Fig. 2

S. stercoralis free-living adults are associated with a specific microbiome. a Schematic of the experimental design. Gerbils were infected with S. stercoralis iL3s, and infested feces were harvested from the gerbils on days 14–27 post-infection. Collected feces were made into fecal-charcoal plates and incubated at either 20 °C for 2 days to obtain S. stercoralis free-living adults, or 23 °C for 7 days to obtain S. stercoralis iL3s. Nematodes were then isolated from the fecal-charcoal plates using a Baermann apparatus and washed in buffer before DNA extraction. Sequencing samples included DNA isolated from nematodes; wash buffer control 1, consisting of the buffer used to wash the nematodes prior to DNA extraction; and wash buffer control 2, consisting of the buffer supernatant after washing the nematodes. b–e Results from the amplicon sequencing analysis for Experiments 1 and 2. b, c Principal coordinates analysis (PCoA) of the different sample categories from Experiments 1 (b) and 2 (c). Free-living adult samples clustered separately from the other samples, suggesting a specific microbiome associated with S. stercoralis free-living adults. In contrast, some of the iL3 samples in Experiment 1 clustered with the control samples. In (b), an empty well control was also included. Ellipses representing the 95% confidence region for the sample groups with more than 3 samples were calculated using the ggplot2 and ellipse packages [73]. d, e Relative abundance of ASVs that showed a significant difference across sample categories in Experiments 1 (d) or 2 (e). Box plots are standard Tukey representations. ASVs with an ANCOM W score in the top 40% of all tested features were selected as significant; individual W scores are displayed below the ASV identifiers. ASV identifiers in bold are those that showed a significant enrichment in S. stercoralis free-living adults vs. controls in both sequencing experiments. ASVs in the genera Escherichia-Shigella, Lactobacillus, and Solibacillus were significantly enriched in the free-living adult samples relative to the other samples