). Many other animals, including some nematodes, acquire fitness benefits by relying on microbes for nutrients, protection from pathogen colonization, and detoxification of harmful xenobiotics. Indeed, C. elegans adults have been observed to harbor undigested microbes in their intestines under some circumstances. In any case, C. elegans has certainly adapted to be highly tuned to microbial cues in order to evaluate their food quality or potential to support growth. However, little is known about how this complex microbial calculus occurs with natural microbes. To more broadly examine the microbes encountered by wild C. elegans populations, we performed both culture-based and comprehensive culture-independent sequencing of microbial SSU rDNA from habitats with proliferating and non-proliferating (mostly dauer) wild C. elegans populations. In addition, we isolated animals away from their habitats to directly sequence their more closely associated microbes and potential natural microbiota. Preliminary analyses of all habitats indicate that these animals most commonly encounter bacteria belonging to four phylogenetic divisions (phyla), Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, along with seven more rare divisions. However, there are also great variations in the diversity and richness of bacteria found within the habitats: some rotting apples have more simple microbial communities (<10 phylotypes) while other rotting vegetation harbors nearly 100 phylotypes. Additionally, several phylotypes were identified from quite disparate habitats (e.g., a snail, a rotting apple and a rotting orange), which could indicate their close association with C. elegans animals themselves. Further analyses of enrichment of specific microbes in isolated worms compared to their habitats (natural or culture-based mimics) will inform these correlations. Determination of what microbes C. elegans associate with naturally is a first step in elaborating the ecological pressures that they may both instigate and ameliorate. Further, by comparing responses of lab-raised strains that have been domesticated on E. coli and wild strains with their naturally associated microbes, we hope to identify conserved host-microbial response pathways that influence C. elegans physiology and metabolism.
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[
Worm Breeder's Gazette,
1993]
DIFFERENTIAL EFFECTS OF DAUER-DEFECTIVE MUTATIONS ON L1- SPECIFIC SURFACE ANTIGEN SWITCHING. David G. Grenache and Samuel M. Politz, Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA.
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[
International Worm Meeting,
2021]
The animal gut is critical for both acquisition and interpretation of nutrients and other molecules in the environment, in addition to cultivating a robust community of microbes. Communication of nutritional and microbial information between the gut and the brain, referred to as the gut - brain axis, occurs via signaling molecules like host-derived neuropeptides and neurotransmitters, in addition to microbially produced metabolites. Thus, altering the gut - brain axis and the communication within can have influence host physiology and metabolism, but also impact the regulation of the host's gut microbiome. Studies conducted within the last decade have highlighted the importance of balanced relationships within the microbiome, but the molecular mechanisms of microbiome regulation of the gut - brain axis remain largely uncharacterized due to the complexities in other systems and difficulties with high-throughput testing. We use C. elegans to address this because it contains a comparable gut whose composition is well defined, is amenable to various high - throughput techniques, and is transparent, which allows us to view differences in composition using fluorescent bacteria. Insulin signaling members, specifically the insulin receptor and downstream pathway factors, have previously been shown to alter microbiome composition. To determine which neuropeptides, and their respective receptors, are associated with gut microbiome regulation, we have conducted RNAi experiments coupled with 16s rRNA sequencing to assess gut composition. The resulting data has shown that roughly 20 of 104 RNAi clones assayed alter composition of the microbiome, broadly or in taxa-specific manner, in N2 animals when knocked - down via RNAi in a tissue - specific manner. Specifically, flp -14, ins -1, ins -11, ins -18, ins -21, and ins -24, promote colonization of Ochrobactrum when knocked down in the gut alone using an intestinal - specific RNAi strain. These knockdown findings will be further validated using fluorescent bacteria colonization phenotypes as a proxy for sequencing. Insulin signaling members, specifically the insulin receptor and downstream pathway factors, have previously been shown to alter microbiome composition. The data obtained from the RNAi screening suggests that the normal function of these 20 or so neuropeptides is to relay information about the microbiome or otherwise that could influence microbe's ability to colonize the gut. Through these and related studies, we hope to better characterize the gut - brain axis signaling pathways and identify novel molecular factors that regulate the microbiome in C. elegans.
