Distinct microbial environments exert diverse effects on the physiology and survival of the nematode Caenorhabditis elegans. Here, we show that C. elegans grown on two Escherichia coli strains exhibit different survival dynamics. Wild-type C. elegans on the B type OP50 exhibit more early deaths compared to C. elegans on K-12 type CS180. These early deaths on OP50 are characterized by swollen pharynges (P-deaths) due to bacterial accumulation within the tissue. In contrast, animals on CS180 are more resistant to P-deaths. These bacteria-dependent differences in P-deaths depend on bacterial lipopolysaccharide structures and the activities of the C. elegans neuropeptide neuromedin U receptor NMUR-1, which reduces P-deaths on OP50, but not on CS180. Surprisingly, however, NMUR-1 promotes the opposite response when the insulin receptor DAF-2 has reduced function-where NMUR-1 now stimulates P-deaths on OP50, but again with no effect on CS180. We also find that NMUR-1 acts in sensory neurons to promote its bi-directional effects on longevity, which depend on the FOXO transcription factor DAF-16. In addition, NMUR-1 downregulates the expression of the insulin-like peptide daf-28, but only when DAF-2 function is not reduced. This suggests a regulatory mechanism through which NMUR-1 maintains insulin receptor DAF-2 signaling at a suitable level. Thus, our studies reveal that NMUR-1 serves to buffer the dynamic range of DAF-2 receptor signaling, thereby optimizing pharyngeal health and survival in response to specific bacteria.

An animal’s responses to environmental cues are critical for its reproductive program. Thus, a mechanism that allows the animal to sense and adjust to its environment should make for a more efficient reproductive physiology. Here, we demonstrate that in Caenorhabditis elegans specific sensory neurons influence onset of oogenesis through insulin signaling in response to food-derived cues. The chemosensory neurons ASJ modulate oogenesis onset through the insulin-like peptide (ILP) INS-6. In contrast, other sensory neurons, the olfactory neurons AWA, regulate food type-dependent differences in C. elegans fertilization rates, but not onset of oogenesis. AWA modulates fertilization rates at least partly in parallel to insulin receptor signaling, since the insulin receptor DAF-2 regulates fertilization independently of food type, which requires ILPs other than INS-6. Together our findings suggest that optimal reproduction requires the integration of diverse food-derived inputs through multiple neuronal signals acting on the C. elegans germline.

Abstract Insulin signaling has been implicated in many physiological processes, such as development, metabolism and stress survival, thereby ensuring an animal’s homeostasis. Thus, to enable the pathway to function with high precision, its ligands can undergo multiple levels of regulation—at the transcriptional, post-transcriptional and post-translational levels. Interestingly, we now find that the endogenous mRNA transcripts of the C. elegans insulin-like peptide ins-6 also become localized to specific structures. ins-6 transcripts are present in the axonal nerve ring of the stress-resistant dauer larva. In contrast, non-dauer and post-dauer larvae have ins-6 transcripts that are localized to the somas of the ASI and ASJ sensory neurons and not to the nerve ring. The absence of other neuronal mRNAs in the dauer axonal nerve ring suggests specificity in this mRNA localization of ins-6, which has previously been shown to be important during the switch to and from dauer physiology.

While C. elegans males are known to induce demise of hermaphrodites, the mechanism underlying this phenomenon is poorly understood. Through transcriptomic analyses, Booth et al. identify specific hermaphrodite gene sets that respond to male pheromones, sperm and seminal fluid and can mediate premature death of hermaphrodites.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Preferably, lifespan-extending therapies should work when applied late in life without causing undesired pathologies. Reducing insulin/insulin-like growth factor (IGF)-1 signaling (IIS) increases lifespan across species, but the effects of reduced IIS interventions in extreme geriatric ages remains unknown. Using the nematode Caenorhabditis elegans, we engineered the conditional depletion of the DAF-2/insulin/IGF-1 transmembrane receptor using an auxin-inducible degradation (AID) system. This allowed for the temporal and spatial reduction in DAF-2 protein levels at time points after which interventions such as RNAi become ineffective. Using this system, we found that AID-mediated depletion of DAF-2 protein surpasses the longevity of daf-2 mutants. Depletion of DAF-2 during early adulthood resulted in multiple adverse phenotypes, including growth retardation, germline shrinkage, egg retention, and reduced brood size. By contrast, AID-mediated depletion of DAF-2 post-reproduction, or specifically in the intestine in early adulthood, resulted in an extension of lifespan without these deleterious effects. Strikingly, at geriatric ages, when 75% of the population had died, AID-mediated depletion of DAF-2 protein resulted in a doubling in lifespan. Thus, we provide a proof-of-concept that even close to the end