Extended Data Fig 6.

daf-2 is required for various forms of C. elegans associative learning^2,27,30–33. daf-16 is required for daf-2’s improvements and extensions of abilities with age². daf-2 mutants are defective for salt chemotaxis learning^27,30,31, and daf-16 is not involved in salt chemotaxis learning^27,30,31. Furthermore, salt learning utilizes a unique daf-2c isoform²⁷ in a daf-16-independent manner³⁰, suggesting a learning mechanism distinct from the associative memory paradigms studied here. We are specifically interested in understanding how activation of DAF-16 results in the improved and extended abilities of daf-2 mutants to carry out olfactory associative learning², short-term associative memory^2,33, and long-term associative memory², all of which require daf-16. A) Chemotaxis index profile of wild type (N2) and daf-2 animals at time points following memory training. B) RNAi knockdown of sod-3, a non-neuronal DAF-16-regulated target that influences lifespan, has no effect on the extended short-term associative memory (STAM) of daf-2 mutants when treated with RNAi-feeding bacteria throughout the whole life (B) or only the post-developmental (adult-only) period (C, D) of the animal. daf-2 worms treated with daf-16 RNAi have defective STAM, as previously reported². E) Knockdown of the neuronal IIS candidate genes zip-5 and best-23 does not affect STAM. Time-courses showing the chemotaxis index for each time point are shown in D and E. Learning indices are shown in B, C, F, and G. B–E) Two-way repeated measures ANOVA, Bonferroni post hoc tests. F) Treatment of daf-2 worms with neuronal DAF-16 target RNAi does not affect short-term associative learning. G) Neuronal-RNAi sensitive worms (Punc-119::sid-1) in a wild-type background were treated only during adulthood with RNAi targeted against the neuronal DAF-16 target genes. (0h) Learning and 1 h short-term associative memory time points are shown. A–G) Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.