Evolution Under Dietary Restriction Decouples Survival From Fecundity in Drosophila melanogaster Females

Evolution Under Dietary Restriction Decouples Survival From Fecundity in Drosophila melanogaster... Abstract One of the key tenets of life-history theory is that reproduction and survival are linked and that they trade-off with each other. When dietary resources are limited, reduced reproduction with a concomitant increase in survival is commonly observed. It is often hypothesized that this dietary restriction effect results from strategically reduced investment in reproduction in favor of somatic maintenance to survive starvation periods until resources become plentiful again. We used experimental evolution to test this “waiting-for-the-good-times” hypothesis, which predicts that selection under sustained dietary restriction will favor increased investment in reproduction at the cost of survival because “good-times” never come. We assayed fecundity and survival of female Drosophila melanogaster fruit flies that had evolved for 50 generations on three different diets varying in protein content—low (classic dietary restriction diet), standard, and high—in a full-factorial design. High-diet females evolved overall increased fecundity but showed reduced survival on low and standard diets. Low-diet females evolved reduced survival on low diet without corresponding increase in reproduction. In general, there was little correspondence between the evolution of survival and fecundity across all dietary regimes. Our results contradict the hypothesis that resource reallocation between fecundity and somatic maintenance underpins life span extension under dietary restriction. Drosophila melanogaster, Nutrition, Adaptation, DR, Experimental evolution Understanding the relationship between nutrition, reproduction, and survival, on the genetic and the phenotypic levels, is thought to be essential for health-span and life span extension (1). Research on genes involved in the modulation of these traits has revealed a network of nutrient and energy sensing signaling pathways that govern reproduction and survival (2), with substantial evolutionary conservation across the tree of life (3,4). Life span extending effects of dietary restriction (DR)—the most successful intervention to prolong life to date (3)—is a case of phenotypic plastic response that generally not only increases survival but also decreases reproduction (5). Evolutionary life-history theory and the antagonistic pleiotropy theory for the evolution of aging state that early- and late-life fitness components are generally trading off against each other and that these negative correlations between traits are genetically based (6,7). Within this framework, the plastic response to DR can be understood as the consequence of a shift in the energy trade-off between reproduction and survival. The disposable soma theory of aging is built around this theoretical conjecture (8), and it states that resource requirements for reproduction directly compete with those required for somatic maintenance and that this relationship should be observed both on the physiological level and on the genetic level (see the distinction between “physiological” and “genetic” [evolutionary] trade-off discussed in ref. (9)). Under the disposable soma theory, if the observed plasticity in this trade-off is adaptive, living longer and reproducing less under short-term DR (within an individual’s life span) should confer an evolutionary advantage (10,11) and can be understood as a short-term emergency solution to cope with nutritional stress (12). This prediction was tested in a formal life-history DR model parameterized using house mouse data by Shanley and Kirkwood (13), who found that under certain assumptions (ie, an extra cost before successful reproduction and lower juvenile survival under DR), the classic DR response can evolve (discussed in ref. (8)). Although there is suggestive evidence from a recent meta-analysis that DR might act differently on mortality rates in rodents, compared with D. melanogaster (ref. (14), but see ref. (15)), the main pathways leading to reduced aging seem to be evolutionary conserved between phyla (3). Nevertheless, one fundamental assumption of the disposable soma theory is that organisms can reallocate resources (mainly regarded in terms of energy units in this context) from reproduction to somatic maintenance and survival, and vice versa (16). While allocating more resources to survival, away from reproduction, is adaptive under short-term DR, this response should be maladaptive if resources are restricted permanently. If food shortage is permanent, spanning adult lifetimes over many generations, individuals that switch to a strategy of increased reproductive output at the cost of decreased survival will have a selective advantage. One way this could happen is when the ability to respond plastically to DR erodes over evolutionary time (ie, when the reaction norm for reproductive output across nutritional environments becomes less steep) or when either already segregating alleles or de novo mutations that confer higher reproductive output under DR are favored (ie, evolutionary adaptation). If a negative genetic correlation (evolutionary trade-off) between reproduction (especially during early life) and survival exists, as has often been observed (17–23), higher levels of reproductive output under DR (regardless if short-term and transient, or evolved) should at the same time decrease life span, to an extent that depends on the strength of the correlation. On the other hand, even if reproduction and life span are decoupled, we would still expect an increase in reproduction after sufficient numbers of generations under chronic DR, independent of a response in life span. In the present study, we test this prediction using experimental evolution in D. melanogaster, by manipulating