of an individual’s lifespan, it is possible to slow aging and promote longevity. eLife digest The goal of geroscience, or research into old age, is to promote health during old age, and thus, to increase lifespan. In the body, the groups of biochemical reactions, or ‘pathways’, that allow an organism to sense nutrients, and regulate growth and stress, play major roles in ensuring healthy aging. Indeed, organisms that do not produce a working version of the insulin/IGF-1 receptor, a protein involved in one such pathway, show increased lifespan. In the worm Caenorhabditis elegans, mutations in the insulin/IGF-1 receptor can even double their lifespan. However, it is unclear whether this increase can be achieved once the organism has reached old age. To answer this question, Venz et al. genetically engineered the nematode worm C. elegans so that they could trigger the rapid degradation of the insulin/IGF-1 receptor either in the entire organism or in a specific tissue. Venz et al. started by aging several C. elegans worms for three weeks, until about 75% had died. At this point, they triggered the degradation of the insulin/IGF-1 receptor in some of the remaining worms, keeping the rest untreated as a control for the experiment. The results showed that the untreated worms died within a few days, while worms in which the insulin/IGF-1 receptor had been degraded lived for almost one more month. This demonstrates that it is possible to double the lifespan of an organism at the very end of life. Venz et al.’s findings suggest that it is possible to make interventions to extend an organism’s lifespan near the end of life that are as effective as if they were performed when the organism was younger. This sparks new questions regarding the quality of this lifespan extension: do the worms become younger with the intervention, or is aging simply slowed down? Introduction The goal of aging research or geroscience is to identify interventions that promote health during old age (Kennedy et al., 2014; López-Otín et al., 2013; Partridge et al., 2018). Nutrient-sensing pathways that regulate growth and stress resistance play major roles as conserved assurance pathways for healthy aging (Kenyon, 2010; López-Otín et al., 2013). One of the first longevity pathways discovered was the insulin/insulin-like growth factor (IGF)-1 signaling pathway (reviewed in Kenyon, 2010). Reducing insulin/IGF-1 signaling (IIS) increases lifespan across species (Kenyon, 2010). Mice heterozygous for the IGF-1 receptor, or with depleted insulin receptor in adipose tissue, are stress-resistant and long-lived (Blüher et al., 2003; Holzenberger et al., 2003), for example, and several single-nucleotide polymorphisms in the IIS pathway have been associated with human longevity (Kenyon, 2010). Moreover, gene variants in the IGF-1 receptor have been associated and functionally linked with long lifespans in human centenarians (Suh et al., 2008). This suggests that a comprehensive understanding of this pathway in experimental, genetically tractable organisms has promising translational value for promoting health in elderly humans. However, whether or not reducing IIS during end-of-life stages can still promote health and longevity in any organism is unknown. Therefore, we turned to the model organism Caenorhabditis elegans to investigate whether reducing IIS during old age was sufficient to increase lifespan. The groundbreaking discovery that a single mutation in daf-2, which is the orthologue of both the insulin and IGF-1 receptors (Kimura et al., 1997), or mutations in ‘downstream’ genes in the IIS pathway, could double the lifespan of an organism was made in the nematode C. elegans (Friedman and Johnson, 1988; Kenyon et al., 1993). Since its discovery, over 1000 papers on daf-2 have been published, making it one of the most studied genes in this model organism (Source: PubMed). Genetic and genomics approaches have revealed that the DAF-2 insulin/IGF-1 receptor signaling regulates growth, development, metabolism, inter-tissue signaling, immunity, stress defense, and protein homeostasis, including extracellular matrix remodeling (Ewald et al., 2015; Gems et al., 1998; Kimura et al., 1997; Murphy and Hu, 2013; Wolkow et al., 2000). Much of our knowledge of the effects of daf-2 on aging has come from the study of reduction-of-function alleles of daf-2. Several alleles of daf-2 have been isolated that are temperature-sensitive with respect to an alternative developmental trajectory. For instance, most daf-2 mutants develop into adults at 15°C and 20°C but enter the dauer stage at 25°C (Gems et al., 1998), which is a facultative and alternative larval endurance stage in which C. elegans spends most of its life cycle in the wild (Hu, 2007). Under favorable conditions, C. elegans develops through four larval stages (L1–L4). By contrast, when the animals are deprived of food and experience an overcrowded environment and/or thermal stress (above 27°C), the developing larvae molt into an alternative pre-dauer (L2d) stage. If conditions do not improve, C. elegans enter the dauer diapause instead of the L3 stage (Golden and Riddle, 1984; Hu, 2007; Karp, 2018). A major limitation in using daf-2 mutants is that several of them show L1 larval and pre-dauer stage (L2d) arrest (Gems et al., 1998). Furthermore, the daf-2 alleles have been categorized into two mutant classes depending on the penetrance of dauer-like phenotypes during adulthood, such as reduced brood size, small body size, and germline shrinkage, as observed in the daf-2 class II mutants (Arantes-Oliveira et al., 2003; Ewald et al., 2018; Ewald et al., 2015; Gems et al., 1998; Hess et al., 2019; Patel et al., 2008; Podshivalova and Kerr, 2017). RNA interference of daf-2 can be applied, which increases lifespan without dauer formation during development and circumvents induction of daf-2 class II mutant phenotypes during adulthood (Dillin et al., 2002; Ewald et al., 2018; Ewald et al., 2015; Kennedy et al., 2004). However, the increase in lifespan by RNAi of daf-2 is only partial compared to strong alleles such as daf-2(e1370) (Ewald et al., 2015). Furthermore, adult-specific RNAi knockdown of daf-2 quickly loses its potential to increase lifespan and does not extend lifespan when started after day 6 of adulthood (Dillin et al., 2002), that is, after the reproductive period of C. elegans. Whether this is due to age-related functional decline of RNAi machinery or residual DAF-2 protein levels, or whether the late-life depletion of daf-2 simply does not extend lifespan remains unclear. As such, using an alternative method to reduce DAF-2 levels beyond RNAi or daf-2 mutation may allow us to more clearly uncouple the pleiotropic effects of reduced IIS during development from those that drive daf-2-mediated longevity during late adulthood. To this end, we used an auxin-inducible degradation (AID) system to induce the depletion of the degron-tagged DAF-2 protein with temporal precision (Zhang et al., 2015). The Arabidopsis thaliana IAA17 degron is a 68-amino acid motif that is specifically recognized by the transport inhibitor response 1 (TIR1) protein only in the presence of the plant hormone auxin (indole-3-acetic acid; Dharmasiri et al., 2005). Although cytoplasmic, nuclear, and membrane-binding proteins tagged with degron have been recently shown to be targeted and degraded in C. elegans (Beer et al., 2019; Zhang et al., 2015), to our knowledge, the AID system has not been used previously to degrade transmembrane proteins, such as the DAF-2 insulin/IGF-1 receptor. We find that using AID effectively degrades DAF-2 protein and promotes dauer formation when applied early in development. Dauer-like phenotypes are present in adults when AID of DAF-2 is applied late in development. Some of these adulthood dauer traits are induced by the loss of daf-2 in neurons, but others seem to be caused by the systemic loss of daf-2. More importantly, the post-developmental, conditional degradation of DAF-2 protein extends lifespan without introducing dauer-like phenotypes. Remarkably, we demonstrate that when more than half of the population has died at day 25 of adulthood, AID of DAF-2 in these remaining aged animals is sufficient to promote longevity. Our work suggests that therapeutics applied at even extremely late stages of life are capable of increasing longevity and healthspan in animals. Results Generation and validation of a degron-tagged DAF-2 receptor To monitor and conditionally regulate protein levels of the C. elegans DAF-2 insulin/IGF-1 receptor, we introduced a degron::3xFLAG tag into the 3' end of the daf-2 open reading frame (Figure 1—figure supplement 1). This degron::3xFLAG insertion into the genome was designed to tag the DAF-2 receptor at the cytosolic part for two reasons: first, to minimize any interference by the 81-amino acids large degron::3xFLAG-tag with the DAF-2 receptor function; and second, to ensure accessibility of the degron for targeted degradation by the TIR1 ubiquitin ligase expressed in the cytoplasm (Figure 1A). We endogenously tagged the DAF-2 receptor using CRISPR, and the resulting daf-2(bch40) CRISPR allele was verified by PCR (Figure 1—figure supplement 1). We performed western blot analysis against the 3xFLAG-tag and detected a specific band in daf-2(bch40) animals. This band was absent in wild type (N2) and animals carrying only the eft-3p::TIR1::mRuby::unc-54 3'UTR transgene (Figure 1B), which expresses TIR1 in all somatic cells, including neurons (Tomioka et al., 2016). To promote degradation of the degron::3xFLAG-tagged DAF-2 receptor, we crossed daf-2(bch40) into TIR1-expressing C. elegans. The strain obtained from this cross will be called ‘DAF-2::degron’ throughout this paper (i.e., Si57 [Peft-3::TIR1::mRuby::unc-54 3'UTR+ Cbr-unc-119(+)] II; daf-2(bch40 [degron::3xFLAG::STOP::SL2-SV40-degron::wrmScarlet-egl-13 NLS]) III). This strain showed no obvious phenotypes and exhibited a normal developmental progression at 20°C (Figure 1—figure supplement 1). To verify whether the band from the western blot was indeed DAF-2::degron::3xFLAG, we treated DAF-2::degron animals with daf-2 RNAi. The band nearly completely disappeared after 48 hr of daf-2(RNAi) feeding (Figure 1C and D, Source data 1 and Source data 2). Collectively, these results suggested that the tagged transmembrane receptor DAF-2 did not interfere with normal DAF-2 function. Figure 1 with 1 supplement see all Download asset Open asset Degron-tagged transmembrane insulin/insulin-like growth factor-1 (IGF-1) receptor