Effects of a natural defined microbiota on acute osmotic and oxidative stress in C. elegans

Animal resilience to acute environmental stress is shaped not only by host genetics and physiology but also by the natural microbiota, which can modulate how an animal survives. This emerging field still holds many open questions, from the host pathways at play to the ways microbiota influence survival and recovery to acute stress, underscoring ample room for discovery. In Caenorhabditis elegans, a defined 11-member Experimental Microbiota (E.M.), composed of 11 commensal bacteria isolated from natural worm microbiota. The E.M. was compared with the standard Escherichia coli OP50 laboratory diet to test how the experimental microbiota influences survival after acute stress. Wild-type (WT N2) worms and mutants in hlh-30/TFEB and the p38 MAPK cascade (pmk-1, nsy-1) were exposed to osmotic stress (1 M NaCl, 1 h), and acute oxidative stress (juglone, 250 μM, 6 h) was tested in WT N2 worms only. Survival was scored at 1 h and 24 h. Assay conditions were optimised to yield partial, reproducible lethality and refined from day 1 of adulthood to L4 larvae stage to avoid confounds such as internal hatching. In WT N2, the E.M. consistently increased survival after osmotic stress at both time points, demonstrating robust, diet-dependent protection. This benefit required HLH-30 to be present: hlh-30 mutants lost the E.M. advantage and instead survived better on OP50 than on E.M., identifying HLH-30 as essential for microbiota-derived protection. Strikingly, loss of p38 MAPK signalling (pmk-1, nsy-1) improved survival under osmotic stress and abolished diet dependence, indicating that p38 activity is not required to have E.M. benefits and that its disruption alone enhances resilience. WT N2 worms under acute oxidative stress, the E.M. conferred no advantage relative to OP50, revealing stressor specificity. Overall, the E.M. enhances acute osmotic-stress resilience via HLH-30, and the mutation of genes in the p38 MAPK pathway independently improves survival to osmotic stress, regardless of diet. With no observed E.M. protection under acute oxidative stress induced by juglone in WT N2 worms.