Smelling diacetyl, a microbial‐fermentation byproduct, is a key food cue for Caenorhabditis elegans. Chemotaxis assays serve as a traditional model for olfactory behavior. However, the N2 reference strain may not reflect species-wide sensitivity. We first sought to establish straightforward, replicable conditions to enhance the reproducibility of the volatile chemotaxis response, so that we could use the assay to compare diacetyl responses across wild strains. After systematically optimizing hermaphrodite age, lay duration, brood size, and incubation temperature, we found that four gravid day-1 adults and a 16-hour lay at 20 °C produced abundant, synchronized progeny and highly reproducible chemotaxis responses. We established a robust dose–response curve for diacetyl and identified 3 × 10⁻⁴ as the dilution that elicited an N2 chemotaxis index (CI) of ~0.6 so that both diminished and elevated CIs relative to N2 could be observed. Applying this protocol to 48 wild strains (the 12 divergent + 36 mapping strains) together with N2 showed that across the 49-strain panel, CIs ranged from approximately 0.20–0.85. Several strains exceeded N2 while others underperformed, placing N2 in the upper-middle rather than at the maximal end of the species’ olfactory spectrum. In sum, our streamlined conditions enable sensitive, comparable measurements across strains and set the stage for genome-wide association (GWA) studies to explore how natural genetic variation between wild strains contributes to differences in olfactory behavior.

¹Department of Biological Sciences, University at Buffalo, The State University of New York, Buffalo, NY, USA

Funding Support: This work was supported by the National Science Foundation (IOS-2334104 to DMF 3 and DEB-2049947 to OG), the National Institutes of Health (R35GM156519 to OG), and 4 a Human Frontiers Science Foundation grant (RGP0001/2019) to ECA.