a, Loss of function of SPR-3 and SPR-4 reduces the lifespan extension induced by daf-2 RNAi. Left, representative lifespan analysis of spr-4(by105);spr-3(ok2525) double mutant and wild-type worms following daf-2 or empty vector control RNAi. WT + EV, n = 54 worms; spr-4;3 + EV, n = 58 worms; WT + daf-2, n = 26 worms; spr-4;3 + daf-2, n = 54 worms. Right, values represent mean ± s.e.m. per cent lifespan extension (daf-2 RNAi versus EV control) in the indicated genotypes. WT, n = 6 independent experiments; spr-4(by105), n = 3, *P = 0.017 versus WT; spr-4(tm465), n = 4, **P = 0.0062 versus WT; spr-3(ok2525), n = 4, **P = 0.0018 versus WT; spr-4(by105);spr-3(ok2525), n = 4, **P = 0.0016 versus WT; Students t-test. See Supplementary Table 22 for individual lifespan data and statistics. b, Lifespan is unaffected by spr-4 and spr-3 mutations in a wild-type background. WT, n = 50 worms; spr-3(ok2525), n = 31; spr-4(by105);spr-3(ok2525), n = 32; spr-4(by105), n = 34; spr-4(tm465), n = 33.There were no reproducibly significant changes by the log-rank test in 3–6 independent experiments per genotype (see Supplementary Table 22). c, Quantification of lifespan extension in daf-2 mutant worms shown in Fig. 3b attributable to neuronal expression of spr-3 and spr-4. RNAi was targeted to neurons by neuronal expression of a sid-1 transgene in otherwise sid-1-null daf-2(1370) mutants (daf-2;p[neuron]:sid-1), and compared with untargeted RNAi in sid-1 wild-type daf-2(1370) mutants (daf-2). Values represent mean ± s.e.m. lifespan extension relative to the control sid-1(pk3321);p[neuron]:sid-1 worms treated with empty vector (n = 3 independent experiments). Significant lifespan effects were not observed for RNAi in the absence of the daf-2 mutation. *P < 0.05; **P < 0.01 by Student’s t-test. d, Gene expression determined by RNA-seq in day 2 adult worms. Differentially expressed genes (rows) and the indicated worm genotypes (columns) were clustered, and gene expression, transformed to a z-score per gene, is represented in a heat map. n = 3 independent replicates per genotype. e, Venn diagram illustrating the overlap in differentially expressed genes in daf-2 single mutant versus WT and spr-4;spr-3;daf-2 triple mutant versus daf-2 single mutant worms. P = 7 × 10–30, Fisher’s exact test with a one-sided alternative hypothesis. f, Long-lived daf-2 mutants do not show an age-related increase in neural excitation. Shown are maximum ASH GCaMP intensity changes in day 1–2 (n = 39) and day 14–16 (n = 20) daf-2(e1370) mutant worms. Note the absence of the age-related increase in excitation observed in wild-type ageing worms (Fig. 1e). P = 0.93, Mann–Whitney U-test. g, The spr-4;spr-3 double mutation in a wild-type background does not significantly affect neural excitation in ASH neurons. WT, n = 15 worms; spr-4;spr-3, n = 15 worms. P = 0.62, Mann–Whitney U-test. h, DAF-16 does not mediate suppression of neural excitation in the daf-2 mutant. RNAi against daf-16 was performed in daf-2(e1370) mutant worms on a sid-1(pk3321);p[neuron]:sid-1 background to augment RNAi in neurons (daf-16 RNAi, n = 20 worms, EV control, n = 12 worms). P = 0.33, Mann–Whitney U-test. i, Descriptions of the genes targeted by RNAi in Fig. 4d. Source Data