The goal of aging research is to extend healthy, active life. For decades, C. elegans daf-2 insulin/insulin-like growth factor 1 (IGF-1) receptor mutants have served as a model for extended lifespan and youthfulness. However, a recent report suggested that their longevity is associated with an undesirable phenotype: a disproportionately long period of decrepitude at the end of life. In the human population, such an outcome would be a burden to society, bringing into question the relevance of daf-2 mutants as a model for life extension. However, here we report that, following an extended period of movement, daf-2 mutants survive longer in a decrepit state because of a beneficial trait: they are resistant to colonization of the digestive tract by dietary bacteria, a condition that leads to premature death in the wild-type and prevents their manifestation of decrepitude. If bacterial colonization is prevented, then daf-2 mutants lead both chronologically and proportionately healthier lives relative to the wild-type.

In this study, we set out to accomplish three goals: to undertake a quantitative large-scale analysis to corroborate the reported disproportionately extended end-of-life decrepitude in a daf-2 mutant, to determine whether this phenotype could be due to behavioral particularities of the specific daf-2 allele that was examined, and, if not, to elucidate the cause of this apparently undesirable phenotype.

C. elegans with partial loss-of-function mutations in daf-2, the C. elegans insulin/IGF-1-receptor gene, not only live longer but also maintain more youthful characteristics, such as active movement (), neuronal function (), and memory (), indicating an extension of healthspan as well as lifespan. However, a recent study followed the functional ability of daf-2 mutants and found that the daf-2 healthspan, although chronologically longer than that of the wild-type, did not scale with lifespan, resulting in a disproportionately extended period of age-related decrepitude (). This report was disconcerting because such an outcome would be undesirable in a human society, where population aging has already increased healthcare costs substantially (). It also brought into question the validity of C. elegans as a model organism to study healthy life extension.

Caenorhabditis elegans has been an invaluable experimental organism for the discovery and characterization of conserved pathways that extend lifespan. In particular, reduced signaling through the stress and nutrient-sensing insulin/insulin-like growth factor 1 (IGF-1) pathway was first shown to double the lifespan of C. elegans and was later found to increase the longevity of other species, including mammals (). Moreover, polymorphisms in IGF-1-pathway genes and low plasma IGF-1 levels are associated with extreme longevity in humans ().

Aging research aims not simply to increase lifespan but, rather, to increase the duration of a healthy, disability-free life, or healthspan. In theory, lifespan can be extended by decreasing the rate of aging, postponing its onset, or eliminating a cause of mortality in old individuals. These mechanisms would affect the duration of healthspan and age-related deterioration differently.

Results and Discussion

Swierczek et al., 2011 Swierczek N.A.

Giles A.C.

Rankin C.H.

Kerr R.A. High-throughput behavioral analysis in C. elegans. Figure 1 daf-2(e1370) but Not daf-2(e1368) Mutants Exhibit Several Behavioral Phenotypes During Early Adulthood Show full caption (A) Data collection using the Multi-Worm Tracker. As an example, the speed of N2 (wild-type) young adults on a single plate over the course of a tracking session is shown: 0–900 s, no stimulation; starting at 900 s, a mechanical tap stimulus was delivered every 10 s 30 times. (B) Average speed of stimulated and unstimulated locomotion. (C) Speed of unstimulated locomotion in forward and backward directions. (D and E) Average time spent performing each of the four stereotypical behaviors (D) and fraction of the population spontaneously reversing movement direction during unstimulated locomotion (measured over a 60 s window) (E). Omega turn - a sharp turn that utilizes only forward body motion. (F) Fraction of animals responding (i.e., reversing) and average response time to a mechanical tap stimulus (the first tap). A reversal following the tap cannot be interpreted as a response to the tap if the time to a reversal is equal to the time between spontaneous reversals. When this happens, tap response curves are no longer shown (i.e., in older animals). (G) Distance traveled backward following a tap stimulus before resuming forward locomotion. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; n.s., not significant. Error bars represent SEM. Age, adult age (0–young adult). n > 200.p < 0.05;p < 0.005;p < 0.0005; n.s., not significant. Error bars represent SEM. Figures S1 A–S1C show behavioral phenotypes in the absence of FUDR. Figures S2 E and S2F show young adult behavior in additional daf-2 mutants. First, we used the Multi-Worm Tracker (MWT) computer vision system () to carefully measure the locomotory speed and behaviors of these two daf-2 mutants during young adulthood ( Figure 1 A). In the lab, C. elegans are typically observed responding to stimulation; for example, to the jolt of their culture dishes landing on the stage of a dissecting microscope ( Figure 1 A). When measured after delivering a mechanical stimulus in a controlled manner, the stimulated movement speed of daf-2(e1370) mutants was modestly but significantly slower than that of the wild-type (22%; Figure 1 B). When the speed of normal explorative locomotion was measured in the absence of external stimulation, daf-2(e1370) mutants moved markedly more slowly than the wild-type (53% slower; Figure 1 B). The impairment was particularly pronounced for the backward movement ( Figure 1 C). In contrast, both the stimulated and unstimulated movement speed of the daf-2(e1368) mutant closely resembled that of the wild-type ( Figure 1 B-C).