adult dietary yeast levels and testing for an evolved response in female flies after approximately 50 generations. We previously found a response in male reproduction to this experimental evolution regime, with males evolved on DR having increased reproduction when tested on DR, standard or enriched diets, but no reduction in survival (24). Methods Experimental Design Experimental flies (D. melanogaster) originated from experimental evolution lines that evolved on three distinct diets with different yeast contents as adults (low diet [LD], standard diet [SD], high diet [HD]; specific diet characteristics are given in Supplementary Table S1). Flies in the experimental evolution lines were kept in four replicate mixed-sex population cages per diet treatment, containing 150 adult males and 150 adult females each. All larvae were reared on SD, and only adults were exposed to the experimental evolution diets in the population cages. More specific details on the experimental setup of the lines can be found in Zajitschek and colleagues (24). In short, our experimental flies were derived from Dahomey, a large outbred laboratory population that originally was sampled in 1970 from the wild in Benin, West Africa. Ever since the population has been maintained in mixed-sex population cages with overlapping generations under constant environmental conditions (25°C, 60% humidity, 12:12 light–dark cycle, on standard yeast–sugar diet). Recent studies on this population showed that it hosts substantial levels of genetic variation for life span (25,26). We tested for an evolutionary response in females after approximately 50 generations of experimental evolution. Sample sizes are given in Supplementary Table S2. To remove any parental effects from the diet treatments before the start of the experiment, experimental flies were passed through two generations of common garden. To accomplish this, females from the experimental population cages were allowed to lay eggs in wide plastic vials (28.5 mm × 95 mm used for all experimental work) with SD overnight. Eggs were trimmed to 100 eggs per vial, and eclosing adults were allowed to mate for the 2 days after eclosion before females were allowed to lay eggs in new SD vials for 2 hours. Eggs were again trimmed to 100 eggs per vial and eclosing adult females were used in assays. Each vial was populated with around 50 female flies. Assay flies were provided with one of the three experimental evolution diets, with two replicate vials per cage and evolution diet × assay diet combination (total number of individual females per evolution diet × assay diet treatment: N = 400). For weekly matings, females of each vial were transferred to new SD vials and given the matching number of 2-day-old males that were bred in a separate stock sourced from the same population cage, once every week for 12 hours. Eggs laid during this period were counted. Total fecundity was calculated by summing eggs laid over all vials and weeks. Survival was checked every Monday, Wednesday, and Friday until all flies had died. We measured dry adult body mass of groups of 10 individual female flies and replicated 10 times per cage per evolutionary diet treatment (N = 400 per treatment). Prior to weighing, all flies were raised for two generations on SD medium, as described earlier. Statistical Analysis To analyze survival, we used mixed Cox proportional hazard models (function coxme, R package coxme) (27). As the interaction term between assay diet and evolution diet was significant in a global analysis (χ2 = 104.63, degrees of freedom [df] = 4, p < .001), we performed (a) post hoc analyses for assay diet effects within evolution diet groups, using Tukey’s Honest Significant Difference method to adjust for multiple testing (function glht in R package multcomp) (28) and (b) separate analyses for each assay diet, with evolution diet as a fixed effect and experimental vial and population cage fitted as a random intercept. Models containing evolution diet were compared with models that only contained an intercept, using log-likelihood ratio tests, with twice the difference in log-likelihoods of the models taken as chi-square distributed, and a .05 significance level. Untransformed life span and body mass were tested in linear mixed models (using maximum likelihood estimation), after testing residuals for normal distribution, with the same random effects as specified for Cox proportional hazards analyses (using function lmer in R package lme4) (29). We used the R package lmerTest to calculate p values for linear mixed models, with df based on the Satterthwaite approximation (30), and performed post hoc analyses as described earlier. To test for differences in hazard rates, we fitted exponential and Gompertz models, using Bayesian methods implemented in the R package BaSTA (31). The exponential model assumes a constant mortality rate at all ages, whereas the Gompertz model assumes an increase in mortality rate at later ages (ie, aging):  μx=b0eb1x with instantaneous mortality rate (hazard rate) at age x is given by µx, parameter b0 is the intercept and is interpreted as the initial or baseline mortality rate, parameter b1 is the increase of mortality rate with advancing age (the aging parameter). We compared exponential and Gompertz model fits using the deviance information criterion (32). For all reported analyses, diet was treated as a categorical variable. Life span summary statistics and sample sizes are given in Supplementary Table S2, and median life span is plotted in Supplementary Figure S3. Female reproductive fitness was estimated as the sum of all weekly fecundity measurements of each population of females in a vial, scaled by the initial number of females in a vial. Total fecundity was analyzed in linear mixed effects models following the same process as in the analysis for survival and life span, with population cage fitted as a random