DAF-2 is susceptible to auxin-mediated degradation. (A) Schematic illustration of auxin-inducible degradation (AID)-mediated DAF-2 receptor depletion in daf-2(bch40) Caenorhabditis elegans. (B) Immunoblot of eft-3p::TIR1::mRuby::unc-54 3’UTR, daf-2(bch40), wild type (N2), and DAF-2::degron (eft-3p::TIR1::mRuby::unc-54 3’UTR; daf-2(bch40)). (C) Immunoblot of ‘DAF-2::degron’ animals that were grown on OP50 NGM and at L4 stage shifted to either empty vector control RNAi (L4440) or daf-2(RNAi) plates containing either DMSO or 1 mM auxin. After 48 hr on the second day of adulthood, animals were harvested for western blotting. (D) Densitometric quantification of (C) from n = 3 independent experiments. Error bars represent s.d. Two-sided t-test was used for statistical analysis. *: p < 0.05, **: p < 0.01, ***: p < 0.001. (E) Immunoblot of DAF-2::degron animals showed no decrease of DAF-2 levels at high temperatures. Animals were raised at 15°C and put as L4 for 24 hr at the indicated temperatures. (F) A representative immunoblot of ‘DAF-2::degron’ animals after 1% glucose and 36–48 hr starvation on the second day of adulthood. L4 DAF-2::degron animals were either placed on OP50 NGM plates with or without 1 mM auxin, or containing 1% glucose, or on empty (no bacteria) NGM plates. Animals were harvested 36–48 hr later. (G) Densitometric quantification of (F) from n = 3 independent experiments. Error bars represent s.d. Two-sided t-test was used for statistical analysis. *: p < 0.05, **: p < 0.01, ***: p < 0.001. (H) Immunoblot analysis of starved DAF-2::degron animals. Animals were raised on OP50 NGM at 20°C and shifted from L4 to L4440 containing FUdR. After 2 days, they were washed off, and either frozen as control or put on empty plates and harvested after 24 or 48 hr, respectively. (I) Immunoblot analysis of 1-day-old adult DAF-2::degron animals treated with 1 mM auxin for the indicated time periods. (J) Quantification of (I) from n = 3 independent experiments. Error bars represent s.d. One-sided t-test was used for statistical analysis. *: p < 0.05, **: p < 0.01, ***: p < 0.001. For (B–J), see Source data 1 and Source data 2 for raw data, full blots, and statistics. Dietary changes modulate endogenous DAF-2/insulin/IGF-1 receptor abundance Next, we monitored endogenous DAF-2 protein levels under different environmental conditions, such as temperature and diet. Previously, Kimura and colleagues used DAF-2 antibody immunostaining of whole animals and reported that mutant DAF-2(e1370) protein is present at 15°C but barely detected at 25°C, whereas mutant DAF-2(e1370) protein in a daf-16 null background or wild-type DAF-2 protein persists at both 15°C and 25°C (Kimura et al., 2011). By contrast, upon 24 hr of starvation, the DAF-2 receptor is no longer detectable by using immunofluorescence in fixed C. elegans (Kimura et al., 2011). Since the FOXO transcription factor daf-16 is the transcriptional output of daf-2 signaling (Ewald et al., 2018; Gems et al., 1998), these results suggest that DAF-2 protein levels may be autoregulated by IIS and might be influenced by temperature and food availability. We first asked whether our DAF-2::degron::3xFLAG tag allows quantification of endogenous DAF-2 levels. We observed comparable wild-type DAF-2::degron::3xFLAG levels across a range of temperatures (15–28°C; Figure 1E), indicating that temperature does not influence DAF-2 levels in wild type. Intriguingly, however, we found that using FLAG-HRP antibodies to monitor protein levels, DAF-2 protein almost completely disappeared after 36–48 hr of starvation (Figure 1F and G). In keeping with this result, well-fed animals, for which we added 1% glucose into the bacterial diet (OP50), increased the DAF-2 protein levels (Figure 1F and G). Curiously, we noted that this starvation-induced degradation of DAF-2 did not happen when, during development, C. elegans were fed another bacterial strain, HT1115, used for RNAi (L4440). When DAF-2::degron animals were grown on L4440 and then shifted on empty NGM plates for 24 or 48 hr of starvation, DAF-2 levels did not decrease (Figure 1H). This observation suggests a hypothesis that the nutritional composition of the animal’s diet prior to starvation influences DAF-2 stability, which will be interesting to test in future research. We conclude that food availability controls not only the secretion of insulin-like peptides to regulate DAF-2 activity (Pierce et al., 2001) but also DAF-2 receptor abundance. Auxin-induced degradation of degron-tagged insulin/IGF-1 receptor In C. elegans, cytosolic degron-tagged proteins are almost completely degraded after 30 min of auxin treatment (Zhang et al., 2015). However, the degradation of transmembrane proteins using AID in vivo has not been previously reported. We hypothesized that C. elegans might exhibit similar kinetics of degradation of a transmembrane protein following auxin treatment. In keeping with that hypothesis, after 30 min of 1 mM auxin treatment, we observed a dramatic decrease in transmembrane DAF-2 protein abundance (Figure 1I and J). Levels of DAF-2 were only slightly further reduced by continued auxin treatment, as indicated at 4 and 24 hr time points (Figure 1I and J). After 24 hr of 1 mM auxin treatment, we observed only a 