a, ORO quantification. Mean ± s.d., n ≥ 17 worms per condition. b, ORO images. Scale bar: 100 μm. c, ORO quantification. Mean ± s.d., n ≥ 9 dissected intestines per condition. d-e, Nile Red staining and quantification. Mean ± s.d., n ≥ 7 dissected intestines (d) or n ≥ 11 worms (e) per condition. Scale bar: 100 μm. f, Autofluorescence and quantification. Mean ± s.d., n ≥ 17 worms per condition. Scale bar: 100 μm. g, Fertility quantification of live brood size (i), fertilized eggs (ii), and unfertilized oocytes (iii) laid per worm. Mean ± s.d., n ≥ 25 worms per condition. h, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. i, RT-qPCR. Mean ± s.e.m. from 2 independent experiments, each with 2-3 biological replicates. j, ORO quantification. Mean ± s.d., n ≥ 26 worms per condition. k, ORO quantification. Mean ± s.d., n ≥ 29 worms per condition. l, RT-qPCR. Mean ± s.e.m. of 3 biological replicates. m, D.I.C. (Nomarski) and GFP fluorescence images. n, ORO quantification. Mean ± s.d., n ≥ 26 worms per condition. a-f, k (rescue line #1), n, Representative of 2 experiments. P values: a, c-g, Two-tailed Mann-Whitney; h-i, Two-tailed Mann-Whitney with Benjamini-Hochberg correction; j-k, n, Kruskal-Wallis with Dunn's correction; l, Two-tailed t-test with Benjamini-Hochberg correction. * P < 0.05, ** P < 0.01.

a, GC-MS quantification of MUFAs. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. b-c, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. d, GC-MS quantification of MUFAs. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. e, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. P values: a-e, Two-tailed Mann-Whitney with Benjamini-Hochberg correction. * P < 0.05, ** P < 0.01.

a, RNA-seq tissue sample collection pipeline. b, Principal component analysis (PCA) with both intestine and germline samples (left), only intestine samples (middle), or only germline samples (right). c, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. d-g, ORO quantification. Mean ± s.d., n ≥ 15 worms per condition. h, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. i-j, ORO quantification. Mean ± s.d., n ≥ 21 (i) and n ≥ 27 (j) worms per condition. k, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. l, GC-MS. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. m, ORO quantification. Mean ± s.d., n ≥ 19 worms per condition. n, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. o, Lifespan extension by ash-2 RNAi is reduced in rsks-1(ok1255) mutants (13.12%) compared to wild-type worms (29.20%) (P < 0.0001, two-way ANOVA). p, let-363/mTOR RNAi and daf-15/Raptor RNAi extend lifespan in wild-type worms (P < 0.0001, log-rank), but not in set-2(ok952) mutants. d (except rrf-1 data), i-j, Representative of 2 experiments. P values: c, k, l, n, Two-tailed Mann-Whitney with Benjamini-Hochberg correction; h, Two-tailed Mann-Whitney; d-g, i-j, m, Kruskal-Wallis with Dunn's correction. * P < 0.05, ** P < 0.01.

a-d, Images and quantification of SBP-1 nuclear accumulation. Mean ± s.d. of 2 independent experiments, each with 4-6 nuclei per worm of ≥ 8 worms per condition. e, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. f, GC-MS quantification of MUFAs. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. g, ORO quantification. Mean ± s.d., n ≥ 21 worms per condition. h, ash-2 RNAi extends lifespan in wild-type worms (P < 0.0001, log-rank), but not in mdt-15 RNAi worms. i, ORO quantification, Mean ± s.d., n ≥ 24 worms per condition. j, ash-2 RNAi extends lifespan in both wild-type worms and nhr-49(nr2041) mutants (P < 0.0001, log-rank). k, ORO quantification. Mean ± s.d., n ≥ 27 worms per condition. l, ash-2 RNAi extends lifespan in both wild-type worms and nhr-80(tm1011) mutants (P < 0.0001, log-rank). i-k, Representative of 2 experiments. P values: a, Two-tailed Mann-Whitney; b-d, g, i, k, Kruskal-Wallis with Dunn's correction; e-f, Two-tailed Mann-Whitney with Benjamini-Hochberg correction. * P < 0.05.

