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Insulin-like Peptides and the mTOR-TFEB Pathway Protect Caenorhabditis elegans Hermaphrodites From Mating-Induced Death


Insulin-like Peptides and the mTOR-TFEB Pathway Protect Caenorhabditis elegans Hermaphrodites From Mating-Induced Death

Cheng Shi et al. Elife.


Lifespan is shortened by mating, but these deleterious effects must be delayed long enough for successful reproduction. Susceptibility to brief mating-induced death is caused by the loss of protection upon self-sperm depletion. Self-sperm maintains the expression of a DAF-2 insulin-like antagonist, INS-37, which promotes the nuclear localization of intestinal HLH-30/TFEB, a key pro-longevity regulator. Mating induces the agonist INS-8, promoting HLH-30 nuclear exit and subsequent death. In opposition to the protective role of HLH-30 and DAF-16/FOXO, TOR/LET-363 and the IIS-regulated Zn-finger transcription factor PQM-1 promote seminal-fluid-induced killing. Self-sperm maintenance of nuclear HLH-30/TFEB allows hermaphrodites to resist mating-induced death until self-sperm are exhausted, increasing the chances that mothers will survive through reproduction. Mothers combat males' hijacking of their IIS pathway by expressing an insulin antagonist that keeps her healthy through the activity of pro-longevity factors, as long as she has her own sperm to utilize.

Keywords: C. elegans; TFEB; genetics; genomics; insulin signaling; lifespan; mTOR; mating.

Conflict of interest statement

CS, LB, CM No competing interests declared


Figure 1.
Figure 1.. Brief mating-induced death is dependent on self-sperm and acts through male seminal fluid.
(A) Schematic and representative survival curves illustrating survival after a brief (2 hr) mating; C. elegans hermaphrodite lifespan is dependent on the presence of self-sperm. (B) Wild-type N2 hermaphrodites have reduced lifespan after 2 hr mating on day 7 of adulthood, but have normal lifespan when such brief mating occurs on day 3. Day 3 N2 unmated: 14.1 ± 0.7 days; mated: 13.9 ± 0.6 days, p=0.8481; Day 7 N2 unmated: 13.8 ± 0.5 days; mated: 10.0 ± 0.4 days, p<0.0001. In this study, we define day 0 as the beginning of the adulthood for all lifespans. Kaplan-Meier analysis with log-rank (Mantel-Cox) method was performed to compare the lifespans of different groups in this study. See Supplementary file 1 for all lifespan data summary. (C) Self-spermless fog-2(q71) are susceptible to 2 hr mating on both day 3 and day 7 of adulthood. Day three fog-2 unmated: 19.7 ± 0.7 days; mated: 15.4 ± 0.7 days, p<0.0001; Day seven fog-2 unmated: 21.0 ± 0.6 days; mated: 14.8 ± 0.5 days, p<0.0001. (D–E) Mating with fog-2 males (which are functionally wild type) or with fer-6 males (which lack sperm) leads to a similar magnitude of lifespan decrease in fog-2 self-spermless hermaphrodites. Day 3 fog-2 unmated: 18.2 ± 0.8 days; mated with fog-2 males: 12.6 ± 0.7 days, p<0.0001; mated with fer-6 males: 14.1 ± 0.7 days, p=0.0002 (compared to unmated). Day 7 fog-2 unmated: 20.3 ± 1.0 days; mated with fog-2 males: 14.6 ± 0.9 days, p<0.0001; mated with fer-6 males: 15.6 ± 1.0 days, p=0.0016 (compared to unmated). (F–G) N2 hermaphrodites are not short-lived after 2 hr exposure to male pheromone-conditioned plates (conditioned by 30 wild-type males for 2 days) either on day 3 or day 7 of adulthood. Only old day 7 N2 hermaphrodites have a shorter lifespan after 2 hr mating with daf-22 male pheromone production defective males (F). Day 3 N2 unmated: 11.9 ± 0.6 days; mated with daf-22 males: 11.1 ± 0.4 days, p=0.3058; unmated on male-conditioned plates (MCP; pre-conditioned with 30 males, see Materials and methods for details): 11.7 ± 0.5 days, p=0.9696 (compared to unmated). Day 7 N2 unmated: 14.3 ± 0.7 days; mated with daf-22 males: 11.4 ± 0.4 days, p=0.0001; unmated on male-conditioned plates (MCP): 13.7±0.7 days, p=0.5984 (compared to unmated). (H–I) Like N2 hermaphrodites, the lifespans of fog-2 hermaphrodites are not affected by 2 hr male pheromone exposure. However, both young (G) and old (H) fog-2 hermaphrodites live significantly shorter after brief mating with daf-22 males. Day 3 fog-2 unmated: 15.3 ± 0.8 days; mated with daf-22 males: 10.4 ± 0.6 days, p<0.0001; unmated on male-conditioned plates (MCP): 16.5 ± 0.8 days, p=0.3459 (compared to unmated). Day 7 fog-2 unmated: 15.5±0.6 days; mated with daf-22 males: 13.1 ± 0.4 days, p=0.0017; unmated on male-conditioned plates (MCP): 14.7±0.6 days, p=0.4493 (compared to unmated).
