Detlef Weigel

eLife Assessment
This useful study reports on lifespan extension in C. elegans males that carry a mutation in a gene for an insulin receptor; while the observations are striking, the strength of evidence is currently incomplete. The central claim of male-specificity is undermined by the absence of direct hermaphrodite healthspan comparisons, and the reliance on a single mutant allele leaves open the possibility that background mutations, rather than daf-2 loss-of-function, drive the phenotype. Methodological details critical for reproducibility are also lacking, particularly regarding male housing density, censoring of plate-leaving animals, and the adequacy of replication for the key epistasis experiment. The work could be substantially strengthened by a targeted set of additional experiments and fuller engagement with the existing literature on sex-specific aging in C. elegans.

anonymous

Reviewer #1 (Public review):
Summary:
This paper provides interesting observations about the effects of a classical mutation in the daf-2 insulin-like receptor in male C. elegans. The observations are a contribution in and of themselves; however, the conclusions reached about these observations are not supported by the work presented. Most importantly, male-specific effects on healthspan measures are asserted without direct comparison to hermaphrodites. Perhaps more fundamentally, essential features of the methods and experimental design are lacking, which makes formal assessment of the results impossible, especially given our knowledge of negative male-male interactions, which have gone completely unacknowledged here. Indeed, there is a general lack of context for known sex differences in C. elegans, especially in terms of the core elements of longevity, which are presented here as entirely novel but in fact are not.
Major comments:
(1) The main overall criticism of the premise of the paper is that it lacks a clear hypothesis that would lead to explicit experimental tests. Instead, many of the results are observational, and the conclusions reached go beyond the actual experiments conducted. The goal should be explicit and consistent between the introduction/ discussion, and the findings should directly address the goal.
The overall focus appears to be that daf-2 males have an extended lifespan for reasons that are different from hermaphrodites. This conclusion is apparently based on the observation of lipid reserves in mutant animals. However, none of the healthspan measures were conducted in parallel with identical measures in hermaphrodites. How can the authors then claim that males are unique? This is especially problematic since other studies have demonstrated that daf-2 hermaphrodites also have altered lipid composition (Vrablik 2015 Biochim Biophys Acta; Horikawa 2010 Mol Cell Endocrin).
(2) The authors make unwarranted claims about causation from observational data that is correlative in nature. Again, they claim that male longevity is caused by increased lipid reserves. This may in fact be the case, but there is no evidence to show that this is causal, only that lipid reserves are increased in mutant animals. Causation requires an actual experiment, in this case, disrupting lipid maintenance in daf-2 males (e.g., Lapierre 2013 Autophagy). Their conclusions are consistent with their results, but their conclusions are much too strong given the nature of the evidence, especially given the concerns about proper comparisons to hermaphrodites.
(3) With these concerns in mind, all conclusions related to male-specific effects should be statistically tested using a sex-by-treatment interaction term in the statistical model. This is obviously impossible for the healthspan data, but for lifespan, this can be directly tested using (genotype x sex interaction in the CPH analysis). Further, it is unclear why each of the replicates is shown separately in Figure 1.
It is nice that the authors do not directly pool them, as most longevity studies do, but the replicate effects can be included in a more comprehensive model, which would yield an appropriate "average" effect curve.
(4) There is an inadequate review of pre-existing literature and findings that predate the observations presented here. While this is not an issue in general, the authors present their work as entirely novel when it is not.
In addition to Gems and Riddle (2000), which is tangentially cited in the discussion, the following papers should be cited and discussed in the introduction to clarify what is currently known and what remains to be explored:
Partridge and Gems (2002) Mechanisms of aging: public or private?
McCulloch and Gems (2007) Sex‐specific Effects of the DAF‐12 Steroid Receptor on Aging in Caenorhabditis elegans
Hotzi et al (2018) Sex‐specific regulation of aging in Caenorhabditis elegans
Al-Saadi et al (2025) Disruption of the insulin signaling pathway in C. elegans dramatically increases male longevity and enhances reproductive health late in life
In addition, the authors assert that the study of sex differences is unstudied. If the authors are specifically referring to the sex differences in aging research, they should explicitly state that and revise their language to reflect that it is "understudied" rather than "unstudied". But as stated below, there are many studies that look at sex-specific differences in behavior, physiology, development, etc. This is most important in the context of sexual conflict, of which there are many studies that are directly relevant to the work presented here. The authors are encouraged to review some of these papers.
(5) This is particularly important in the context of how the experiments presented here were actually conducted. The methods are inadequate to assess this, and the results would therefore be impossible to replicate in the absence of additional details. Exactly how many individuals were raised on each plate during the longevity assays (and other work) is critical to understanding the results of this study. This is because males have direct, chemically and physically mediated negative impacts on one another (see many papers from the Brunet and Murphy labs). Further, it is not even clear whether males and hermaphrodites were reared separately from one another. Males are known to leave plates without hermaphrodites, which requires appropriate inclusion of censoring criteria in studies such as these. It is unclear whether and how this was handled. Censoring is an essential feature of any longevity study and so needs to be explicitly described in the statistical methods.
The methods describe the use of heat shock to induce the production of males, but it is unclear which generation is being used here. Ordinarily, males would be induced, and then male populations would be maintained by forced mating (picking to ensure that there is a high relative frequency of males) for several generations to eliminate any carryover effects of the heatshock itself. Were the heatshock males put directly into the longevity assays? If so, were hermaphrodites subject to identical treatment? This is confusing, and a potentially critical confound is not performed correctly.