a, ORO quantification. Mean ± s.d., n ≥ 29 worms per condition. b, ash-2 RNAi leads to lifespan extension in control (P < 0.0001, log-rank), but not in fat-6(tm331);fat-7(wa36) mutants. c, ash-2 RNAi extends lifespan in both wild-type worms and fat-7(wa36) single mutants (P < 0.0001, log-rank). d, ash-2 RNAi extends lifespan in fat-6(tm331) single mutants (P = 0.0162, log-rank), but lifespan extension by ash-2 RNAi is reduced in fat-6(tm331) mutants (9.26%) compared to wild-type worms (20.46%) (P = 0.0072, two-way ANOVA). e, ash-2 RNAi extends lifespan in fat-5(tm420);fat-7(wa36) double mutants (P < 0.0001, log-rank). f, ash-2 RNAi extends lifespan in fat-5(tm420);fat-6(tm331) worms (P = 0.0002, log-rank), but lifespan extension by ash-2 RNAi is reduced in fat-5(tm420);fat-6(tm331) mutants (14.03%) compared to wild-type worms (20.46%) (P = 0.0358, two-way ANOVA). g-h, GC-MS quantification of oleic acid. Mean ± s.e.m. of 3 biological replicates. i, ORO quantification. Mean ± s.d., n ≥ 13 worms per condition. Boxed conditions are identical to . j, Oleic acid supplementation extends lifespan (P < 0.0001, log-rank), which is not further extended by ash-2 RNAi. Oleic acid supplementation extends lifespan in fat-7;ash-2 RNAi worms (P < 0.0001, log-rank). Boxed conditions are identical to . k, ORO quantification. Mean ± s.d., n ≥ 21 worms per condition. l, GC-MS quantification of MUFAs. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. m, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. n, ORO quantification. Mean ± s.d., n ≥ 25 worms per condition. o, ash-2 RNAi extends lifespan in control (P < 0.0001, log-rank) but not fat-2 RNAi worms. c, i-j, n-o, Representative of 2 experiments. P values: a, g-i, k, n, Kruskal-Wallis with Dunn's correction; l-m, Two-tailed Mann-Whitney with Benjamini-Hochberg correction. * P < 0.05, ** P < 0.01.

a, GC-MS. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. b, Cis-vaccenic acid supplementation extends lifespan in wild-type worms (P < 0.0001, log-rank). Inset: ORO quantification. Mean ± s.d., n ≥ 20 worms per condition. c, GC-MS. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. d, Linoleic acid supplementation does not extend lifespan in wild-type worms. Inset: ORO quantification. Mean ± s.d., n ≥ 23 worms per condition. e, GC-MS. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. f, Alpha-linolenic acid supplementation does not increase the lifespan of wild-type worms. Inset: ORO quantification. Mean ± s.d., n ≥ 23 worms per condition. g, RT-qPCR. Mean ± s.e.m. of 3 biological replicates. h, ORO quantification. Mean ± s.d., n ≥ 54 worms per condition. i, Overexpression of FAT-7 extends lifespan (P < 0.0001, log-rank), which is not further extended by dietary oleic acid. Boxed regions are identical to . j, Proposed model by which ash-2 deficiency in the germline could lead to the fat metabolic switch in the intestine. b, d, f, Representative of 2 experiments. P values: a, c, e, Two-tailed Mann-Whitney with Benjamini-Hochberg correction; b inset, d inset, f inset, Two-tailed Mann-Whitney; g, Kruskal-Wallis test with Dunn's correction (non-significant, likely due to small sample size). One-way ANOVA with Bonferroni's correction. h, Kruskal-Wallis with Dunn's correction; * P < 0.05, ** P < 0.01, *** P < 0.001.

