Developmental phenotypes and reproducibility

We chose 22 Caenorhabditis natural isolates spanning three species (C. briggsae, C. elegans and C. tropicalis) to maximize sampling of genetic and geographic diversity within each species27,28. In terms of total divergence, the genetic differences encompassed among these strains is comparable to sampling genomes from mice to humans27. We first tested whether the three independent labs could reproducibly score two major life history traits; the developmental time to reproductive adulthood (egg laid to adult deposition of the first egg), referred to as α-time and hermaphrodite self-fertility (total number of viable progeny). These studies also served to inform on the general health of the natural variant strains under the culture conditions we established to test compound-based ageing interventions.

We determined the α-time for the 22 strains (>1,800 observations; see Supplementary Data 1 and 6), finding that the developmental rate at 20 °C is slightly delayed in C. tropicalis strains relative to C. briggsae and C. elegans, with good agreement among labs (Fig. 1a). Indeed, partitioning variation among potential sources of error using a general linear model (GLM) indicated that there was very little systematic difference among outcomes at the three laboratories (Table 1 and Supplementary Table 1), although there were some lab-specific differences among species and strains (roughly 4% of the total variance attributable to each source). The key finding was that the vast amount of variation (79%) was attributed to genetic variation among strains and species. These data suggest that growth conditions and practices for developmental time analysis were uniform across labs.

Figure 1: Summary of developmental time and fertility of 22 Caenorhabditis strains. (a,b) Graphical representation of the mean developmental time (a) and mean fertility (b) for 22 Caenorhabditis strains under the test culture conditions (see Methods). Each point represents the average of 20 individual animals, scored in one of the three CITP labs. Middle bar represents the mean with smaller bars indicating the s.e. Graphs are segregated by species such that eight C. briggsae strains are shown in grey, eight C. elegans strains are shown in black and six C. tropicalis strains are shown in off white. Statistical summaries of the parent data used to generate these graphs is presented in Table 1 and Supplementary Table 1, with the per-replicate estimates and sample sizes provided in Supplementary Data 1 and 2. Full size image

Table 1 Variation attributable to different sources. Full size table

We also assayed hermaphrodite self-fertility under our culture conditions. Each of the species used here reproduces as self-fertilizing hermaphrodites and are limited by sperm production during development. The self-fertility of each strain was determined by counting the number of viable progeny born from individual animals. In general the C. elegans strains exhibited the highest fertility, with strain QX1211 the clear outlier (Fig. 1b). QX1211 exhibited lower fertility than any other strain and was observed to lay many eggs that failed to hatch (unquantified observations), indicating some level of embryonic lethality. As with the developmental rate results, we found that self-fertility scores were reproducible among labs (1% variance among labs), with slightly larger lab-specific differences among strains than observed for α-time (Table 1). Variation in fertility under our lab conditions can be assigned primarily to genetic background (63%) and ‘random’ (unexplained) differences among individual animals (23%). Importantly, the data again support little systematic difference in scores (and therefore in culture conditions) across the three CITP laboratories.

Some Caenorhabditis strain differences are due to genetics

We cultured each of the 22 natural isolates under standardized conditions and scored survival, with each trial being initiated with ≥105 adults distributed over three technical replicate plates. All CITP sites collected data from three biological replicate trials (21,143 observations; experimental details in Methods section and Supplementary Data 3 and 8). We observed broad differences in longevity among test strains spanning a twofold range at 20 °C (Fig. 2). We found strong, repeatable differences in the average mortality dynamics among the three species, as well as among strains within species (Fig. 2). In general, both C. briggsae and C. tropicalis live longer than C. elegans. However, whereas both C. briggsae and C. tropicalis have reduced early life mortality relative to C. elegans, C. briggsae continues a pattern of reduced mortality throughout life; the mortality rate in C. tropicalis tends to increase late in life such that its maximum lifespan ends up being fairly comparable to C. elegans. Natural isolates within each species also differed from one another in terms of their lifespan, with differences in median lifespan generally ranging from 3 to 7 days within each species (Fig. 2 and Supplementary Fig. 1). The clear outlier is C. briggsae strain HK104, whose median lifespan is 30% longer than any other Caenorhabditis natural isolate in our analysis, including other C. briggsae strains. Genetic differences account for roughly 20% of the total variation in longevity observed (12% among species and 8% among strains within species; Table 2).