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. The presence of self-sperm protects the hermaphrodites from brief mating-induced death.
(A) Both self-spermless fog-2(q71) and fem-1(hc17) hermaphrodites are susceptible to 2 hr mating on day 3 of adulthood. fog-2 unmated: 16.3 ± 0.7 days; mated: 13.4 ± 0.5 days, p=0.0010. fem-1 unmated: 14.2 ± 0.4 days; mated: 11.7 ± 0.3 days, p<0.0001. (B) Young N2 hermaphrodites’ resistance to brief mating-induced death is not due to low mating efficiency. Total male progeny from 2 hr mated N2 were counted (we only counted male progeny because we did not want to include progeny from self-fertilization. 2x male progeny from mated N2 hermaphrodites is a good estimate of the total number of cross-progeny) as well as total progeny (fog-2 does not have self sperm, so all the progeny produced after mating are cross-progeny) from 2 hr mated fog-2 hermaphrodites (All hermaphrodites were mated on Day 3 of adulthood). Mated N2 hermaphrodites produce at least two-fold more male progeny than total progeny produced by mated fog-2. Therefore, potentially more male sperm and seminal fluid were transferred to N2 hermaphrodites. Still, young N2 are resistant to brief mating-induced death, suggesting that young N2 hermaphrodites’ resistance to brief mating-induced death is not due to lower mating efficiency or the transfer of less male sperm or seminal fluid.
Figure 2.
Figure 2.. IIS/FOXO in brief mating-induced death.
(A) daf-16(mu86) mutants lived shorter after 2hr mating on Day 3 of adulthood. Unmated: 12.2±0.5 days; mated: 9.5±0.5 days, p=0.0003. (B) daf-2(e1370) mutants are resistant to 2 hr mating in the absence of self-sperm on Day 8 of adulthood. Unmated: 33.2±2.6 days; mated: 32.7±2.9 days, p=0.7519. (C–D) Nuclear DAF-16::GFP in self-spermless fog-2 hermaphrodites becomes diffuse after mating. (C) Representative images of DAF-16::GFP in unmated and mated fog-2 hermaphrodites (Day 3). (D) Quantitation of DAF-16::GFP, p=0.0006. Each worm was assigned a category based on DAF-16::GFP localization. Chi-square test was used to determine the significance.
Figure 3.
Figure 3.. INS-37 is required for self-sperm protection from brief mating-induced death.
(A) Heatmap of all the insulin genes from microarray-based transcriptome comparison between N2 and fog-2 L4 hermaphrodites. (Four biological replicates) (B) qRT-PCR reveals over 25 fold up-regulation of ins-37 in N2 L4 hermaphrodites compared to fog-2 L4 hermaphrodites, p=0.0043. Reference gene pmp-3 was used for normalization. RNA was extracted from three additional biological replicates (different from the sample used for microarray in Figure 3A). (C) Knocking down ins-37 in N2 hermaphrodites makes them susceptible to 2 hr mating on day 3 of adulthood. (see Figure 3—figure supplement 2B for results of N2 on control RNAi vector) N2 ins-37 RNAi unmated: 13.6 ± 0.4 days; N2 ins-37 RNAi mated: 12.0 ± 0.4 days, p=0.0040.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Transcriptomic comparison between self-spermless, wild type, and excess self-sperm mutants reveals HLH-30 binding motif.