The daf-2(e1370) mutant displayed a number of additional behavioral phenotypes. It spent less time moving forward and more time taking pauses during explorative locomotion ( Figure 1 D). During young adulthood, but not after day 4, daf-2(e1370) animals spontaneously reversed the direction of locomotion more frequently than did the wild-type ( Figure 1 E). Furthermore, although the probability of initiating an escape response following a tap stimulus and the amount of time it took to react were normal ( Figure 1 F), the magnitude of the response (i.e., the distance traveled during the reversal) was reduced ( Figure 1 G). All of these behaviors were unaffected in the daf-2(e1368) mutant ( Figures 1 D–1G). Together, these results support and extend previous reports that daf-2(e1370) mutants have movement impairments as young adults, suggesting a mild dauer-like phenotype, and show that these early-life impairments are completely absent in the daf-2(e1368) mutant. In contrast, these animals exhibited completely normal behaviors but still lived long.

Figure 2 daf-2 Mutants Maintain Vigor Longer but Have an Extended Period of Immobility at the End of Life Show full caption (A) Survival analysis of daf-2(e1368) and daf-2(e1370) mutants under standard growth conditions. (B) Unstimulated movement speed of detected animals as a function of adult age. Excess speed of daf-2 mutants is calculated by subtracting wild-type speed from mutant speed. Locomotion of young, middle-aged, and old animals is also plotted separately below. Horizontal bars indicate duration of life and proportion of maximal lifespan without any detectable movement in the population. See Figure S1 D for analysis of stimulated movement. n > 200 on day 0. (C) Fraction of live worms exhibiting detectable movement at a given age. The data are averages of eight independent replicate plates with 30–50 worms per plate on day 0. (D) The same as (C) but plotted versus survival probability (i.e., normalized to lifespan). If every animal exhibited movement right until death regardless of whether it died early or late (i.e., if there were no late-life decrepitude), then this plot would be a straight horizontal line. A local polynomial regression (LOESS) fit with a 95% confidence interval is shown. Error bars represent SEM. Figures S1 E and S1F show stimulated and unstimulated speed scaled to maximal lifespan. Figures S2 A–S2E show early and late-life movement phenotypes in additional daf-2 mutants. Table S1 shows survival statistics for all lifespan experiments. To test the hypothesis that the early-life dauer-like phenotypes are related to the extension of late-life immobility, we measured the behavior of daf-2(e1370) and daf-2(e1368) mutants throughout life. Both mutants had extended lifespans ( Figure 2 A) and had a greater average movement speed than did the wild-type starting on day 9 of adulthood, as wild-type movement declined ( Figure 2 B; Figure S1 D). Because the MWT only detects animals that can move at least half a body length within a 1-min interval, any live but immobile animals would not be taken into account for speed calculation, resulting in the average speed of a population with immobile animals to be overestimated. Thus, we quantified the fraction of live animals that were able to move (i.e., were detectable) at a given age. This analysis also showed that the two daf-2 mutants preserved movement ability longer than did wild-type ( Figure 2 C). Thus, both daf-2 mutants gain additional healthy days of life, indicating a slower rate of behavioral aging.

Bansal et al., 2015 Bansal A.

Zhu L.J.

Yen K.