40% total decrease in DAF-2 protein abundance rather than a complete loss (Figure 1C and D). Similar kinetics in the degradation of DAF-2::degron::3xFLAG levels were confirmed using additional FLAG and degron antibodies (Source data 2). Taken together, these results suggest that our AID system allows for the partial, rapid degradation of the transmembrane DAF-2 receptor. Inactivation of DAF-2::degron by the AID inhibits downstream IIS We wondered whether the reduction of DAF-2 levels by AID would have consequences consistent with reduced IIS. Activation of DAF-2/insulin/IGF-1 receptor induces a downstream kinase cascade to phosphorylate the transcription factors DAF-16/FOXO and SKN-1/NRF, causing their retention in the cytoplasm (Figure 2A; Ewald et al., 2015; Henderson and Johnson, 2001; Lin et al., 2001; Murphy et al., 2003; Ogg et al., 1997; Tullet et al., 2008). Genetic inhibition of daf-2 results in less DAF-16 and SKN-1 phosphorylation and promotes nuclear translocation to induce the expression of target genes, such as sod-3 (superoxide dismutase) and gst-4 (glutathione S-transferase), respectively (Ewald et al., 2015; Henderson and Johnson, 2001; Lin et al., 2001; Murphy et al., 2003; Tullet et al., 2008). Within 1 hr of 1 mM auxin treatment, we found that most DAF-16::GFP translocated into the nuclei in DAF-2::degron animals (Figure 2B), with observable translocation already after 30 min (Figure 2—figure supplement 1). This DAF-16::GFP nuclear localization in DAF-2::degron animals was time- and auxin-concentration-dependent and did not occur in DAF-16::GFP animals with wild-type DAF-2 (Figure 2B, Figure 2—figure supplement 1). Similarly, SKN-1- or DAF-16-target gene expression of gst-4 or sod-3 was only induced upon auxin treatment in DAF-2::degron animals (Figure 2C and D). Thus, expectedly, IIS is reduced upon AID DAF-2 degradation. Figure 2 with 2 supplements see all Download asset Open asset Inactivation of DAF-2::degron by the auxin-inducible degradation (AID) upregulated downstream reporters and caused dauer entry at any temperature. (A) A schematic illustration of the DAF-2 signaling pathway. DAF-2 phosphorylates the transcription factors DAF-16 and SKN-1 through a cascade of kinases and sequesters them to the cytosol. Lower DAF-2 levels or activity leads to de-phosphorylation and translocation of DAF-16 and SKN-1 to the nucleus and expression of target genes like sod-3 and gst-4, respectively. (B) One hour exposure to auxin led to nuclear translocation of DAF-16::GFP in a concentration-dependent manner in DAF-2::degron; daf-16::gfp animals, but not in daf-16::gfp at the L4 stage. n > 94 from two (for daf-16::gfp) or three (for DAF-2::degron; daf-16::gfp) independent experiments. **: p < 0.01, ****: p < 0.0001. (C) Exposure to 1 mM auxin activates the reporter sod-3::GFP in DAF-2::degron; sod-3::gfp but not in animals that carry sod-3::gfp alone. L4 animals were exposed to 1 mM auxin or DMSO for 24 hr and GFP levels were scored (i.e., at day 1 of adulthood). n > 140 from two (for sod-3::gfp) or four (for DAF-2::degron; sod-3::gfp) independent experiments. ****: p < 0.0001. (D) Exposure to 1 mM auxin activates the reporter gst-4::GFP in DAF-2::degron; gst-4::gfp but not in animals that carry gst-4::gfp alone. L4 animals were exposed to 1 mM auxin or DMSO for 24 hr and GFP levels were scored (i.e., at day 1 of adulthood). n > 111 from two (for gst-4::gfp) or four (for DAF-2::degron; gst-4::gfp) independent experiments. ****: p < 0.0001. (E) Auxin treatment of DAF-2::degron affected development. Synchronized L1 put at low concentrations (1–50 μM auxin) shows reduced growth speed, and their offspring enters dauer. At high concentrations (100 and 1000 μM), the L1 animals arrest and enter the dauer stage after a few days. (F) Representative pictures of growth impairment caused by auxin-mediated degradation of DAF-2 in DAF-2::degron animals at different concentrations. Bar = 1 mm. (G) Dauer entry of DAF-2::degron animals at 1 mM auxin was temperature-independent, but the time needed for dauer entry is temporally scaled. To distinguish dauer animals from pre-dauer animals, they were treated for 15 min with 1% SDS. Only dauer animals survived SDS treatment. (H) Microscope pictures of DAF-2::degron animals after dauer entry (right column) and control animals (left column) kept for the same time at 15°C, 20°C, and 25°C. Control animals were on their second day of adulthood when the auxin-treated counterparts entered the dauer stage. Bar = 1 mm. For (B–D), see Source data 1 for raw data and statistics. AID of DAF-2::degron promotes dauer entry at any temperature Reduced IIS during development promotes dauer entry. Dauer formation at 15°C has been observed for a variety of strong loss-of-function daf-2 alleles, such as the class I alleles e1369 and m212, the class II allele e979, the null alleles m65, m646, m633, and a variety of unclassified alleles discovered by Malone and Thomas (Gems et al., 1998; Kimura et al., 2011; Malone and Thomas, 1994; Patel et al., 2008). For the commonly used reference alleles e1368 and e1370, penetrant dauer formation only occurs at 25°C (Gems et al., 1998). By contrast, knockdown of daf-2 by RNAi does not cause dauer formation at any temperature (Dillin et al., 2002; Ewald