Figure 2: Natural and experimental variation in longevity among natural isolates of Caenorhabditis. (a) The set of survivorship curves displaying the total range of observed longevity for each of the 728 experimental replicates (plates) from three laboratories measured across the three species and 22 natural isolates measured in this study. The cause of plate-to-plate differences in responses can be attributed to different sources using a hierarchical analysis that partitions the total observed variation to known sources of genetic differences and replication error. Overall, the average longevity across the entire experiment did not differ across the three laboratories (b), although there were species- and strain-specific responses that varied from lab to lab (c). There were also distinct differences among species (d), but in fact more variation among strains within species (e). Relative percentages of the total variation attributable to each source are given in Table 2. Orange lines are C. elegans, tan are C. briggsae and purple are C. tropicalis. Dashed lines (blue) are replicates from the Buck Institute, solid lines (green) are from Oregon and dotted lines (red) are from Rutgers. Sample sizes and per-replicate estimates for means and medians are provided in Supplementary Data 3. Full size image

Table 2 Reproducibility of longevity estimates within and between labs. Full size table

Minimal among-lab variation in lifespan

Given that a critical component of the CITP plan is to reproduce findings among labs, we examined reproducibility of longevity data at three primary levels: variability in outcomes among labs, variability of outcomes among replicates within labs and sensitivity of outcomes to genetic variation within and among species. Partitioning variation in longevity to different potential sources of variation in a hierarchical manner using a GLM (see Methods), we determined that reproducibility for longevity measurements across the three laboratories is extremely high, with on average there being no differences at all among labs (Fig. 2, Table 2 and Supplementary Table 2). Although there were effectively no systematic differences among laboratories, there is some indication of heterogeneous, strain-specific differences among laboratories, with laboratory-specific species and laboratory-specific strain results, respectively, accounting for <1% and 7% of the total variation. As the variation among laboratories was minor relative to other sources of variation, we infer that our strict adherence to uniform procedures was successful in largely eliminating systematic differences in lifespan outcomes among labs.

Replicate variation within each laboratory is relatively high

Interestingly, although we found that systematic differences among labs were minor, we calculated replicate-to-replicate variation within each lab to be relatively high. After accounting for other sources of variation, strong among-replicate differences remained, representing roughly 15% of the total variation in individual lifespan observed in our study (9% derived from the trial-specific effects, 6% from the among-plate in same trial differences and none from experimenter-specific differences). Thus, although the results obtained on any given day of a replicate trial tended to be fairly consistent with one another, conducting the same assay a month later could yield results as different as looking at a strain from a different species.

Given that we observe a relatively large amount of variation among trials across each of the three labs, despite strict adherence to standardized procedures and culture conditions, we conclude that a major challenge to reproducibility in this system may arise from trial-specific cohort responses to unidentified and apparently subtle differences in the assay environment, which vary similarly within each laboratory.

Bimodal ageing for C. briggsae replicates

The observed among-trial variation could simply be a random byproduct of tracking a phenotype (longevity) that is unlikely to be under tight regulatory control. Indeed, the large amount of residual variation in longevity (57%) is consistent with longevity being an inherently variable trait. However, the strikingly discrete nature of among-replicate variation for some lines, especially within C. briggsae strains, suggests that fundamental biology may underlie the observed trial-specific differences in lifespan outcomes (Fig. 3). For example, strain JU1264 exhibited distinct clusters of longevity trajectories across the different trials: cohorts either showed high early mortality or long life (Fig. 3). The absence of intermediate outcomes suggests that even populations reared under tightly controlled conditions may shift between discrete physiological states, perhaps induced by some unmeasured/unknown environmental factor. The propensity of a given strain to display distinct longevity trajectories varied from lab to lab, although all labs observed this phenomenon for one strain or another. C. elegans and C. tropicalis also displayed some degree of strain-specific differences of among-trial variation, although distinct differences in longevity trajectories are less obvious in these species than in C. briggsae (Supplementary Figs 2 and 3). If this state-shift is a general feature of this system, then reproducibility ‘error’ at this level might actually reflect an inherent property of these species that cannot be eliminated without further knowledge of its root causes. At present, our controlled studies suggest that investigator, site, plate or reagent batch, overall temperature and humidity, and generational epigenetic factors associated with food availability are not factors in bimodal outcome; likewise, strains that generate males with highest frequency did not uniformly show this bimodal response. Our observations thus introduce an unexpected area for mechanistic investigation.