Tissenbaum H.A. Uncoupling lifespan and healthspan in Caenorhabditis elegans longevity mutants. However, we were surprised to find that, following this extended period of motility, both mutants spent a relatively long time (that is, a greater proportion of the maximal lifespan relative to the wild-type) without any spontaneous or stimulated movement ( Figure 2 B; Figure S1 D). Accordingly, in the daf-2(e1370) population, and, to a lesser extent, in the daf-2(e1368) population, a larger fraction of live animals was immobile at a given survival probability (i.e., proportion of lifespan; Figure 2 D). For example, with half of the population still alive, 42% of the live wild-type exhibited movement, whereas only about 15% of daf-2(e1368) and 0% of daf-2(e1370) did. Together, these data show that, although they have an extended chronological healthspan relative to the wild-type, daf-2 mutants end up spending a greater fraction of life in a state of decrepitude (which we define as a severely impaired spontaneous and stimulated movement ability that follows a gradual age-dependent behavioral decline). This extended period of decline at the end of life results in daf-2 mutants' appearing less healthy compared with the wild-type at a corresponding percentile of maximal lifespan ( Figures S1 E and S1F), consistent with what has been reported for daf-2(e1370) (). Because disproportionate extension of late-life decrepitude was observed in both daf-2(e1368) and daf-2(e1370) mutants, it can be genetically uncoupled from the early-life slow movement phenotypes seen in daf-2(e1370) mutants, arguing against a causal connection. Consistent with this, we analyzed two additional daf-2 perturbations and found no correlation between vigor in young adults and the duration of decrepitude at the end of life ( Figure S2 ).

Evans et al., 2008 Evans E.A.

Chen W.C.

Tan M.W. The DAF-2 insulin-like signaling pathway independently regulates aging and immunity in C. elegans. Garsin et al., 2003 Garsin D.A.

Villanueva J.M.

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Ruvkun G.

Ausubel F.M. Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens. Samuel et al., 2016 Samuel B.S.

Rowedder H.

Braendle C.

Félix M.-A.

Ruvkun G. Caenorhabditis elegans responses to bacteria from its natural habitats. Garigan et al., 2002 Garigan D.

Hsu A.L.

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Kenyon C. Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Gems and Riddle, 2000 Gems D.

Riddle D.L. Genetic, behavioral and environmental determinants of male longevity in Caenorhabditis elegans. In summary, we found that two very different daf-2 mutants both remain active longer and age more slowly than the wild-type, at least through mid-life, but then go on to stay alive but decrepit for a long time. We wanted to understand what might cause this extended decrepitude. Theoretically, eliminating a cause of death that kills relatively young individuals would result in a population's growing older and frailer. We wondered whether resistance to bacterial toxicity might play a role. daf-2 mutants are resistant to a wide variety of environmental stresses, including bacterial pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa (). Bacteria are the natural food source of C. elegans in the wild (), and in the lab, worms are fed an O-antigen-negative strain of Escherichia coli called OP50 that is considered to be non-pathogenic. However, wild-type worms fed dead OP50 have been shown to live longer than worms fed live OP50 (). Therefore, we hypothesized that the pathogenicity of this E. coli strain is a significant cause of death in wild-type C. elegans and that daf-2 mutants are more resistant to this pathogenicity. If so, the still-alive daf-2 mutants might continue to age, falling into a state of decrepitude. This situation would extend the overall proportion of life spent in a decrepit state.

Garigan et al., 2002 Garigan D.

Hsu A.L.

Fraser A.G.

Kamath R.S.

Ahringer J.