L4 hermaphrodites of each genotype were collected for RNA extraction and microarray-based transcriptome analysis. One-class SAM was used to determine genes with significant changes in expression between each comparison. The gene lists from SAM (FDR = 1%) were then imported to g:Profiler for GO terms and TF binding motifs analysis.
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. Ins-7 is highly up-regulated in L4 hermaphrodites with self-sperm.
(A) qRT-PCR of ins-37. Ins-37 has an over 25-fold increase in N2 L4 hermaphrodites compared to fog-2 L4 hermaphrodites (same figure shown in Figure 3B). Mating has very little impact on ins-37 expression compared to the presence of self-sperm. Expression level of ins-37 increased about twofold in Day 2 mated fog-2 compared to unmated fog-2 (p=0.0137). (B) Control of Figure 3C,N2 hermaphrodites do not live shorter after 2 hr mating on day 3 of adulthood. N2 vector RNAi unmated: 12.1 ± 0.5 days; N2 vector RNAi mated: 11.6 ± 0.5 days, p=0.6335. .
Figure 4.
Figure 4.. HLH-30 protects against brief mating-induced death.
(A) hlh-30(tm1978) mutants live shorter after brief mating on day 3, unlike wild type (N2) hermaphrodites, which are resistant to 2 hr mating on day 3 of adulthood (Figure 4—figure supplement 1L). hlh-30 unmated: 11.7 ± 0.3 days; mated: 9.8 ± 0.3 days, p=0.0002. (B) Nuclearly localized HLH-30::GFP becomes diffuse with age. Representative images of HLH-30::GFP (BC11288) in unmated hermaphrodites at various ages. Worms were imaged under free moving conditions; no anesthetic was used, to prevent possible artificial perturbation of HLH-30 localization. (C–D) Quantitation of HLH-30::GFP in B). In C), each worm was assigned a category based on HLH-30::GFP localization. Chi-square test was used to determine the significance. In D), number of nuclei with nuclearly localized HLH-30::GFP was counted for each worm. The average of each group was compared to L4 using the unpaired t-test. (E–F) The amount of self-sperm is positively correlated with the number of worms having strongly nuclearly localized HLH-30::GFP. (Three biological replicates per condition; average was compared by t-test). In F), fem-3 worms were grown at 25C to induce excess self-sperm phenotype, control fem-3 were grown at 20 C. (G–I) Knockdown of ins-37 reduces nuclearly localized HLH-30::GFP in unmated hermaphrodites. (G) Representative images of day 3 HLH-30::GFP hermaphrodites on control RNAi (left) and ins-37(RNAi) (right). (H–I) Quantitation of HLH-30::GFP in G), same method used in C-D. (J–K) HLH-30::GFP strain is more resistant to longer mating, compared to wild-type (N2) hermaphrodites. Wild-type are already susceptible to 12 hr mating (J), whereas HLH-30::GFP worms are resistant (K). 24 hr mating reduces lifespan of both strains. N2 unmated: 13.2 ± 0.5 days; 12 hr mated: 11.0 ± 0.3 days, p=0.0005; 24 hr mated: 9.0 ± 0.4 days, p<0.0001 (compared to unmated). HLH-30::GFP unmated: 11.9 ± 0.4 days; 12 hr mated: 11.6 ± 0.4 days, p=0.4471; 24 hr mated: 9.7 ± 0.4 days, p=0.0001 (compared to unmated). (L) Summary of nuclear HLH-30::GFP quantitation of worms with age and after mating for various time on day 3. (See Figure 4—figure supplement 1A–K for more detailed information).
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Quantitation of HLH-30::GFP in worms at different ages and after mating for various lengths of time.
(A–K) HLH-30::GFP becomes more diffuse with age, even if the hermaphrodites have not been mated; however, mating accelerates this process. All worms were mated on Day 3 of adulthood. (B, C; E–K) 2 hr and 8 hr mating seem to have no impact on HLH-30::GFP localization (same as unmated control). However, starting from 16 hr mating, HLH-30::GFP become less nuclearly localized. (L) Control of Figure 4A,N2 hermaphrodites do not live shorter after 2 hr mating on day 3 of adulthood. N2 unmated: 13.4 ± 0.5 days; N2 mated: 12.7 ± 0.4 days, p=0.2263.
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Mating also decreases nuclear HLH-30::GFP in fog-2 self-spermless hermaphrodites.