Reviewer #2 (Public review):
Summary:
The manuscript presents interesting observations regarding the exceptional longevity and improved healthspan of male daf-2 mutants. Given the comparatively limited focus on male aging in C. elegans, the study provides a potentially useful characterization of sex-specific effects associated with reduced IIS signaling.
Strengths:
The 4-fold increase in lifespan of male daf-2 mutants is a striking and unexpected observation. The altered fat metabolism between older daf-2 mutant males and hermaphrodites provides further evidence of sex-specific effects.
Weaknesses:
(1) A major limitation of the current study is that the conclusions rely primarily on a single daf-2 allele. It would strengthen the manuscript to validate at least the major observations using an independent daf-2 allele or through daf-2 RNAi. This is particularly relevant for the proposed male-specific enhancement of longevity and healthspan, as it remains unclear whether the observed effects broadly reflect reduced IIS signaling or may be influenced by allele-specific effects or background mutations.
(2) The methods for male lifespan assays require additional detail. Although the authors state that males were generated and transferred every three days, it is not clear whether males were maintained singly or in groups, how many males were placed per plate, or how many were censored by fleeing. These details are particularly important for male aging assays, as male lifespan in C. elegans is known to be influenced by social interactions. Factors such as population density can affect survival and healthspan measurements. Clarifying these procedures would improve reproducibility and interpretation of the reported male-specific lifespan effects.
(3) Because reduced IIS signaling in daf-2 mutants is known to alter metabolism, physiology, and potentially male-derived signaling, it would be interesting to determine whether the enhanced longevity of daf-2 males is influenced by altered male-male interactions or resistance to male-associated toxicity. In this context, clarification of whether lifespan assays were performed with grouped or individually maintained males would be valuable. If not already tested, lifespan analysis under isolated single-male conditions could help distinguish intrinsic longevity effects from potential contributions of population-dependent signaling or social interactions.
(4) In Figure 2D, the body-length measurements in WT males appear somewhat unexpected, particularly the apparent increase between Day 14 and Day 20. Since adult worms are not typically expected to exhibit substantial growth at advanced ages, additional clarification regarding the measurement methodology would be helpful, including confirmation that the scale bars and image scaling were applied consistently across conditions.
(5) The use of palmitic acid barriers following Beydoun et al. (2024) is appropriate; however, it would be helpful to clarify whether WT and daf-2 males exhibited comparable fleeing behavior under these assay conditions. Because male worms are highly prone to plate leaving and censoring, genotype-dependent differences in fleeing behavior could potentially influence survival analyses and the number of censored animals. In addition, as Beydoun et al. primarily characterized these barrier conditions using hermaphrodites, it would be useful to clarify whether comparable barrier effectiveness was observed in male lifespan assays.
(6) Separately, Beydoun et al. (2024) also reported that palmitic acid barrier conditions can influence body-size measurements, whereas PEG-based barriers did not show similar effects on body size. It would therefore be useful to know whether comparable body-length trends were observed under alternative barrier conditions, particularly given the unexpected increase in WT male body length at later ages.

