The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model

Abstract

Background: Host-microbe associations underlie many key processes of host development, immunity, and life history. Yet, none of the current research on the central model species Caenorhabditis elegans considers the worm's natural microbiome. Instead, almost all laboratories exclusively use the canonical strain N2 and derived mutants, maintained through routine bleach sterilization in monoxenic cultures with an E. coli strain as food. Here, we characterize for the first time the native microbiome of C. elegans and assess its influence on nematode life history characteristics.

Results: Nematodes sampled directly from their native habitats carry a species-rich bacterial community, dominated by Proteobacteria such as Enterobacteriaceae and members of the genera Pseudomonas, Stenotrophomonas, Ochrobactrum, and Sphingomonas. The C. elegans microbiome is distinct from that of the worm's natural environment and the congeneric species C. remanei. Exposure to a derived experimental microbiome revealed that bacterial composition is influenced by host developmental stage and genotype. These experiments also showed that the microbes enhance host fitness under standard and also stressful conditions (e.g., high temperature and either low or high osmolarity). Taking advantage of the nematode's transparency, we further demonstrate that several Proteobacteria are able to enter the C. elegans gut and that an Ochrobactrum isolate even seems to be able to persist in the intestines under stressful conditions. Moreover, three Pseudomonas isolates produce an anti-fungal effect in vitro which we show can contribute to the worm's defense against fungal pathogens in vivo.

Conclusion: This first systematic analysis of the nematode's native microbiome reveals a species-rich bacterial community to be associated with C. elegans, which is likely of central importance for our understanding of the worm's biology. The information acquired and the microbial isolates now available for experimental work establishes C. elegans as a tractable model for the in-depth dissection of host-microbiome interactions.

Keywords: Antifungal defense; Caenorhabditis elegans; Holobiont; Metaorganism; Microbiome; Ochrobactrum; Pseudomonas; Sphingomonas; Stenotrophomonas.

Fig. 1

The native microbiome of the nematode C. elegans. a–c Frequency spectra of the bacterial classes based on MiSeq genotyping analysis for C. elegans (a), C. remanei (b), and C. briggsae (c), including results for natural worms (single worms), lab-enriched worms (nematode populations), and the corresponding substrates. d Differential interference contrast micrograph of C. elegans inhabited by its native microbiome. The anterior end of the worm is to the left. e–g Ordination by canonical correspondence analysis of bacterial operational taxonomic unit abundance in natural Caenorhabditis isolates and their substrates from German and French locations (indicated by colors and symbols; see legend), showing the three most significant axes. A three-dimensional visualization is given in Additional file 2. Statistics are provided in Additional file 1: Tables S1-6, S1-7, and S1-8. Detailed information about the samples is provided in Additional file 1: Tables S1-1 and S1-2. See also Additional file 3

Fig. 2

C. elegans cultured in the laboratory with a mix of 14 wild-caught bacteria retains a specific microbiome. a–b Average frequencies of bacterial taxa of the experimental microbiome for the lawns at different time points and two developmental stages (L4 and adult) of three C. elegans strains (N2, MY316, MY379; Additional file 1: Tables S1-12 and S1-15). b Shows the results for an independent experiment with N2 only. c Fluorescence micrograph of C. elegans inhabited by its experimental microbiome, visualized through fluorescence in situ hybridization of the bacteria with the general eubacterial probe EUB338 in red and DAPI staining of nematode cell nuclei in blue. The anterior end of the worm is to the left. See Additional file 6 for a three-dimensional illustration. d–f Canonical correspondence analysis of the experimental microbiome of C. elegans, showing the first three axes and including nematode stage (L4 or adult), sample type (nematode or lawn), and nematode strain (N2, MY316, or MY379) as factors (indicated by colors and symbols; Additional file 1: Tables S1-13, S1-14, and S1-16). A three-dimensional visualization is given in Additional file 8; see also Additional file 7 and Additional file 9. For all treatments, we considered at least six replicates. The only exception referred to the treatment combination worm-L4-MY379, for which only three replicates remained after quality control and which was thus excluded from further statistical analysis. Further details are given in Tables S1-10 and S1-11

Fig. 3

Interaction of individual bacterial isolates with C. elegans. a Persistence of bacterial isolates in the C. elegans gastrointestinal tract. We assessed the presence of selected bacteria at the beginning of the experiment (0 h, black color) and after 24 h on either a lawn of the same bacterium (dark red colour) or an empty plate (light red colour), using fluorescence in situ hybridization with eubacterial probe EUB338. Bacterial load was quantified in four categories: (0) absent, (1) single cells, countable, (2) clumps of cells, too many to count, and (3) region is filled. Results are shown for an E. coli control and seven bacterial isolates: Pseudomonas MY11b, Comamonas MY131b, Pseudomonas MY187b, Pseudomonas MY193b, Stenotrophomonas MY57b, Ochrobactrum MY71b, and Achromobacter MY9b. Barplots show the mean bacterial load of 10 worms with standard error of the mean of a total of three independent replicates (raw data in Additional file 1: Table S1-17). b, c Nematode population size on either the experimental microbiome community (b) or individual bacteria (c). Population size was measured as total offspring of three N2 hermaphrodites after 5 days. In (b), population size was determined under standard and different stress conditions, including normal nematode growth medium (NGM), peptone-free medium (PFM) at three temperatures (15 °C, 20 °C, 25 °C), and five salt concentrations (0–200 mM NaCl). Please note that the standard laboratory growth conditions for C. elegans in our lab consist of either PFM or NGM at 20 °C and 50 mM NaCl. In (c), all experiments were performed on PFM at 20 °C and 50 mM NaCl. The dashed lines indicate the median worm fitness on E. coli OP50 under the respective control conditions of the respective experiments. Asterisks denote significant differences from the E. coli control (*, α ≤ 0.05, false discovery rate-corrected for (c), Additional file 1: Tables S1-19 and S1-21, n ≥ 10; the raw data is provided in Additional file 1: Tables S1-18 and S1-20). Colors highlight the different bacterial groups

Fig. 4

Pseudomonas isolates from the C. elegans microbiome inhibit fungal growth. a In vitro effect of three Pseudomonas isolates (relative to E. coli OP50) on growth of six selected fungi, isolated from natural Caenorhabditis samples, after 3 days (n = 3, statistics in Table S1-23 and raw data in Additional file 1: Table S1-22). Each fungus is indicated by a different colour. b Effect of the Pseudomonas isolate MY11b on C. elegans mortality induced by the fungal pathogen Drechmeria coniospora (n = 6, statistics in Table S1-25 and raw data in Additional file 1: Table S1-24). Worms were either grown on Pseudomonas MY11b or E. coli (indicated on X axis) and then exposed to the fungal pathogen either in the presence of Pseudomonas MY11b or E. coli (given by the two panels)