et al., 2015; Kennedy et al., 2004). We hypothesized that dauer formation would not happen because the decrease of DAF-2::degron levels after auxin treatment is only around 40%. However, synchronized L1 treated with 0.1 or 1 mM auxin all formed dauers at 25°C (Figure 2E and F). We observed dose-dependent retardation of the developmental speed when using lower auxin concentrations (1, 10, and 50 μM), but the offspring of retarded animals, grown on plates containing auxin, became dauers (Figure 2E and F). The dependence on auxin concentration for dauer formation of L1 animals suggests a threshold of DAF-2 receptor levels for the decision or commitment to dauer diapause. Even more surprising was the observation that dauer formation was also observed at 15°C and 20°C with complete penetrance (Figure 2G and H). We found that dauer formation was related to the developmental speed at a given temperature: At 15°C, it took 6 days; at 20°C, it took 4 days; and at 25°C, it took 3 days to form dauers (Figure 2H). We verified that all auxin-induced DAF-2::degron dauers showed dauer-specific characteristics, such as SDS resistance, cessation of feeding, constricted pharynxes, and dauer-specific alae (Figure 2G, Figure 2—figure supplement 1), suggesting a complete dauer transformation. Thus, the AID of DAF-2 promotes complete dauer formation independent of temperature but dependent on DAF-2 protein abundance. Dauer commitment at mid-larval stage 1 upon DAF-2 degradation Wild-type animals enter the pre-dauer L2d stage, where they keep monitoring their environment before completely committing to dauer formation (Golden and Riddle, 1984; Hu, 2007; Karp, 2018). Treating wild type with dauer pheromone suggested mid-L1 as the stage when the dauer decision is made (Golden and Riddle, 1984). By contrast, previous temperature-shifting experiments (from 15°C to 25°C) with daf-2 mutants suggested a dauer decision time window from L1 to L2 stage, before the L2d stage (Swanson and Riddle, 1981). Since AID allows for precise temporal degradation of DAF-2, we pinpointed the dauer entry decision to the mid-L1 stage. Specifically, we shifted synchronized L1s at different time points to plates containing 1 mM auxin and counted the number of cells in developing gonads to determine the developmental stage, when 50% of the population committed to becoming dauers (Figure 3—figure supplement 1). We found that when DAF-2 levels are below a given threshold at the mid-L1 stage, the animals commit to becoming dauers. AID-degraded DAF-2 resembles a non-conditional and severe loss-of-function Daf-2 allele The FOXO transcription factor DAF-16 is required for dauer formation in daf-2 mutants. We crossed DAF-2::degron with DAF-16::degron (Aghayeva et al., 2021) and found that daf-16 was required for dauer formation and developmental speed alterations after DAF-2::degron depletion (Figure 2—figure supplement 2). Previous reports suggest that many daf-2 alleles show low to severe penetrance of embryonic lethality and L1 arrest at higher temperatures (Collins et al., 2008; Ewald et al., 2016; Gems et al., 1998; Patel et al., 2008). Although the constitutive dauer formation of the proposed null allele daf-2(m65) is suppressed by daf-16 null mutations, the embryonic lethality and L1 arrest are not daf-16-dependent (Patel et al., 2008). We observed no embryonic lethality nor L1 arrest in the progeny of animals placed on 1 mM auxin as L4s. Similar results were seen using either the DAF-2::degron or DAF-2::degron; germline TIR1 strains. Higher concentrations of auxin lead to toxicity in both wild-type and DAF-2::degron animals (Figure 2—figure supplement 2). A lack of embryonic lethality could be explained by insufficient DAF-2 degradation or earlier decision stages. Taken together, the inactivation of DAF-2 by AID is 100% penetrant for dauer formation at any temperature. Still, the absence of embryonic lethality or L1 arrest at 1 mM auxin suggests that DAF-2::degron functionally is more similar to a non-conditional and severe loss-of-function mutation than a null allele. Enhanced lifespan extension by AID of DAF-2 in adult animals Given the strong phenotypic effects of DAF-2 AID on animal development, we next explored whether DAF-2 degradation by AID could affect the function of adult animals. Previous studies indicate that reducing IIS, either by daf-2 RNAi knockdown or in genetic mutants, increases lifespan at any temperature (15–25°C) (Ewald et al., 2018; Gems et al., 1998). We hypothesized that AID-dependent degradation of DAF-2 would have similar effects on the lifespan of animals. We found that auxin supplementation of DAF-2::degron animals, starting from L4, resulted in a 70–135% lifespan extension (Figure 3A; Supplementary file 1). Impressively, DAF-2 degradation using 1 mM auxin surpassed the longevity of commonly used daf-2(e1368) and daf-2(e1370) mutants (Figure 3A, Supplementary file 1). By contrast, auxin treatment at 0.1 or 1 mM concentration had little or no effect on wild-type lifespan (Figure 3A; Supplementary file 1). These results suggest that auxin-induced degradation of daf-2 is a powerful tool to promote longevity. Figure 3 with 2 supplements see all Download asset Open asset Depletion of DAF-2::degron resulted in daf-2 class II