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[
International Worm Meeting,
2019]
The gut microbiome both extends the capabilities of its host and alters its physiology. Host genetics, diet, environment, and microbe-microbe interactions influence microbiome form and function. In humans, microbiome community function is important for resilience to disease. Despite its importance, the essential functions that drive microbiome assembly and stability in remain largely unaddressed in any host. To address this challenge, we leverage the nematode Caenorhabditis elegans to explore how microbiomes of distinct scales and functional complexity assemble in different host genetic backgrounds. This system has several advantages, including (1) a simple microbiome that can be removed (bleaching) and manipulated in the lab; (2) highly conserved intestinal physiology and innate immunity; and (3) comparable microbial mechanisms for host gut persistence. Previous studies in the lab have identified host-dependent variation in the microbiome: around 40 C. elegans wild strains were exposed to a model microbiome (BIGbiome) and they form three distinct gut types that differ in dominant taxa [Ochrobactrum, Bacteroidetes or a mix]. To examine the essential functions for colonization of the C. elegans gut, we first sequenced more than 100 bacterial genomes of its microbiome to determine their functional potential. Next, bacteria in each C. elegans microbiome were mapped to their corresponding genomes and scaled by abundance (16S) to construct virtual metagenomes. In-silico metabolic reconstructions and comparative metagenomic analyses both indicate broad variation in potential functions and provide many candidate genes important for host association. Unique functions may also allow for host selection of specific taxa. Analyses of each of the genomes indicate a wide range of potential metabolic capacities from "generalists" (like Enterobacteria) that have genes for utilization of a wide range of carbohydrate substrates to "specialists" (like Ochrobactrum) that have a narrower nutrient usage potential. Interestingly, specialists thrive within the worm gut whereas generalists are less successful in a community context. To explore this further, we exposed C. elegans strains to microbiomes of increasing complexity in function and taxa diversity. Together, these studies highlight key molecular functions under host selection that may underlie assembly and stability of the microbiome.
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[
International Worm Meeting,
2021]
Teasing apart the dense network of molecular mechanisms that link digestive tract microbial community members to each other and their host is challenging in most systems due to complexity and tractability. Recently, Caenorhabditis elegans has emerged as a powerful model system to study host-microbe interactions. The simplicity of the C. elegans digestive tract, together with the nematode's genetic amenability, and the availability of relevant microbial collections make it ideal for the study of the fundamental mechanisms of host-microbiota interactions. To characterize those mechanisms, we focused on identifying genetic features important for bacteria colonizing the C. elegans gut. We developed "WormBiome" a pipeline that predicts the combined functional potential of defined C. elegans gut microbiome. The "Wormbiome" pipeline match predicted microbial abundance from 16S rRNA amplicon datasets to functional genomic annotations and output functional profiles for each microbial community present in the submitted dataset. Our functional annotations rely on curated metabolic (Metacyc) and functional annotations (KEGG) databases build on known C. elegans related bacteria. Then the pipeline predicts features significantly different between user-defined sample groups. We tested the pipeline using a defined and fully sequenced 12-member model microbiome (CeMbio) grown with and without N2 animals. With 10 replicates for each condition, we identified 1700 significantly different features, distributed across 180 KEGG and 63 Metacyc categories. The most abundant features belong to the lipid, amino acid, and cofactor metabolisms. Among genes predicted to be more abundant in worm-associated communities, we found the de novo synthesis of vitamin B12 and metabolic pathways for host-essential amino acids, such as proline, alanine, and arginine. We verified the pipeline's prediction by examining the impact of nutrient depletion on gut microbiome composition by selectively supplementing or removing amino acids individually or altogether. Our results show that a single change in amino acid can affect how bacteria interact with each other and promote the growth of certain community members and that complete removal of amino acids promotes colonization of metabolically flexible members of the microbiome like Ochrobactrum. This study establishes a robust framework for identifying microbial functions that govern affect host-microbe associations and beneficial interactions.