intercept. To specifically compare early-, mid-, and late-life fecundity, we also tested effects on mean fecundity in age classes (early-life fecundity = fecundity in week 1, mid-life fecundity = fecundity in weeks 2 and 3, late-life fecundity = fecundity in week 4 and later). Post hoc tests were conducted using function difflsmeans in R package lmerTest. Effects of evolution diet on age-dependent fecundity trajectories across life span were tested in general additive mixed models to account for nonlinear relationships, with vial fitted as a random effect, and correcting for initial number of females in a vial by including it as a fixed effect. We used a tensor product smooth function of age at measuring fecundity (weekly), and thin plate regression splines. Effects of evolution diet within assay diet were tested by comparing a model fitting separate curves to evolution diet groups, with a model without accounting for evolution diet, using Akaike’s information criterion (AIC). All models were fitted and predicted trajectories visualized in R package mgcv (33). All analyses were run in the software R, version 3.3.1 or higher (34). Results Survival We report effects of long-term experimental evolution under low, standard, and high yeast adult diets, on survival and reproduction of D. melanogaster females that were mated once every week. In contrast to male flies that were tested previously (24), female survival responded to the experimental evolution regimes. The effect of assay diet on survival rates and mean life span was dependent on evolution diet (survival: χ2 = 104.63, df = 4, p < .001; life span: F4,2941 = 21.20, p < .001; Figures 1 and 2). Figure 1. View largeDownload slide Female fruit fly mean life span. Each graph shows mean life span for AD groups. Error bars show ±2 SE. Asterisks indicate the statistical significance of differences between groups: ***p < .001. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Figure 1. View largeDownload slide Female fruit fly mean life span. Each graph shows mean life span for AD groups. Error bars show ±2 SE. Asterisks indicate the statistical significance of differences between groups: ***p < .001. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Figure 2. View largeDownload slide Female fruit fly survivorship. Each panel shows Kaplan–Meier survival curves for AD treatment groups. Separate curves depict survivorship of ED populations, tested on different ADs. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Figure 2. View largeDownload slide Female fruit fly survivorship. Each panel shows Kaplan–Meier survival curves for AD treatment groups. Separate curves depict survivorship of ED populations, tested on different ADs. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Evolution diet regime affected survival and life span when tested on LD (survival: χ2 = 110.89, df = 2, p < .001; life span: χ2 = 131.57, df = 2, p < .001) and SD (survival: χ2 = 32.15, df = 2, p < .001; life span: χ2 = 43.93, df = 2, p < .001), but not on HD (survival: χ2 = 0.43, df = 2, p = .808; life span: χ2 = 0.84, df = 2, p = .658). On LD assay diet, SD evolution diet group survival and mean life span was higher than LD evolution diet (survival: z = 4.34, p < .001; Figure 2; mean life span: z = −4.76, p < .001; Figure 1), and of flies evolved on HD evolution diet (survival: z = 10.57, p < .001; Figure 2; mean life span: z = −11.78, p < .001; Figure 1). When tested on LD, flies evolved on SD lived on average 6.5 days longer than flies evolved on LD and 14.5 days longer than flies evolved on HD (Supplementary Table S2). On SD assay diet, LD and SD evolution diet group survival and mean life span were not different (survival: z = 1.99, p = .116; Figure 2; mean life span: z = −1.38, p = .352; Figure 1), and both were higher than that of flies on HD evolution diet (LD vs. HD: survival: z = 3.95, p < .001; Figure 2; mean life span: z = −5.11, p < .001; Figure 1; SD vs. HD: survival: z = 5.65, p < .001; Figure 2; mean life span: z = −6.37, p < .001; Figure 1). Our control treatment females (evolution diet SD) showed the classic DR life span extension effect when assayed on LD, with females on low assay diet living on average 5 days longer than females on SD (survival: z = 7.55, p < .001; Figure 2; life span: z = −3.93, p = .003; Figure 1; Supplementary Table S2). This DR effect was not observed in females evolved on LD, where no significant difference in life span between standard and restricted assay diet was found (z = 0.72, p = .999; shape of survival curves did marginally not differ: z = 3.05, p < .057; Figure 2), nor in females evolved on high-protein diet (life span: z = −0.84, p = .996; survival: z = 3.02, p = .064). All groups showed an exponential increase in hazard rate—a signature of aging (see Supplementary Table S3; Supplementary Figure S2). Differences between evolution diet regimes in age-dependent hazard rate occurred when tested on LD, with SD evolution regime flies having the lowest baseline hazard rate, and the highest aging rate, compared with LD and HD evolution regimes (Supplementary Table S3; Supplementary Figure S2). When tested on SD, the lower life span of HD evolution regime flies was caused by a higher baseline hazard rate, compared with LD and SD evolution regime flies, despite a lower aging rate (Supplementary Table S3). Although the DR life span extension effect that was observed only in SD evolution diet flies was based on a decrease in baseline hazard rate, aging rate was decreased and baseline hazard rate increased in LD and HD evolution diet flies tested on LD, compared with when tested on SD (Supplementary Table S3). Reproduction Effects of evolution diet and assay diet on reproduction were significant, but not their interaction (evolution diet: F2,71 = 4.29, p = .017; assay diet: F2,71 = 319.36, p < .001; assay diet × evolution diet: F4,71 = 1.23, p = .305), with richer assay diet having a positive effect on fecundity (Figure 3). In separate analyses for each assay diet, the effect of evolution diet was not significant (LD: F2,9 = 1.28, p = .324; SD: F2,20 = 1.83, p = .187; HD: F2,21 = 2.08, p = .150). Figure 3. View largeDownload slide Female fruit fly fecundity, compared between ED populations. Bars show fecundity as total egg numbers (sum of weekly counts, scaled by initial number of flies in each vial), averaged across vials in each treatment. Error bars show ±2 SE. