Larval crowding accelerates C. elegans development and reduces lifespan

Abstract

Environmental conditions experienced during animal development are thought to have sustained impact on maturation and adult lifespan. Here we show that in the model organism C. elegans developmental rate and adult lifespan depend on larval population density, and that this effect is mediated by excreted small molecules. By using the time point of first egg laying as a marker for full maturity, we found that wildtype hermaphrodites raised under high density conditions developed significantly faster than animals raised in isolation. Population density-dependent acceleration of development (Pdda) was dramatically enhanced in fatty acid β-oxidation mutants that are defective in the biosynthesis of ascarosides, small-molecule signals that induce developmental diapause. In contrast, Pdda is abolished by synthetic ascarosides and steroidal ligands of the nuclear hormone receptor DAF-12. We show that neither ascarosides nor any known steroid hormones are required for Pdda and that another chemical signal mediates this phenotype, in part via the nuclear hormone receptor NHR-8. Our results demonstrate that C. elegans development is regulated by a push-pull mechanism, based on two antagonistic chemical signals: chemosensation of ascarosides slows down development, whereas population-density dependent accumulation of a different chemical signal accelerates development. We further show that the effects of high larval population density persist through adulthood, as C. elegans larvae raised at high densities exhibit significantly reduced adult lifespan and respond differently to exogenous chemical signals compared to larvae raised at low densities, independent of density during adulthood. Our results demonstrate how inter-organismal signaling during development regulates reproductive maturation and longevity.

Fig 1. Population density dependent acceleration of development (Pdda) in C. elegans hermaphrodites.

A) Time point of first egg laying on plates with different population densities, set up according to protocol A (see Methods). First egg laying was scored from 60 h after plating. B) Schematic of experimental protocol B used for measuring the effect of population density on the time point of first egg laying. C) Time point of first egg laying of worms isolated throughout development (ISO) or kept at high density for 48 h or 59 h (HD) before transfer onto new plates at one worm per plate. D) Developmental stage of isolated worms and groups of 100 worms at 52 h. E) Pdda is unaffected by mutation of eat-2 and egl-4, (see Methods for calculating percent wildtype Pdda). Error bars, STD; ***P < 0.0001; **P < 0.001; *P < 0.05.

Fig 2. Pdda is counteracted by ascaroside signaling.

A) Pdda is increased in peroxisomal β-oxidation mutants compared to wildtype. B) DIC images of developing daf-22(ok693) mutant worms at 56 h, grown under HD (left panels) and ISO (right panels) conditions, showing complete animals (upper panels) and the gonadal tract (lower panels); lower left panel: gonadal arms and vulva (L4.6 stage) in an HD animal (enlargement: magnified vulva); lower right panel: L3 stage gonad, left arm (red highlight) in an ISO animal; C) Chemical structures of major ascarosides produced by wildtype hermaphrodites. D, E) Ascarosides reduce Pdda in wildtype (D) and daf-22 mutant (E) worms. Synthetic ascarosides were added at 400 nM. F) Pdda in chemosensory and mechanosensory mutants; G) Time point of first egg laying of daf-22(ok693) mutants, comparing HD worms, ISO worms, and ISO worms on HD pre-incubated plates. Box plot showing medians, upper and lower quartiles, whiskers at 1.5 IQR. Error bars, STD (panels A, D, E, F); ***P < 0.0001; **P < 0.001; *P < 0.05.

Fig 3. Pdda interacts with steroid hormone and nuclear hormone receptor signaling.

A) Chemical structures of endogenous DAs. B) Pdda in insulin signaling mutants. C) Pdda in nuclear hormone receptor signaling mutants. D) Effect of dafa#3 (at 100 nM) on Pdda in wildtype and daf-9; daf-12 double mutants. E) Effect of dafa#3 (at 100 nM) on Pdda nhr-8 mutant worms compared to wildtype. F) Effect of dafa#3 (at 100 nM) on Pdda in nhr-8;daf-12 double mutant worms compared to wildtype. G) DIC images of developing nhr-8;daf-12 double mutant worms at 65 h grown under HD (left panels) and ISO (right panels) conditions, showing complete animals (upper panels) and the gonadal tract (lower panels); lower left panel: right gonadal arm and proliferating oocytes (young adult stage) in an HD animal, right lower panel: L4.6 stage gonad, right gonad arm and vulva in an animal raised under ISO conditions (enlargement: magnified vulva). H) Time point of first egg laying of nhr-8;daf-12 double mutants, comparing HD worms, ISO worms, and ISO worms on plates pre-incubated with daf-22(ok693) worms. Box plot showing medians, upper and lower quartiles, whiskers at 1.5 IQR. Error bars, STD (panels B-E); ***P < 0.0001; **P < 0.001; *P < 0.05. The Bonferroni correction was used to assess significance for multiple comparisons in panels D-F.

Fig 4. Population density affects lifespan of C. elegans hermaphrodites and a model for Pdda.

A) Mean lifespan of hermaphrodites at different population densities, set up as eggs on mock treated plates (black bars) or set up as eggs on plates containing 100 nM dafa#3 (grey bars), or raised on plates of >100 animals and moved onto mock-treated assay plates at indicated densities at 62 h (red bars). B) Aging curves for hermaphrodites kept at 1 worm/plate, set up by isolating worms from plates with >100 hermaphrodites at 62 h (protocol I, red) or as eggs (protocol II, black [control] and grey dashed [with 100 nM dafa#3]). Error bars, SEM; ***P < 0.0001; **P < 0.001; *P < 0.05. C) Model for the roles of small molecule signaling in Pdda. Population density controls development via two competing small-molecule cascades. High population density results in accumulation of ascarosides that downregulate DA biosynthesis and thereby retard developmental progression. This effect is antagonized by a second chemical signal, here named compound X, that accelerates development and represents the proximal cause of Pdda.