mutant phenotypes at 15°C. (A) Auxin treatment extended the lifespan of DAF-2::degron animals. Animals were shifted as L4 to plates containing DMSO or 1 mM auxin at 20°C. (B) 1 mM auxin treatment leads to the ‘dauer-like’ egg retention phenotype in DAF-2::degron animals. The animals were raised at 15°C, then shifted as L4 to 25°C or kept at 15°C. Two days later at 25°C and 3 days later at 15°C, they were examined for egg retention. The experiment was performed three independent times. Error bar represents s.d. ****: p < 0.0001. (C) Gonads were shrunk after 1 mM auxin treatment in DAF-2::degron animals. Animals were raised at 15°C and shifted as L4 to 25°C or kept at 15°C. Two days later at 25°C and 3 days later at 15°C, the animals were checked for gonad size. The experiment was performed three independent times. ****: p < 0.0001. (D) 1 mM auxin treatment decreased the body size of DAF-2::degron animals at 15°C and 25°C. Animals were raised at 15°C and shifted to 1 mM auxin or DMSO plates at the L4 stage. The experiment was performed three independent times. ****: p < 0.0001. (E) 1 mM auxin treatment of DAF-2::degron resulted in a smaller brood size at 15°C and 25°C. Animals were shifted to 1 mM auxin or DMSO plates at the L4 stage and either kept at 15°C or moved to 25°C. The experiment was performed three independent times. ****: p < 0.0001. (F) Tissue-specific depletion of DAF-2 in neurons caused egg retention phenotype. Animals were raised at 15°C and shifted from L4 to 25°C. Two days later, the animals were checked for egg retention. The experiment was performed three independent times. Error bar represents s.d. ****: p < 0.0001. (G) Gonads were shrunk after neuronal depletion of DAF-2. Animals were raised at 15°C and shifted from L4 to 25°C. Two days later, the animals

The coordination between the animal's external environment and internal state requires constant modulation by chemicals known as neuromodulators. Neuromodulators, such as biogenic amines, neuropeptides and cytokines, promote organismal homeostasis. Over the past several decades, Caenorhabditiselegans has grown into a powerful model organism that allows the elucidation of the mechanisms of action of neuromodulators that are conserved across species. In this perspective, we highlight a collection of articles in this issue that describe how neuromodulators optimize C. elegans survival.

Aberrations in insulin or insulin-like peptide (ILP) signaling in the brain causes many neurological diseases. Here we report that mRNAs of specific ILPs are surprisingly mobilized to the axons of C. elegans during stress. Transport of the ILP ins-6 mRNA to axons facilitates recovery from stress, whereas loss of axonal mRNA delays recovery. In addition, the axonal traffic of ins-6 mRNA is regulated by at least two opposing signals: one that depends on the insulin receptor DAF-2 and a kinesin-2 motor; and a second signal that is independent of DAF-2, but involves a kinesin-3 motor. While Golgi bodies that package nascent peptides, like ILPs, have not been previously found in C. elegans axons, we show that axons of stressed C. elegans have increased Golgi ready to package peptides for secretion. Thus, our findings present a mechanism that facilitates an animal9s rapid recovery from stress through axonal ILP mRNA mobilization.

ABSTRACTAberrations in insulin or insulin-like peptide (ILP) signaling in the brain causes many neurological diseases. Here we report that mRNAs of specific ILPs are surprisingly mobilized to the axons ofC. elegansduring stress. Transport of the ILPins-6mRNA to axons facilitates recovery from stress, whereas loss of axonal mRNA delays recovery. In addition, the axonal traffic ofins-6mRNA is regulated by at least two opposing signals: one that depends on the insulin receptor DAF-2 and a kinesin-2 motor; and a second signal that is independent of DAF-2, but involves a kinesin-3 motor. While Golgi bodies that package nascent peptides, like ILPs, have not been previously found inC. elegansaxons, we show that axons of stressedC. eleganshave increased Golgi ready to package peptides for secretion. Thus, our findings present a mechanism that facilitates an animal’s rapid recovery from stress through axonal ILP mRNA mobilization.

Social environment modulates learning through unknown mechanisms. Here, we report that a pheromone mixture that signals overcrowding inhibits C. elegans from learning to avoid pathogenic bacteria. We find that learning depends on the balanced signaling of two insulin-like peptides (ILPs), INS-16 and INS-4, which act respectively in the pheromone-sensing neuron ADL and the bacteria-sensing neuron AWA. Pheromone exposure inhibits learning by disrupting this balance: it activates ADL and increases expression of ins-16, and this cellular effect reduces AWA activity and AWA-expressed ins-4. The activities of the sensory neurons are required for learning and the expression of the ILPs. Interestingly, pheromones also promote the ingestion of pathogenic bacteria while increasing resistance to the pathogen. Thus, the balance of the ILP signals integrates social information into the learning process as part of a coordinated adaptive response that allows consumption of harmful food during times of high population density.