Kenyon C. Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Figure 3 Colonization by the E. coli Food Source Is a Risk Factor for Death in C. elegans Show full caption (A and B) Representative low (A) and high (B) magnification images of GFP-labeled OP50 E. coli accumulating in the terminal pharyngeal bulb and anterior intestine (dashed ovals) of wild-type worms with age. For quantification using >15 animals, see Figures 4 B and 4C. Worms were grown on GFP-OP50 starting at L1. Images of dead worms were taken at a lower exposure to avoid saturation. Arrows point to the terminal bulb of the pharynx. Scale bars, 100 μm. (C) Survival of wild-type animals that did or did not display colonization of the terminal bulb and anterior intestine on day 9 of adulthood. (D) Worms that displayed colonization on day 9 were treated with gentamicin starting on day 9 and for the remainder of their lifespan. The solid black curve indicates a population of worms that did not display any colonization on day 9 and from which any live worm that developed colonization was physically removed. Survival curves ended when there were no more animals left in this group because all wild-type worms eventually developed colonization. Note that there were no deaths in worms without colonization. (E) Gentamicin effectively reduces the bacterial load in colonized worms. Worms colonized by GFP-OP50 on day 9 were isolated and transferred to plates containing gentamicin (because dead OP50 do not make GFP, bacterial lawns on these plates were non-green). Twenty worms were singled out and, if alive, scored in the days following treatment as strongly colonized (same GFP intensity as on day 9), moderately colonized (less than on day 9), or not colonized (no GFP detectable by eye). To address the possibility that green bacteria are cleared by the worm and simply replaced by non-green bacteria independently of gentamicin, control animals were transferred to plates seeded with live non-GFP-OP50 without any gentamicin. (F) Animals were treated as in (E), and more than ten animals were picked randomly from gentamicin-treated and untreated plates and imaged. Representative images as well as mean GFP intensity per worm (mean ± SD) are shown. Scale bars, 100 μm. ∗p < 0.05, ∗∗∗p < 0.0005. (G) C. elegans lifespan is not affected by life-long gentamicin treatment when grown on gentamicin-resistant OP50 E. coli. Figures S3 A–S3C show that gentamicin-killed OP50 do not induce dietary restriction. To test this hypothesis, we first asked whether the E. coli food source is a risk factor for death in C. elegans that is reduced in daf-2 mutants. To visualize bacteria in the animal, we imaged GFP-expressing OP50 E. coli over the course of life. We found that bacteria accumulate in old wild-type worms ( Figures 3 A and 3B ). In particular, we observed an age-dependent increase in bacterial density in the terminal bulb of the pharynx and in the proximal intestine (100 μm from the pharyngeal-intestinal valve). Every dead wild-type worm in our study displayed this pattern of colonization ( Figures 3 A and 3B). During mid-life, we observed a mixture of colonized and non-colonized worms. We found that worms that did not display detectable colonization during mid-life (day 9 of adulthood) lived significantly longer than worms that did ( Figure 3 C). In fact, in these experiments, we never observed a worm dying prior to colonization (an affliction that eventually affects all worms). This association suggested that bacterial colonization, or something tightly linked to colonization, kills the animal, which is consistent with our previously published results (). To address causality more directly, we “cured” colonized worms of bacteria with the antibiotic gentamicin on day 9 and found that curing significantly increased their lifespans ( Figure 3 D). Gentamicin effectively penetrated the animal ( Figures 3 E and 3F) and did not affect lifespan on its own ( Figure 3 G). In addition, gentamicin-killed bacteria did not extend lifespan via dietary restriction ( Figures S3 A–S3C). Together, these data show that colonization is strongly associated with death in wild-type C. elegans and that reducing the bacterial load in colonized animals significantly improves their survival.

Youngman et al., 2011 Youngman M.J.

Rogers Z.N.

Kim D.H. A decline in p38 MAPK signaling underlies immunosenescence in Caenorhabditis elegans. Murphy et al., 2003 Murphy C.T.

McCarroll S.A.

Bargmann C.I.

Fraser A.

Kamath R.S.

Ahringer J.

Li H.

Kenyon C. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Hahm et al., 2011 Hahm J.H.

Kim S.

Paik Y.K. GPA-9 is a novel regulator of innate immunity against Escherichia coli foods in adult Caenorhabditis elegans. Gelino et al., 2016 Gelino S.

Chang J.T.

Kumsta C.

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Davis A.

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Hansen M. Intestinal Autophagy Improves Healthspan and Longevity in C. elegans during Dietary Restriction. Figure 4 daf-2 Mutants Resist Bacterial Colonization Show full caption (A and B) Representative images (A) and quantification (B) of GFP-OP50 accumulation in the terminal bulb and anterior intestine (100 μm from the terminal bulb, dashed ovals) in N2 and daf-2(e1368) with age. Scale bar, 100 μm. Each dot represents a worm, and horizontal bars represent medians. n > 15. The extent of colonization appears to be biphasic. This could be due to bacteria that accumulate in relatively young adults being cleared by the animal or due to a subpopulation of highly colonized worms dying, whereas the remaining, less colonized worms go on to develop further colonization later in life. We favor the second idea, given that there is little clearance of colonizing bacteria in day 9 animals ( Figure 3 E) and given the wide distribution of colonization severities in day 9–16 animals. (C) Local regression of data for live worms in (B) plotted as a function of age (days of adulthood) or lifespan. Note that the daf-2 curve ends when 88% of animals are dead rather than 100%. Figure S5 shows colonization in daf-2(e1370) mutants. We then measured bacterial colonization of a daf-2 mutant, focusing initially on the daf-2(e1368) mutant. As shown in Figures 4 A and 4B , colonization of the upper digestive tract was delayed and never reached the same maximum as in the wild-type. daf-2(e1368) animals had reduced colonization relative to both an age-matched wild-type and a wild-type with the same survival probability ( Figure 4 C), demonstrating that they spend both a larger amount of time and a larger proportion of life with less colonization. This finding is consistent with the idea that resistance to colonization allows daf-2 mutants to survive into old age. Why bacterial colonization occurs in old C. elegans and how exactly it causes death remains unknown. (Interestingly, the kinetics of colonization look biphasic, so there could be more than one reason [see Figure 4 , legend]). Decreased immune function with age () could contribute to bacterial accumulation and proliferation, and daf-2 mutants have higher expression of some antimicrobial genes () that curtail the rate of bacterial proliferation in the intestine (). One possibility for why bacterial accumulation kills worms is that metabolites produced by live metabolically active bacteria reach toxic levels when bacterial density increases inside the animal. Because the intestine becomes permeable with age (), another possibility is that worms die when bacteria leak out of the lumen and enter the body cavity. Although we did not observe any bacteria within tissues of live old worms (data not shown), it is possible that death occurs immediately after bacteria invade.