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Figure 3. View largeDownload slide Female fruit fly fecundity, compared between ED populations. Bars show fecundity as total egg numbers (sum of weekly counts, scaled by initial number of flies in each vial), averaged across vials in each treatment. Error bars show ±2 SE. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Testing age-dependent (vial-based) fecundity trajectories, we found an overall difference between evolution diet regimes when tested on LD (∆AIC = 11.38; Supplementary Figure S1) and SD (∆AIC = 15.81; Supplementary Figure S1), but not on HD assay diet (∆AIC = 7.73; Supplementary Figure S1). Visual inspection of fitted splines suggests lower early-life fecundity of LD evolution flies tested on LD when compared with SD and HD evolution diet flies (Supplementary Figure S1), lower early-life fecundity of SD evolution flies on SD assay diet when compared with LD and HD evolution diet, and no difference due to evolution diet when tested on HD. Analysis of age classes (week 1, weeks 2 and 3, older than 3 weeks [week 4 up]; see Methods) showed that evolution diet affected age-classed fecundity in females tested on LD diet (age class × evolution diet: F4,73.3 = 2.92, p = .027), but not on SD (age class × evolution diet: F4,32.7 = 2.39, p = .071) and HD assay diet (age class × evolution diet: F4,31.1 = 2.35, p = .077). The effect on LD assay diet was driven by lower initial fecundity of flies evolved on LD (Supplementary Figure S1), compared with flies evolved on SD (week 1: t23 = −3.16, p = .004) and HD (week 1: t24.3 = −2.35, p = .027). This supports the visual difference in spline shapes on low evolution diet, but not on standard evolution diet. Body Mass Female body mass did not differ between evolution diet regimes (F2,2.53 = 5.77, p = .114). Discussion The life span extending effect of DR is often explained as an adaptive plastic response, which reallocates energy from reproduction to somatic maintenance to survive temporary periods of food shortage (16). When DR becomes chronic, such strategy becomes maladaptive, and selection is predicted to favor reproduction over somatic maintenance and longevity. In accordance with this prediction, we found decreased life span of females that evolved on LD, compared with females evolved on SD, when populations from both evolutionary regimes were tested on low assay diet. However, the evolution of shorter life span under LD was not accompanied by the evolution of increased reproduction, as predicted by the disposable soma hypothesis. On the contrary, early fecundity was reduced in lines that evolved on the LD and were tested on the LD, compared with the SD. We previously tested this prediction in males, using the same experimental evolution lines as in the present study (24). In contrast to females, male reproduction increased when evolving on low-protein diet. However, we did not observe a simultaneous decrease in survival, as would be expected from a negative correlation between reproduction and survival. Together, our results from this long-term DR experiment show that although both sexes evolved in response to different dietary regimes, there was no detectable correlated response between reproduction and survival in either sex. The evolutionary response of the sexes to dietary regimes differed considerably, but the lack of genetic correlation between survival and reproduction across populations was, perhaps, one unifying feature. A previous experimental evolution study that manipulated larval diet, instead of adult diet as in the present study, found a negative effect of low nutrient food (restricted in protein and carbohydrates) on adult body mass (35). However, there is no indication that our results were affected by differences in female body mass because we observed no evolutionary response of body mass in either of our dietary regimes. Although empirical studies often support a trade-off between reproduction and survival—the so-called cost of reproduction (6,36,37)—including in D. melanogaster females (5,15,36), there are many studies in which no trade-off has been detected (reviewed for example in refs (36,37)). For example, recent studies show that ratios of dietary amino acids can be manipulated to produce the standard DR life span extension, without any reduction in reproductive output (38,39). This reveals that survival and reproduction can be uncoupled to a substantial extent. In the study by Grandison and colleagues (38), the level of only one amino acid, methionine, was increased in a DR diet to result in the apparent resolution of a potential trade-off between reproduction and life span. Another line of evidence for a substantially decoupled effect of DR on reproduction and survival comes from studies that show a DR-induced increase in life span when reproduction is experimentally inhibited (40,41). It is important to recognize that if no trade-off is detected, there is still a possibility that trade-offs are manifest only with other fitness components, such as immune response, which can have a weak undetectable correlation with fitness under the specific experimental conditions and might not even be measured. Discussing our previous results in males, we invoked insulin/insulin-like signaling/target of rapamycin signaling–dependent autophagy (42). This process is upregulated in low dietary resource environments (43) and could be a potential mechanism to explain higher reproduction without lowered survival in males, which has been previously suggested as a general explanation for DR effects on life span (44). We hypothesized that a sexually antagonistic effect, for example, through the p53 pathway (45) that is involved in regulating autophagy, might explain the positive effect on reproduction in males, trading off with fitness effects in females. If this would be the case, evolving under chronic DR would be expected to have negative effects in females, presumably in reproductive traits, as a more efficient reuse of internal resources through increased autophagy (organelles and long-lived proteins) (46) might also negatively affect processes related to egg production under DR. A certain level of autophagy and apoptosis, targeted at somatic nurse cells and germline follicle cells that are essential during oocyte development, is part of the normal process of oogenesis (47). Although extreme nutrient depletion increases the level of autophagy in ovaries (48,49), it is not clear at this stage whether restricted nutrient regimes have a less pronounced but similar effect on egg production. We did not find a strong effect of multigenerational chronic DR on female reproduction: Evolving on LD decreased early female fecundity, with no significant effect on total reproduction. Females evolved under DR had lower survival compared with females evolved on SD. Together, these responses can be cautiously interpreted as negative effects of multigenerational chronic DR on females, compared with positive effect on male fitness, and thus putatively support a role for sexual antagonistic genetic variation in the observed qualitative sex differences in response to chronic DR. Genetically based metabolic and physiological constraints that are genotype (female/male) and environment (protein rich/protein poor) specific might also constrain the evolution of similar phenotypes in females, compared with males. When tested on LD, flies evolved on SD had a lower baseline hazard rate and therefore lived longer than flies evolved on LD or HD, as observed in other studies (50–52). Flies evolved on LD and tested on LD showed slower actuarial aging, compared with flies evolved on SD. It, therefore, seems that evolution under DR not only removes any life span extension observed in female flies evolved on SD, but is also characterized by an earlier onset of aging. Evolution in a rich resource environment (HD) resulted in low life span not only when tested on DR, but also when assayed on SD. The fact that females evolved on HD and tested on DR had very low survival, but did not show a simultaneous increase in reproduction also does not support a direct reallocation between reproduction and survival. However, the disposable soma theory is generally not very suitable to explain phenotypes in resource-rich environments, as one of its fundamental assumptions is that resources are limited. The negative effect on life span caused by evolving on high-protein diet points to a specific loss of plasticity in the ability to adjust life span to nutrition and to survive longer when assayed in nutritionally less rich environments. Measuring trade-offs is always a difficult endeavor, even in the established model species such as D. melanogaster. We used female fecundity, measured as the number of eggs laid, as our fitness measure. Negative fitness effects could potentially manifest in the quality of the offspring, for example through egg viability, hatching success, and condition of eclosed offspring, which we did not capture in our assay. Another caveat that concerns all experimental evolution and artificial selection studies is the possibility of parental effects through nongenetic transgenerational inheritance. To lower these effects, we allowed one generation of relaxed selection on SD, before assessing treatment effects. In summary, our findings do not support the leading hypothesis that life span extension under DR results from the strategic reallocation of resources from reproduction to survival to survive a temporary famine. It is possible that DR is reducing superfluous nutrient-sensing signaling in late life, as suggested by the hyperfunction theory of aging (53,54). Future studies should aim to test the whole range of new theoretical approaches to solve the paradox of cost-free life span extension. Supplementary Material Supplementary data is available at The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online. Funding This study was supported by a Wenner-Gren Postdoctoral Fellowship to F.Z., a Swedish Research Council grant to U.F., and a European Research Council Starting Grant (AGINGSEXDIFF) and Consolidator Grant (GermlineAgeingSoma 724909) to A.A.M. Conflict of Interest None reported. 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Abstract

Abstract One of the key tenets of life-history theory is that reproduction and survival are linked and that they trade-off with each other. When dietary resources are limited, reduced reproduction with a concomitant increase in survival is commonly observed. It is often hypothesized that this dietary restriction effect results from strategically reduced investment in reproduction in favor of somatic maintenance to survive starvation periods until resources become plentiful again. We used experimental evolution to test this “waiting-for-the-good-times” hypothesis, which predicts that selection under sustained dietary restriction will favor increased investment in reproduction at the cost of survival because “good-times” never come. We assayed fecundity and survival of female Drosophila melanogaster fruit flies that had evolved for 50 generations on three different diets varying in protein content—low (classic dietary restriction diet), standard, and high—in a full-factorial design. High-diet females evolved overall increased