Figure 3: Variation in longevity within labs for each replicate plate for eight natural isolates of C. briggsae. It is noteworthy that in many cases a given natural isolate tends to display distinct patterns of responses under identical laboratory conditions rather than a continuous distribution of ‘error’ among replicates. Among-replicate variation within each lab was a much larger barrier to reproducibility than variation in the average response of a strain across labs (Table 2). Each plate was initiated with n=35 animals. Full size image

Analysis of variation in intervention lifespan outcomes

Our initial characterizations identified a twofold range in median lifespan represented among the strains, which provides a strong substrate of diversity for testing generalized effects of compound interventions. To keep the experimental design logistically tractable for our initial test set of ten compounds, we focused on outcomes with three C. elegans strains and three C. briggsae strains that had fared well under lab growth conditions (46,231 observations; experimental details in Methods and Supplementary Data 4 and 9). The strains we selected captured the range of longevities that we had observed in our initial broad survey. For the first intervention studies we selected ten chemicals that had either attracted particular interest in the ageing field (aspirin29 and resveratrol21), were previously reported to extend the lifespan of the C. elegans strain N2 (α-ketoglutarate (α-KG)30, curcumin31, α-lipoic acid (α-LA)32, propyl gallate (PG)32, quercetin33,34 and valproic acid (VA)35) and/or appeared particularly robust when given to N2 in our own laboratories ((NP1)36 and ThioflavinT (ThT)31).

Another consideration for the initial test set was that the compounds had been predicted to influence ageing by a range of primary mechanisms. For example, ThT promotes protein homeostasis31 and VA extends lifespan via activation of the transcription factor DAF-16 (ref. 35), whereas NP1, α-KG and resveratrol had all been reported to act as dietary restriction (DR) mimetics. To begin to assess variability in lifespan assays involving chemical treatments, we first tested a single concentration of each compound. In general, we chose concentrations previously reported to extend median lifespan in the C. elegans N2 strain.

We identified a similar pattern of variation in the compound trials as we reported above for the baseline, non-intervention studies (Table 2 and Supplementary Tables 2–3). That is to say, partitioning variation among potential sources of error using a GLM again established that little of the observed variation was attributed to differences among labs and minor variation due to combinations of lab-species, lab-strain and lab-compound effects (0.1–0.5% of total variation attributed to each). In contrast, we attributed 9.7% of the total variation to reproducibility within each lab. For interventions however, this among-trial variation was primarily associated with plate-to-plate differences (possibly due to application of the compounds to the plates, which is executed on a per plate basis). Genetic differences and individual differences among animals accounted for most of the variation in the intervention studies (∼44% each; Table 2). Genetic differences were proportionally more important for the compound interventions than in the baseline assays, probably because we intentionally sampled strains covering a wide range of baseline longevities and, as will be seen, treatment by many compounds tends to heighten differences among strains. Overall, then, although the three labs were able to create a high degree of reproducibility across all longevity assays, most replication ‘error’ appears rooted in trial-to-trial differences that impact the three lab outcomes similarly. Collectively, our results indicate that for both chemical and baseline lifespan assays, it is critical to assess the lifespans of discrete cohorts in repeat studies, to capture the expected biological variation.

ThT extended lifespan in a broad range of strains

Having addressed the overall reproducibility in our experiments, we next examined compound-specific effects on lifespan extension. We identified several compounds that had positive effects on lifespan. However, depending on the compound, these effects appeared to be differentially influenced by genetic background (Supplementary Fig. 4). ThT was the most robust of the chemicals we tested, as it significantly and reproducibly extended lifespan in five of the six strains tested, with only JU1348 failing to respond to treatment with significant lifespan extension (Fig. 4a, Supplementary Fig. 5 and Supplementary Table 4). In addition to being the most robust of the treatments, ThT also showed the most potent effect. We found that in some trials, for certain strains, ThT-treated populations exhibited a doubling of the median lifespan relative to control-treated populations (Fig. 4a and Supplementary Fig. 5). The average median lifespan (across all the trials for a given strain) ranged from a small but reproducible effect on HK104, the long-lived C. briggsae strain, to a significantly large effect (70% extension of median lifespan) on C. elegans strain MY16 (Fig. 4a). The potent and robust longevity promoting effect that ThT exhibited on these Caenorhabditis strains suggests that the molecular mechanism targeted by ThT is a major determinant of lifespan that is conserved across divergent genetic backgrounds.