Figure 5 The Extended Period of Decrepitude in daf-2 Mutants Can Be Attributed to a Reduced Risk of Death by Bacterial Colonization Show full caption (A) Lifespans of wild-type and daf-2(e1368) mutants grown on gentamicin-killed OP50 E. coli from hatching. This diet did not affect the time it took the animals to reach adulthood (data not shown). (B) Unstimulated movement speed of wild-type and daf-2(e1368) animals when grown on dead OP50. Excess speed of daf-2 mutants was calculated by subtracting wild-type speed from mutant speed. Horizontal bars indicate duration of life and proportion of maximal lifespan without any detectable movement in the population. n > 200. Error bars represent SEM. (C) Fraction of live worms exhibiting detectable movement at a given survival probability (i.e., normalized to lifespan). LOESS fit of data from eight independent replicate plates with 30–50 worms per plate on day 0 and a 95% confidence interval are shown. Figures S3 D-S3F show rates of behavioral changes on live and dead bacteria. See Figure S4 for additional quantification of healthspan. Figure S5 shows the effect of dead bacteria on daf-2(e1370) mutants. If reduced risk of death due to bacterial colonization allows daf-2 mutants to live long enough to become decrepit, then eliminating bacterial colonization as a cause of “premature” death should allow wild-type worms, too, to live long enough to enter a state of end-of-life decrepitude. To test this hypothesis, we fed wild-type animals OP50 bacteria killed by gentamicin from the time of hatching. Using killed bacteria as a food source extended the wild-type lifespan by 40% ( Figure 5 A). At the end of life, the amount of time (and proportion of lifespan) spent without detectable movement increased from 8 days (24%) on live OP50 ( Figure 2 B) to 16 days (37%) on dead OP50 in wild-type worms ( Figure 5 B). Importantly, feeding dead bacteria did not change the rate of behavioral aging; it specifically extended the period of infirmity ( Figures S3 D–S3F). The likelihood of exhibiting movement at a given survival probability was significantly reduced by a diet of dead OP50 in wild-type worms ( Figure 5 C). In this regard, wild-type animals fed dead OP50 more closely resembled daf-2(e1368) mutants fed the standard diet of live OP50 than the wild-type fed the standard diet. Therefore, eliminating bacterial colonization as a cause of death in wild-type worms phenocopied the extended period of decrepitude seen in daf-2(e1368) mutants. Reducing food pathogenicity in a different way, by feeding worms less pathogenic soil bacteria, Bacillus subtilis, also increased lifespan and the proportion of life spent in a decrepit state, although the increase in the latter was smaller than that achieved by dead OP50 ( Figures S4 A–S4C). The conclusion that feeding worms non-pathogenic bacteria makes the wild-type healthspan more similar to the daf-2 healthspan was supported by a number of additional approaches to quantifying healthspan ( Figures S4 D–S4F).

A diet of dead bacteria also extended the daf-2(e1368) mutant lifespan but by a smaller proportion (16% extension; Figure 5 A), with the mutant's still living longer than the wild-type. Furthermore, when fed dead bacteria, the speed of daf-2 mutant locomotion still declined more slowly than that of the wild-type fed dead bacteria ( Figure 5 B). These two results show that daf-2 mutants model aging/longevity per se and not simply increased immunity/stress resistance. The healthspan of daf-2(e1368) mutants was largely unaffected by dead bacteria. There was a slight decrease in the fraction of live daf-2(e1368) mutant animals exhibiting movement at a given proportion of lifespan in response to dead bacteria (albeit a much smaller one than in the wild-type; Figure 5 C), consistent with the modest lifespan extension achieved by dead bacteria in daf-2(e1368) mutants being due to the elimination of residual susceptibility of these mutants to colonization. Together, these findings provide an explanation for the disproportionately extended period of decrepitude in daf-2 mutants. Moreover, they indicate that daf-2 mutants are not intrinsically unhealthy; instead, they survive a wave of mortality that kills the wild-type, which allows their subsequent manifestation of decrepitude, likely as an extension of the aging process.