fecundity but showed reduced survival on low and standard diets. Low-diet females evolved reduced survival on low diet without corresponding increase in reproduction. In general, there was little correspondence between the evolution of survival and fecundity across all dietary regimes. Our results contradict the hypothesis that resource reallocation between fecundity and somatic maintenance underpins life span extension under dietary restriction. Drosophila melanogaster, Nutrition, Adaptation, DR, Experimental evolution Understanding the relationship between nutrition, reproduction, and survival, on the genetic and the phenotypic levels, is thought to be essential for health-span and life span extension (1). Research on genes involved in the modulation of these traits has revealed a network of nutrient and energy sensing signaling pathways that govern reproduction and survival (2), with substantial evolutionary conservation across the tree of life (3,4). Life span extending effects of dietary restriction (DR)—the most successful intervention to prolong life to date (3)—is a case of phenotypic plastic response that generally not only increases survival but also decreases reproduction (5). Evolutionary life-history theory and the antagonistic pleiotropy theory for the evolution of aging state that early- and late-life fitness components are generally trading off against each other and that these negative correlations between traits are genetically based (6,7). Within this framework, the plastic response to DR can be understood as the consequence of a shift in the energy trade-off between reproduction and survival. The disposable soma theory of aging is built around this theoretical conjecture (8), and it states that resource requirements for reproduction directly compete with those required for somatic maintenance and that this relationship should be observed both on the physiological level and on the genetic level (see the distinction between “physiological” and “genetic” [evolutionary] trade-off discussed in ref. (9)). Under the disposable soma theory, if the observed plasticity in this trade-off is adaptive, living longer and reproducing less under short-term DR (within an individual’s life span) should confer an evolutionary advantage (10,11) and can be understood as a short-term emergency solution to cope with nutritional stress (12). This prediction was tested in a formal life-history DR model parameterized using house mouse data by Shanley and Kirkwood (13), who found that under certain assumptions (ie, an extra cost before successful reproduction and lower juvenile survival under DR), the classic DR response can evolve (discussed in ref. (8)). Although there is suggestive evidence from a recent meta-analysis that DR might act differently on mortality rates in rodents, compared with D. melanogaster (ref. (14), but see ref. (15)), the main pathways leading to reduced aging seem to be evolutionary conserved between phyla (3). Nevertheless, one fundamental assumption of the disposable soma theory is that organisms can reallocate resources (mainly regarded in terms of energy units in this context) from reproduction to somatic maintenance and survival, and vice versa (16). While allocating more resources to survival, away from reproduction, is adaptive under short-term DR, this response should be maladaptive if resources are restricted permanently. If food shortage is permanent, spanning adult lifetimes over many generations, individuals that switch to a strategy of increased reproductive output at the cost of decreased survival will have a selective advantage. One way this could happen is when the ability to respond plastically to DR erodes over evolutionary time (ie, when the reaction norm for reproductive output across nutritional environments becomes less steep) or when either already segregating alleles or de novo mutations that confer higher reproductive output under DR are favored (ie, evolutionary adaptation). If a negative genetic correlation (evolutionary trade-off) between reproduction (especially during early life) and survival exists, as has often been observed (17–23), higher levels of reproductive output under DR (regardless if short-term and transient, or evolved) should at the same time decrease life span, to an extent that depends on the strength of the correlation. On the other hand, even if reproduction and life span are decoupled, we would still expect an increase in reproduction after sufficient numbers of generations under chronic DR, independent of a response in life span. In the present study, we test this prediction using experimental evolution in D. melanogaster, by manipulating adult dietary yeast levels and testing for an evolved response in female flies after approximately 50 generations. We previously found a response in male reproduction to this experimental evolution regime, with males evolved on DR having increased reproduction when tested on DR, standard or enriched diets, but no reduction in survival (24). Methods Experimental Design Experimental flies (D. melanogaster) originated from experimental evolution lines that evolved on three distinct diets with different yeast contents as adults (low diet [LD], standard diet [SD], high diet [HD]; specific diet characteristics are given in Supplementary Table S1). Flies in the experimental evolution lines were kept in four replicate mixed-sex population cages per diet treatment, containing 150 adult males and 150 adult females each. All larvae were reared on SD, and only adults were exposed to the experimental evolution diets in the population cages. More specific details on the experimental setup of the lines can be found in Zajitschek and colleagues (24). In short, our experimental flies were derived from Dahomey, a large outbred laboratory population that originally was sampled in 1970 from the wild in Benin, West Africa. Ever since the population has