Figure 4: Chemical effect on the median lifespan of six Caenorhabditis strains. (a–j) The effect on median lifespan from ten different chemical treatments is shown for six Caenorhabditis strains. The six strains consist of three C. elegans species (N2, MY16 and JU775, black text) and three C. briggsae species (JU1348, AF16 and HK104, grey). The per cent difference in median lifespan was determined by calculating the median lifespan for each plate population (single plate lifespan assays starting with 35–40 animals, each site at least 6 plates in at least 2 biological replicates). Every chemical test plate had a control plate associated with it (diluent only control plate, that was maintained with the test plate), which contained animals from the same egg lay and was always scored by the same technician as the test plate. In all graphs each point represents the percent difference in median lifespan between two single plate populations, one containing the chemical being tested and the other containing the diluent control. Data incorporate censored animals in calculating median lifespan. Points are colour coded to indicate the lab the data was collected in, as indicated in key on a. Middle bar represents the mean with small bars, indicating the s.e. Asterisks represent P-values from the CPH model (Supplementary Table 4; ****P<0.0001, ***P<0.001, **P<0.01 and *P<0.05). Summaries from the statistical analysis of the parent data used to generate these graphs are included in Table 2 and Supplementary Tables 2 and 4. Sample sizes and per-replicate estimates for means and medians are provided in Supplementary Data 4. Full size image

DR mimetics exhibited similar strain specific responses

NP1 is a synthetic compound, which we previously identified as promoting lifespan in the N2 strain through a DR mechanism36. In this study, we found that NP1 exhibited species- and strain-specific effects on lifespan (Fig. 4b and Supplementary Fig. 6). The C. elegans strains all showed significantly extended lifespan when treated with NP1, relative to control-treated animals (Fig. 4b, Supplementary Fig. 6 and Supplementary Table 4). On average, NP1 caused a remarkably consistent and fairly potent effect on lifespan across these strains (∼30% longer median lifespan relative to control treated populations), with the most reproducible effects (among the strain specific replicates) observed in the N2 strain and the most potent effects (across all strain replicates) observed in the wild isolated strains (MY16 and JU775; Fig. 4b). The effect of NP1 on the lifespan of the C. briggsae strains was more varied. NP1 did not have a significant effect on lifespan in either AF16 or JU1348, but it consistently and fairly potently shortened the lifespan of strain HK104 (∼30% shorter median lifespan on average compared with control-treated populations; Fig. 4b, Supplementary Figs 4 and 6, and Supplementary Table 4).

Similar to the effects observed from NP1 treatment, the metabolite α-KG, which was also previously implicated in DR30, significantly extended the lifespan of all the C. elegans strains tested and did not have a significant effect on the lifespan of C. briggsae strains AF16 and JU1348, but shortened the lifespan of strain HK104 (Fig. 4c, Supplementary Figs 4 and 7, and Supplementary Table 4). Although the trends observed were the same for both chemicals, the magnitude of the effect on lifespan from treatment with these chemicals was distinctly different in two of the strains. αKG potently extended the lifespan of the C. elegans strain MY16, with treated populations commonly living 60% longer than control treated animals, whereas its negative effect on the lifespan of strain HK104 was much weaker and less consistent than was observed for NP1 (Fig. 4c, Supplementary Figs 4–7 and Supplementary Table 4). Resveratrol has also been reported to promote DR (Fig. 4d and Supplementary Fig. 8)21. Although there has been considerable controversy surrounding resveratrol and its proposed mechanism of action, most investigators studying resveratrol effects on C. elegans lifespan have documented small but significant beneficial effects on the laboratory standard N2 strain19,37. We also observed this effect and further found that similar to NP1 and α-KG, resveratrol also significantly extended the lifespan of the other C. elegans strains tested. However, unlike NP1 and α-KG, we did not observe any overall significant lifespan effect on any of the C. briggsae strains treated with resveratrol compared to control treated populations (Fig. 4d, Supplementary Figs 4 and 8, and Supplementary Table 4). Overall, our work with candidate DR mimetics suggests that there are promising benefits from such pharmacological interventions, but at the same time our data seem to indicate that these chemicals tend to exhibit highly variable outcomes, dependent on the genetic background of the treated subjects.

Compound that did not reproduce previous longevity outcomes

In previous studies, three compounds that can exhibit antioxidant properties (PG, α-LA and quercetin) extended the lifespan of the standard laboratory strain N2 (refs 32, 33). We found that treatment with PG significantly, but relatively weakly, extended the lifespan of diverse C. elegans strains relative to control-treated populations. However, we did not observe significant effects on the longevity of the C. briggsae strains from treatment with PG (Fig. 4e and Supplementary Table 4). Compared with the other treatments we assessed, this profile appears most similar to the pattern observed in the resveratrol treatments. α-LA-treated populations of the N2 strain exhibited a small but significant lifespan extension relative to control-treated populations. However, none of the wild strains exhibited a significant lifespan response to α-LA (Fig. 4f and Supplementary Table 4). Quercetin did not significantly extend the lifespan of any strain tested (Fig. 4g and Supplementary Table 4). Overall, there was little evidence that compounds with antioxidant properties extended lifespan using the test conditions described here, which might reflect the in vivo pleiotropy of reactive oxygen species, which have been shown to act in pro-longevity signalling cascades, in addition to exhibiting well-known negative impacts on cellular systems38.