Zhang et al., 2016 Zhang W.B.

Sinha D.B.

Pittman W.E.

Hvatum E.

Stroustrup N.

Pincus Z. Extended Twilight among Isogenic C. elegans Causes a Disproportionate Scaling between Lifespan and Health. Sánchez-Blanco and Kim, 2011 Sánchez-Blanco A.

Kim S.K. Variable pathogenicity determines individual lifespan in Caenorhabditis elegans. Interestingly, a recent study that looked at the variability of individual healthspans within a wild-type C. elegans population () found that individuals that die early die looking relatively youthful, whereas individuals that live longer spend a greater proportion of life in a state of decline. Both colonization severity ( Figure 4 B) and pathogen resistance () vary substantially in wild-type animals (likely because of stochastic processes). Therefore, this finding about the healthspans of individual wild-type animals is consistent with greater pathogen resistance's allowing some worms to survive long enough to enter decrepitude.

Gusarov et al., 2013 Gusarov I.

Gautier L.

Smolentseva O.

Shamovsky I.

Eremina S.

Mironov A.

Nudler E. Bacterial nitric oxide extends the lifespan of C. elegans. We also examined bacterial colonization of the daf-2(e1370) mutant, which is longer-lived than daf-2(e1368). This mutant had an even greater delay and reduction in the maximal level of bacterial colonization. Even very late in life (day 55), GFP-OP50 bacteria were barely detectable in these animals ( Figures S5 A–S5C). Feeding dead bacteria to daf-2(e1370) mutants did not further extend their lifespan but, rather unexpectedly, shortened it slightly (by 10%) ( Figure S5 D). We do not know the basis for this phenomenon. One possible explanation is that there may be a beneficial role of live bacteria in lifespan extension (for example, bacteria could provide an essential signaling molecule to old animals), but this beneficial effect is normally preceded by the detrimental effect of the bacteria's establishing colonization and killing susceptible animals. An example of a bacterially derived pro-longevity molecule is nitric oxide, which is produced by B. subtilis () (note, however, that this molecule is not produced by OP50 E.coli). The daf-2(e1370) mutant is highly resistant to bacterial colonization (more so than is daf-2(e1368)), so death associated with colonization is not a hazard to these animals, allowing the beneficial effect of live bacteria to become limiting to lifespan. In other words, to be extremely long-lived (like daf-2(e1370) mutants), C. elegans may need live bacteria, but only if they do not colonize and kill them prematurely. Consistent with this idea, feeding live but less pathogenic B. subtilis did not shorten the lifespan of daf-2(e1370) mutants ( Figure S4 A). It should also be noted that, although dead bacteria shortened the lifespan of daf-2(e1370) mutants, their lifespan was still longer than the lifespan of wild-type or daf-2(e1368) animals fed either live or dead bacteria ( Figure S5 D).

In summary, we find that the level of bacterial colonization predicts wild-type lifespan. The extent of colonization is significantly greater in the wild-type than in daf-2 mutants, and eliminating colonization in wild-type animals allows them to avoid an early death; instead, they remain alive for a longer time in a decrepit, aged state, just like daf-2 mutants. Therefore, we conclude that a beneficial trait (resistance to bacterial colonization) can explain the extended end-of-life frailty of daf-2 mutants. Surviving the hazard from bacterial colonization allows these mutants to grow biologically older and more decrepit than end-of-life wild-type animals. Importantly, when colonization is eliminated as a risk factor for death (i.e., when daf-2(e1368) and wild-type worms are both fed dead bacteria), daf-2 mutants still exhibit a gain in the number of “healthy” days, but they do not exhibit a disproportionately long period of decrepitude relative to the wild-type because extended end-of-life decrepitude can now be manifested in the wild-type as well. This leads to a situation in which daf-2(e1368) mutants appear at least as, or more, vigorous than the wild-type at every day and percentile of life ( Figure 5 B; Figure S4 G).