Left: representative images of HLH-30::GFP in unmated and mated fog-2 hermaphrodites (Day 4). Quantitation of HLH-30::GFP in fog-2. (p=0.0328) Each worm was assigned a category based on HLH-30::GFP localization. Chi-square test was used to determine the significance.
Figure 5.
Figure 5.. TOR signaling components promote Seminal Fluid-induced killing/brief mating-induced death.
(A) Most positive regulators of TOR signaling (e.g., Rictor/rict-1) were modestly downregulated in conditions in which worms have self-sperm and are protected from brief mating-induced death (left block, blue), but rise in mated or short-lived conditions (right block). (B) Independent RNA-seq confirmed the modest downregulation of TOR signaling components in worms that are protected from brief mating-induced death. Note the first two rows are conditions in which the hermaphrodites have self-sperm protection against brief mating. (C) Knockdown of tor/let-363 in self-spermless fog-2 protects the hermaphrodites from SF killing. fog-2;TOR/let-363(RNAi) unmated: 13.3 ± 0.3 days; mated: 13.2 ± 0.4 days, p=0.7368. (D) Control of Figure 5C: fog-2 on vector control RNAi unmated: 13.7 ± 0.6 days; mated: 10.1 ± 0.4 days, p<0.0001. (E) fog-2;raga-1(RNAi) unmated: 15.3 ± 0.5 days; mated: 14.3 ± 0.6 days, p=0.2378. (F) Day 7 self-sperm depleted raga-1(ok701) are also resistant to brief 2 hr mating. Unmated: 18.5 ± 0.7 days; mated: 18.1 ± 0.7 days, p=0.5833. (G) TOR/let-363 knockdown in hlh-30(tm1978) mutants fails to protect the hermaphrodites from brief mating-induced death. hlh-30;TOR/let-363(RNAi) unmated: 13.8 ± 0.3 days; mated: 10.5 ± 0.4 days, p<0.0001. (H) Quantitation of nuclear HLH-30::GFP in mated hermaphrodites treated with let-363 RNAi.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. TOR signaling mediates self-sperm protection from brief mating.
(A–B) Knockdown of let-363 has no impact on lifespan of N2 upon brief mating. (A) N2 on vector control RNAi unmated: 14.9 ± 0.3 days; mated: 14.9 ± 0.4 days, p=0.7888. (B) N2 on TOR/let-363 RNAi unmated: 12.3 ± 0.5 days; mated: 12.1 ± 0.6 days, p=0.8791. (C–F) Individual TOR components knockdown prevents SF killing in spermless females. (D) fog-2;rict-1(RNAi) unmated: 15.2 ± 0.6 days; mated: 14.9 ± 0.6 days, p=0.8375. (E) fog-2;daf-15(RNAi) unmated: 15.9 ± 0.6 days; mated: 15.2 ± 0.6 days, p=0.6903. (C) fog-2; control (RNAi) unmated: 17.4 ± 0.8 days; mated: 11.7 ± 0.6 days, p<0.0001. (F) Knockdown of let-363 in self-spermless fog-2 protects the hermaphrodites from SF killing. fog-2 on TOR/let-363 RNAi unmated: 16.0 ± 0.5 days; mated with fer-6 males: 16.0 ± 0.6 days, p=0.8488. (G–H) Lifespan control for Figure 5G. (G) fog-2 on TOR/let-363 RNAi unmated: 13.0 ± 0.4 days; mated: 12.9 ± 0.4 days, p=0.8275. (H) hlh-30 on vector control RNAi unmated: 10.4 ± 0.2 days; mated: 9.2 ± 0.2 days, p<0.0001.
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. Insulin and mTOR signaling pathways influence HLH-30 and DAF-16 localization in mated hermaphrodites.
(A–B) Knocking down pqm-1, insulins, and Tor/let-363 does not affect HLH-30::GFP localization in unmated worms; (C) By contrast, knocking down pqm-1, ins-7, ins-8, or let-363 largely prevent HLH-30::GFP from localizing to the cytoplasm. (D) Mating-induced DAF-16::GFP localization movement was inhibited in fog-2 after knocking down let-363. (in contrast to Figure 2B–C) .
Figure 6.
Figure 6.. PQM-1 is required for SF and brief mating-induced killing.