Transient increases in mitochondrially-derived reactive oxygen species (ROS) activate an adaptive stress response to promote longevity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce ROS locally in response to various stimuli, and thereby regulate many cellular processes, but their role in aging remains unexplored. Here, we identified the C. elegans orthologue of mammalian mediator of ErbB2-driven cell motility, MEMO-1, as a protein that inhibits BLI-3/NADPH oxidase. MEMO-1 is complexed with RHO-1/RhoA/GTPase and loss of memo-1 results in an enhanced interaction of RHO-1 with BLI-3/NADPH oxidase, thereby stimulating ROS production that signal via p38 MAP kinase to the transcription factor SKN-1/NRF1,2,3 to promote stress resistance and longevity. Either loss of memo-1 or increasing BLI-3/NADPH oxidase activity by overexpression is sufficient to increase lifespan. Together, these findings demonstrate that NADPH oxidase-induced redox signaling initiates a transcriptional response that protects the cell and organism, and can promote both stress resistance and longevity.

Environmental fluctuations influence organismal aging by affecting various regulatory systems. One such system involves sensory neurons, which affect life span in many species. However, how sensory neurons coordinate organismal aging in response to changes in environmental signals remains elusive. Here, we found that a subset of sensory neurons shortens Caenorhabditis elegans' life span by differentially regulating the expression of a specific insulin-like peptide (ILP), INS-6. Notably, treatment with food-derived cues or optogenetic activation of sensory neurons significantly increases ins-6 expression and decreases life span. INS-6 in turn relays the longevity signals to nonneuronal tissues by decreasing the activity of the transcription factor DAF-16/FOXO. Together, our study delineates a mechanism through which environmental sensory cues regulate aging rates by modulating the activities of specific sensory neurons and ILPs.

An animal’s survival strongly depends on a nervous system that can rapidly process and integrate the changing quality of its environment and promote the most appropriate physiological responses. This is amply demonstrated in the nematode worm Caenorhabditis elegans, where its sensory system has been shown to impact multiple physiological traits that range from behavior and developmental plasticity to longevity. Because of the accessibility of its nervous system and the number of tools available to study and manipulate its neural circuitry, C. elegans has thus become an important model organism in dissecting the mechanisms through which the nervous system promotes survival. Here we review our current understanding of how the C. elegans sensory system affects diverse physiological traits, whose coordination would be essential for survival under fluctuating environments. The knowledge we derive from the C. elegans studies should provide testable hypotheses in discovering similar mechanisms in higher animals.

Sensory perception modulates lifespan across taxa, presumably due to alterations in physiological homeostasis after central nervous system integration. The coordinating circuitry of this control, however, remains unknown. Here, we used the Drosophila melanogaster gustatory system to dissect one component of sensory regulation of aging. We found that loss of the critical water sensor, pickpocket 28 (ppk28), altered metabolic homeostasis to promote internal lipid and water stores and extended healthy lifespan. Additionally, loss of ppk28 increased neuronal glucagon-like adipokinetic hormone (AKH) signaling, and the AKH receptor was necessary for ppk28 mutant effects. Furthermore, activation of AKH-producing cells alone was sufficient to enhance longevity, suggesting that a perceived lack of water availability triggers a metabolic shift that promotes the production of metabolic water and increases lifespan via AKH signaling. This work provides an example of how discrete gustatory signals recruit nutrient-dependent endocrine systems to coordinate metabolic homeostasis, thereby influencing long-term health and aging.

Insulin-like peptides (ILPs) play highly conserved roles in development and physiology. Most animal genomes encode multiple ILPs. Here we identify mechanisms for how the forty Caenorhabditis elegans ILPs coordinate diverse processes, including development, reproduction, longevity and several specific stress responses. Our systematic studies identify an ILP-based combinatorial code for these phenotypes characterized by substantial functional specificity and diversity rather than global redundancy. Notably, we show that ILPs regulate each other transcriptionally, uncovering an ILP-to-ILP regulatory network that underlies the combinatorial phenotypic coding by the ILP family. Extensive analyses of genetic interactions among ILPs reveal how their signals are integrated. A combined analysis of these functional and regulatory ILP interactions identifies local genetic circuits that act in parallel and interact by crosstalk, feedback and compensation. This organization provides emergent mechanisms for phenotypic specificity and graded regulation for the combinatorial phenotypic coding we observe. Our findings also provide insights into how large hormonal networks regulate diverse traits.

In Caenorhabditis elegans, a subset of gustatory neurons, as well as olfactory neurons, shortens lifespan, whereas a different subset of gustatory neurons lengthens it. Recently, the lifespan-shortening effect of olfactory neurons has been reported to be conserved in Drosophila. Here we show that the Drosophila gustatory system also affects lifespan in a bidirectional manner. We find that taste inputs shorten lifespan through inhibition of the insulin pathway effector dFOXO, whereas other taste inputs lengthen lifespan in parallel to this pathway. We also note that the gustatory influence on lifespan does not necessarily depend on food intake levels. Finally, we identify the nature of some of the taste inputs that could shorten versus lengthen lifespan. Together our data suggest that different gustatory cues can modulate the activities of distinct signaling pathways, including different insulin-like peptides, to promote physiological changes that ultimately affect lifespan.