a, ORO staining images and quantification. Mean ± s.d., n ≥ 55 worms per condition. b, As in a, n ≥ 16 worms per condition. c, As in a, n ≥ 29 worms (upper panels) or n ≥ 25 intestinal cells (lower panels) per condition. d, Gas chromatography-mass spectrometry (GC-MS) quantification. Mean ± s.e.m of 2 independent experiments, each with 3 biological replicates. e, As in a, n ≥ 26 worms per condition. f, ash-2 RNAi extends lifespan in wild-type (P < 0.0001, log-rank) but not in rde-1(ne219); Is[Pnhx-2::rde-1] worms. g, As in a, n ≥ 18 worms per condition. h, ash-2 RNAi extends lifespan in both wild-type and rde-1(ne219); Si[Pmex-5::rde-1] worms (P < 0.0001, log-rank). b, c, e-h, Representative of 2 experiments. P values: a, b, e, g, Kruskal-Wallis with Dunn's correction; c, d, Two-tailed Mann-Whitney.

a, Fat metabolism pathways with genes used in RT-qPCR. b-c, GC-MS quantification of fatty acids. Mean ± s.e.m. of 2-3 independent experiments, each with 3 biological replicates. d, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. Solid red bars: delta-9 desaturases. e, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. f, Images and quantification of GFP fluorescence. Mean ± s.d., n ≥ 18 worms per condition. Representative of 2 (FAT-5) and 3 (FAT-7) experiments. Scale bar: 100 μm. g-h, GC-MS quantification of MUFAs. Mean ± s.e.m. of 3 (g) and 2 (h) independent experiments, each with 2-3 biological replicates. P values: Two-tailed Mann-Whitney (with Benjamini-Hochberg correction for > 2 conditions). * P < 0.05, ** P < 0.01, *** P < 0.001.

a, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. b, ORO quantification. Mean ± s.d., n ≥ 23 worms per condition. Representative of 2 experiments. c, GC-MS profiles of fatty acids. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. d, RT-qPCR. Mean ± s.e.m. of 2 independent experiments, each with 3 biological replicates. P values: b, Kruskal-Wallis with Dunn's correction; a, c, d, Two-tailed Mann-Whitney with Benjamini-Hochberg correction. * P < 0.05, ** P < 0.01.

a, Conserved transcriptional network regulating delta-9 desaturase genes. b, Images and quantification of SBP-1 nuclear accumulation. Mean ± s.d. of 2 independent experiments, each with n = 4-6 nuclei per worm of ≥ 8 worms per condition. c, RT-qPCR. Mean ± s.e.m. of 3 independent experiments, each with 3 biological replicates. d, GC-MS. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. e, ORO quantification. Mean ± s.d., n ≥ 32 worms per condition. f, Lifespan extension by ash-2 RNAi is reduced in sbp-1 RNAi (9.78%) compared to control worms (21.90%) (P = 0.0003, two-way ANOVA). g, As in e, n ≥ 13 worms per condition. h, Dietary oleic acid extends lifespan in the context of fat-7;ash-2 double RNAi (P < 0.0001, log-rank). g, h, Representative of 2 experiments. P values: b, Two-tailed Mann-Whitney; e, g, Kruskal-Wallis with Dunn's correction; c, d, Two-tailed Mann-Whitney with Benjamini-Hochberg correction. * P < 0.05, ** P < 0.01.

a, Chemical structure of fatty acids used in supplementation experiments. b, GC-MS. Mean ± s.e.m. of 3 independent experiments, each with 2-3 biological replicates. c, Oleic acid supplementation extends lifespan in wild-type worms (P < 0.0001, log-rank). Inset: ORO quantification. Mean ± s.d., n ≥ 27 worms per condition. d, GC-MS. Mean ± s.e.m. of 2 independent experiments, each with 2-3 biological replicates. e, Palmitoleic acid supplementation extends lifespan in wild-type worms (P < 0.0001, log-rank). Inset: as in c, n ≥ 45 worms per condition. f, Mean lifespan changes in independent supplementation experiments (P values: log-rank). g, FAT-7 overexpression extends lifespan (P < 0.0001, log-rank), which is not further extended by dietary oleic acid. P values: Two-tailed Mann-Whitney (Benjamini-Hochberg correction for > 1 comparison). * P < 0.05, ** P < 0.01, *** P < 0.001.