In our study, anticonvulsant drug VA (Fig. 4h) failed to extend lifespan in any strain, an initial surprise, because VA has been previously documented to extend lifespan of C. elegans strain N2 in multiple replicate experiments35. Comparison of the CITP treatment strategy with the published report suggests that differences in outcomes may be due to use of distinctly different treatment protocols (for example, we treated only during adulthood, whereas Evason et al.35 treated the animals from conception). Similarly, under our test conditions, aspirin and curcumin (Fig. 4i,j) did not exert robust positive changes on longevity across the test set. These differences from previously published results may also reflect differences in test conditions.

Species-specific dosage responses

The most robust compounds from our primary test were quite potent in the tested C. elegans strains, yet other than ThT, these same compounds essentially failed to exert positive effects in the C. briggsae strains. One possibility we considered is that C. briggsae may exhibit distinctly different dosage sensitivity than C. elegans. This hypothesis seemed reasonable as the dosages tested were chosen based on published reports of effectiveness in the C. elegans strain N2. To further explore the chemical responsiveness of the C. briggsae strains that failed to respond, we tested a range of doses for the chemicals that were identified as positive in our initial tests. Dose–response experiments with ThT indicated that in fact ThT can have a positive impact on JU1348 but only at lower dosages than we originally examined (Fig. 5a). As in the other strains, the effective dose of ThT is relatively close to the minimum toxic dose (see Discussion). In general, ThT was found to be effective at extending lifespan across all genetic backgrounds tested.

Figure 5: Dosage effects on the lifespan C. briggsae with select positive chemicals. (a–c) Dose response effects on the median lifespan of select C. briggsae strains after treatment with chemicals that exhibited strong positive effects on the C. elegans strains. Dosing was performed only on strains that failed to respond positively in the initial tests (single dose experiments), as we did not attempt to identify peak responses, instead we only sought to identify whether positive effects could be obtained by altering doses. Chemical doses were chosen to center around the effective dose identified for C. elegans strains and were sometimes expanded after preliminary rounds of testing. ThT exhibited a positive effect on strain JU1348 at 25 μM, but was profoundly toxic to all strains at and above 100 μM (a). NP1 exhibited a positive effect on AF16 and JU1348 at 10 μM relative to control treated populations and showed reduced toxicity to HK104 at low micromolar concentrations (b). αKG did not exhibit clear positive effects in any of the C. briggsae strains tested, but exhibited toxicity at high millimolar concentrations (c). None of the resveratrol doses assayed appeared to alter the median lifespan of any of the C. briggsae strains tested (d). PG showed a negative effect on median lifespan for all of the C. briggsae strains tested at low millimolar concentrations (e). Median lifespans were determined from single plate populations. Mean values are plotted here with small bars, indicating the s.e. (sample size and statistical summaries are included in Supplementary Data 5). Full size image

In contrast, the reported DR mimetics (NP1, αKG and resveratrol), which each showed positive effects across the C. elegans strains, resulted in distinct responses from the briggsae species (Fig. 5b–d). For αKG and resveratrol, the three C. briggsae strains all showed similar overall dose-dependent responses. The C. briggsae strains treated with αKG did not clearly respond with lifespan extension at doses in the micromolar range but did exhibit shortened lifespan in the mid-millimolar range (Fig. 5b). In contrast, we did not detect any clear response from the C. briggsae strains treated with resveratrol (Fig. 5d). This lack of response may indicate that the chemical is unavailable to the animals, has low toxicity or that our dose range was too low to observe an effect. The upper limit of our dose testing for resveratrol was 1 mM, an order of magnitude higher than the dose that exhibited positive effects on the C. elegans strains. The C. briggsae strains treated with NP1 displayed more variable responses. Both AF16 and JU1348 showed some indication of a positive effect on lifespan from treatments with NP1 in the low micromolar range, whereas in this same range HK104 exhibited negative effects. The shortening of lifespan effect from NP1 became more pronounced at mid- to high-micromolar concentrations of NP1. We did not identify any dose of NP1 that clearly extended the lifespan of HK104. We also tested PG, which showed positive effects on the C. elegans strains, for dose-dependent effects on the C. briggsae strains. PG showed a similar profile as αKG, with no clear effect at low doses and negative effects observed at low millimolar levels (Fig. 5e). Collectively, these results suggest that the C. briggsae strains exhibit different dosage specificities than C. elegans and further that some of these chemicals have conserved effects, while others show species-specific and strain-specific effects.