Reviewer #3 (Public review):
This manuscript reports a striking sex-specific effect of the daf-2(e1370) mutation on C. elegans lifespan. The authors show that male daf-2 mutants exhibit dramatically extended lifespan relative to wild-type males, wild-type hermaphrodites, and daf-2 hermaphrodites. The study also demonstrates increased lipid accumulation in these long-lived males, which is increased further over time, improved late-life motility, enhanced oxidative stress resistance, and a requirement for the downstream effector of daf-2, daf-16, for the longevity phenotype.
The interest of the work is the magnitude and consistency of the lifespan effect. The authors report large increases in both median and mean lifespan in daf-2 males across independently replicated experiments. This is further supported by healthspan analyses and their finding that male daf-2 mutants maintain improved motility and stress resistance, which argues against the interpretation that lifespan extension merely reflects prolonged frailty. The genetic epistasis experiment demonstrating loss of the longevity phenotype in daf-2;daf-16 double mutants provides evidence that the effect depends on canonical insulin/IGF-1 signalling.
The main limitation is that at least the first figure is rather an incremental increase on previous work examining the lifespan of daf-2 males, although the authors do indeed show that the effects can be much larger (or more 'plastic') than those previously published. While these findings are potentially important, the manuscript would certainly benefit from a more extensive discussion of how the results compare with prior studies of daf-2 mutants and male longevity, including possible explanations for the apparent discrepancies.
The epistasis experiment shows that this exceptional longevity requires the expression of daf-16. However, in contrast to the initial experiments (Figure 1) that show three replicates of the lifespan experiment (the standard in lifespan work in this model), it appears that the daf-2;daf-16 experiment has only been performed once.
In addition, the lifespan data for hermaphrodite daf-2 mutants appear somewhat unusual. Although the mean lifespan is increased, the median lifespan is reported to be only modestly greater than that of wild-type hermaphrodites. I know that this mutant can give lifespan curves that look like this, but either the use of another allele or of the experimental conditions and how these values compare with previously published daf-2(e1370) datasets would help readers interpret the magnitude of the male-specific effect.
The lipid phenotype is intriguing. It would be interesting to expand this to examine somatic vs embryonic fat. In addition, I noted that in the methods section, the authors use palmitic acid to stop the male worms 'fleeing' the plates; is it possible to rule out the possibility that the daf-2 mutants are simply eating and metabolising/storing this fatty acid barrier differently than their wild-type counterparts? This would be worth considering and controlling for, particularly as male C. elegans have been shown to have dramatically altered metabolic transcriptional profiles. If indeed this increased lipid is responsible for the extreme longevity of the daf-2 mutant males, it would be desirable to try to link this mechanistically to the phenotype.
Overall, the evidence convincingly supports the conclusion that male daf-2(e1370) mutants are exceptionally long-lived under the conditions tested and that this phenotype requires DAF-16. The work has the potential to make an important contribution to understanding sex-specific regulation of ageing, although further contextualisation within the existing literature would strengthen the manuscript.

Fourfold lifespan extension in C. elegans daf-2 mutant males

Figures and data

Survival statistics for the aggregated lifespan data from Figure 1.

Loss of daf-2 extends male lifespan fourfold.

Long-lived daf-2 mutant males accumulate and retain lipids in age.

Long-lived daf-2 mutant males retain motility and resist oxidative stress.

Functional daf-16 is required for the extreme daf-2 mutant male lifespan.

Survival statistics for the lifespan data presented in Figure 4.

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