(A–B) pqm-1(ok485) mutant hermaphrodites are resistant to short term (2 hr) mating-induced death (A), as well as long term (24 hr) mating (B) 2 hr pqm-1 unmated: 10.5 ± 0.3 days; mated: 11.1 ± 0.4 days, p=0.2070. 24 hr pqm-1 unmated: 12.5 ± 0.5 days; mated: 12.5 ± 0.5 days, p=0.8407. (See Figure 6—figure supplement 1A–B for more controls) (C) Representative images of PQM-1::GFP hermaphrodites. Left: unmated pqm-1 control, Right: mated pqm-1 (mated on day 1 of adulthood for 24 hr). Images are taken on day 6 of adulthood. White arrows indicate nuclearly localized PQM-1::GFP. (D–E) Quantitation of PQM-1::GFP localization. (D) Non-parametric comparison; E) nuclear and cytoplasmic GFP intensity was measured in the most anterior nuclei (easily recognizable) of the intestine for worms in each group. Averages were compared using a t-test. (F) Knocking down pqm-1 restores nuclearly localized HLH-30 in mated hermaphrodites. Quantitation of nuclear HLH-30::GFP in mated hermaphrodites treated with pqm-1 RNAi (same as Figure 5H). (G) Knocking down pqm-1 blocks the lifespan decrease normally induced by 2 hr mating in hlh-30(tm1978) mutants. pqm-1 on vector control RNAi: unmated:13.2 ± 0.4 days, mated: 13.1 ± 0.5 days, p=0.4169. pqm-1;hlh-30(RNAi): unmated:13.2 ± 0.3 days, mated: 13.1 ± 0.3 days, p=0.9161.
Figure 6—figure supplement 1.
Figure 6—figure supplement 1.. PQM-1 is required for SF killing.
(A–B) pqm-1 mutants are resistant to both long-term (24 hr; B) and brief (2 hr) mating (C). (C) Knocking down hlh-30 in pqm-1 mutants does not make them susceptible to brief mating-induced death. hlh-30 on vector control RNAi: unmated:13.1 ± 0.4 days, mated: 11.6 ± 0.3 days, p=0.0015. hlh-30 on pqm-1 RNAi:unmated:11.7 ± 0.2 days, mated: 12.0 ± 0.2 days, p=0.3601.
Figure 7.
Figure 7.. Insulins mediate seminal fluid killing.
(A) Heatmap of insulin gene expression comparisons between fog-2 mated worms (short-lived after mating) and pqm-1 mated worms (no lifespan decrease after mating). (B) Both mean and maximum Pins-8::gfp expression increased in mated worms (right), correlating with gene expression data (A). (C, D) Knockdown of ins-8 (C) or ins-7 (D) protects hermaphrodites from seminal fluid killing. (C) fog-2;ins-8(RNAi) unmated: 18.4 ± 0.7 days; mated with fog-2 males: 17.2 ± 0.9 days, p=0.2879; mated with fer-6 males: 17.3 ± 0.7 days, p=0.2582 (compared to unmated). (D) fog-2;ins-7(RNAi) unmated: 18.2 ± 0.6 days; mated with fog-2 males: 17.7 ± 1.1 days, p=0.9380; mated with fer-6 males: 17.2 ± 0.7 days, p=0.3994 (compared to unmated). (E) Controls for Figure 7C–D. fog-2 on vector control RNAi unmated: 18.4 ± 0.9 days; mated with fog-2 males: 13.3 ± 0.7 days, p<0.0001; mated with fer-6 males: 15.4 ± 0.6 days, p=0.0032 (compared to unmated). (F) Knocking down ins-7 and ins-8 also restores nuclearly localized HLH-30 in mated hermaphrodites.
Figure 8.
Figure 8.. Model of self-sperm mediated protection from seminal fluid killing Prior to mating, the presence of self-sperm maintains high ins-37 expression.
INS-37 antagonizes DAF-2 activity and promotes DAF-16 and HLH-30 maintenance in the nucleus. Self-sperm also inhibits TOR signaling, which regulates HLH-30 nuclear localization. Seminal fluid transfer upon mating increases expression of ins-8 and ins-7; these agonists activate DAF-2, promoting PQM-1 nuclear localization and DAF-16 and HLH-30 nuclear exit, resulting in premature death. INS-7 acts in a feed-forward loop (Murphy et al., 2007), further accelerating DAF-2 activation.

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