been maintained in mixed-sex population cages with overlapping generations under constant environmental conditions (25°C, 60% humidity, 12:12 light–dark cycle, on standard yeast–sugar diet). Recent studies on this population showed that it hosts substantial levels of genetic variation for life span (25,26). We tested for an evolutionary response in females after approximately 50 generations of experimental evolution. Sample sizes are given in Supplementary Table S2. To remove any parental effects from the diet treatments before the start of the experiment, experimental flies were passed through two generations of common garden. To accomplish this, females from the experimental population cages were allowed to lay eggs in wide plastic vials (28.5 mm × 95 mm used for all experimental work) with SD overnight. Eggs were trimmed to 100 eggs per vial, and eclosing adults were allowed to mate for the 2 days after eclosion before females were allowed to lay eggs in new SD vials for 2 hours. Eggs were again trimmed to 100 eggs per vial and eclosing adult females were used in assays. Each vial was populated with around 50 female flies. Assay flies were provided with one of the three experimental evolution diets, with two replicate vials per cage and evolution diet × assay diet combination (total number of individual females per evolution diet × assay diet treatment: N = 400). For weekly matings, females of each vial were transferred to new SD vials and given the matching number of 2-day-old males that were bred in a separate stock sourced from the same population cage, once every week for 12 hours. Eggs laid during this period were counted. Total fecundity was calculated by summing eggs laid over all vials and weeks. Survival was checked every Monday, Wednesday, and Friday until all flies had died. We measured dry adult body mass of groups of 10 individual female flies and replicated 10 times per cage per evolutionary diet treatment (N = 400 per treatment). Prior to weighing, all flies were raised for two generations on SD medium, as described earlier. Statistical Analysis To analyze survival, we used mixed Cox proportional hazard models (function coxme, R package coxme) (27). As the interaction term between assay diet and evolution diet was significant in a global analysis (χ2 = 104.63, degrees of freedom [df] = 4, p < .001), we performed (a) post hoc analyses for assay diet effects within evolution diet groups, using Tukey’s Honest Significant Difference method to adjust for multiple testing (function glht in R package multcomp) (28) and (b) separate analyses for each assay diet, with evolution diet as a fixed effect and experimental vial and population cage fitted as a random intercept. Models containing evolution diet were compared with models that only contained an intercept, using log-likelihood ratio tests, with twice the difference in log-likelihoods of the models taken as chi-square distributed, and a .05 significance level. Untransformed life span and body mass were tested in linear mixed models (using maximum likelihood estimation), after testing residuals for normal distribution, with the same random effects as specified for Cox proportional hazards analyses (using function lmer in R package lme4) (29). We used the R package lmerTest to calculate p values for linear mixed models, with df based on the Satterthwaite approximation (30), and performed post hoc analyses as described earlier. To test for differences in hazard rates, we fitted exponential and Gompertz models, using Bayesian methods implemented in the R package BaSTA (31). The exponential model assumes a constant mortality rate at all ages, whereas the Gompertz model assumes an increase in mortality rate at later ages (ie, aging):  μx=b0eb1x with instantaneous mortality rate (hazard rate) at age x is given by µx, parameter b0 is the intercept and is interpreted as the initial or baseline mortality rate, parameter b1 is the increase of mortality rate with advancing age (the aging parameter). We compared exponential and Gompertz model fits using the deviance information criterion (32). For all reported analyses, diet was treated as a categorical variable. Life span summary statistics and sample sizes are given in Supplementary Table S2, and median life span is plotted in Supplementary Figure S3. Female reproductive fitness was estimated as the sum of all weekly fecundity measurements of each population of females in a vial, scaled by the initial number of females in a vial. Total fecundity was analyzed in linear mixed effects models following the same process as in the analysis for survival and life span, with population cage fitted as a random intercept. To specifically compare early-, mid-, and late-life fecundity, we also tested effects on mean fecundity in age classes (early-life fecundity = fecundity in week 1, mid-life fecundity = fecundity in weeks 2 and 3, late-life fecundity = fecundity in week 4 and later). Post hoc tests were conducted using function difflsmeans in R package lmerTest. Effects of evolution diet on age-dependent fecundity trajectories across life span were tested in general additive mixed models to account for nonlinear relationships, with vial fitted as a random effect, and correcting for initial number of females in a vial by including it as a fixed effect. We used a tensor product smooth function of age at measuring fecundity (weekly), and thin plate regression splines. Effects of evolution diet within assay diet were tested by comparing a model fitting separate curves to evolution diet groups, with a model without accounting for evolution diet, using Akaike’s information criterion (AIC). All models were fitted and predicted trajectories visualized in R package mgcv (33). All analyses were run in the software R, version 3.3.1 or higher (34). Results Survival We report effects of long-term experimental evolution under low, standard, and high yeast adult diets, on survival and reproduction of D. melanogaster females that were mated once every week. In contrast to male flies that were tested previously (24), female survival responded to the experimental evolution regimes. The effect of assay diet on survival rates and mean life span was dependent on evolution diet (survival: χ2 = 104.63, df = 4, p < .001; life span: F4,2941 = 21.20, p < .001; Figures 1 and 2). Figure 1. View largeDownload slide Female fruit fly mean life span. Each graph shows mean life span for AD groups. Error bars show ±2 SE. Asterisks indicate the statistical significance of differences between groups: ***p < .001. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Figure 1. View largeDownload slide Female fruit fly mean life span. Each graph shows mean life span for AD groups. Error bars show ±2 SE. Asterisks indicate the statistical significance of differences between groups: ***p < .001. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Figure 2. View largeDownload slide Female fruit fly survivorship. Each panel shows Kaplan–Meier survival curves for AD treatment groups. Separate curves depict survivorship of ED populations, tested on different ADs. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Figure 2. View largeDownload slide Female fruit fly survivorship. Each panel shows Kaplan–Meier survival curves for AD treatment groups. Separate curves depict survivorship of ED populations, tested on different ADs. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Evolution diet regime affected survival and life span when tested on LD (survival: χ2 = 110.89, df = 2, p < .001; life span: χ2 = 131.57, df = 2, p < .001) and SD (survival: χ2 = 32.15, df = 2, p < .001; life span: χ2 = 43.93, df = 2, p < .001), but not on HD (survival: χ2 = 0.43, df = 2, p = .808; life span: χ2 = 0.84, df = 2, p = .658). On LD assay diet, SD evolution diet group survival and mean life span was higher than LD evolution diet (survival: z = 4.34, p < .001; Figure 2; mean life span: z = −4.76, p < .001; Figure 1), and of flies evolved on HD evolution diet (survival: z = 10.57, p < .001; Figure 2; mean life span: z = −11.78, p < .001; Figure 1). When tested on LD, flies evolved on SD lived on average 6.5 days longer than flies evolved on LD and 14.5 days longer than flies evolved on HD (Supplementary Table S2). On SD assay diet, LD and SD evolution diet group survival and mean life span were not different (survival: z = 1.99, p = .116; Figure 2; mean life span: z = −1.38, p = .352; Figure 1), and both were higher than that of flies on HD evolution diet (LD vs. HD: survival: z = 3.95, p < .001; Figure 2; mean life span: z = −5.11, p < .001; Figure 1; SD vs. HD: survival: z = 5.65, p < .001; Figure 2; mean life span: z = −6.37, p < .001; Figure 1). Our control treatment females (evolution diet SD) showed the classic DR life span extension effect when assayed on LD, with females on low assay diet living on average 5 days longer than females on SD (survival: z = 7.55, p < .001; Figure 2; life span: z = −3.93, p = .003; Figure 1; Supplementary Table S2). This DR effect was not observed in females evolved on LD, where no significant difference in life span between standard and restricted assay diet was found (z = 0.72, p = .999; shape of survival curves did marginally not differ: z = 3.05, p < .057; Figure 2), nor in females evolved on high-protein diet (life span: z = −0.84, p = .996; survival: z = 3.02, p = .064). All groups showed an exponential increase in hazard rate—a signature of aging (see Supplementary Table S3; Supplementary Figure S2). Differences between evolution diet regimes in age-dependent hazard rate occurred when tested on LD, with SD evolution regime flies having the lowest baseline hazard rate, and the highest aging rate, compared with LD and HD evolution regimes (Supplementary Table S3; Supplementary Figure S2). When tested on SD, the lower life span of HD evolution regime flies was caused by a higher baseline hazard rate, compared with LD and SD evolution regime flies, despite a lower aging rate (Supplementary Table S3). Although the DR life span extension effect that was observed only in SD evolution diet flies was based on a decrease in baseline hazard rate, aging rate was decreased and baseline hazard rate increased in LD and HD evolution diet flies tested on LD, compared with when tested on SD (Supplementary Table S3). Reproduction Effects of evolution diet and assay diet on reproduction were significant, but not their interaction (evolution diet: F2,71 = 4.29, p = .017; assay diet: F2,71 = 319.36, p < .001; assay diet × evolution diet: F4,71 = 1.23, p = .305), with richer assay diet having a positive effect on fecundity (Figure 3). In separate analyses for each assay diet, the effect of evolution diet was not significant (LD: F2,9 = 1.28, p = .324; SD: F2,20 = 1.83, p = .187; HD: F2,21 = 2.08, p = .150). Figure 3. View largeDownload slide Female fruit fly fecundity, compared between ED populations. Bars show fecundity as total egg numbers (sum of weekly counts, scaled by initial number of flies in each vial), averaged across vials in each treatment. Error bars show ±2 SE. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Figure 3. View largeDownload slide Female fruit fly fecundity, compared between ED populations. Bars show fecundity as total egg numbers (sum of weekly counts, scaled by initial number of flies in each vial), averaged across vials in each treatment. Error bars show ±2 SE. AD = assay diet; ED = evolution diet; LD = low-protein diet; SD = standard diet; HD = high-protein diet. Testing age-dependent (vial-based) fecundity trajectories, we found an overall difference between evolution diet regimes when tested on LD (∆AIC = 11.38; Supplementary Figure S1) and SD (∆AIC = 15.81; Supplementary Figure S1), but not on HD assay diet (∆AIC = 7.73; Supplementary Figure S1). Visual inspection of fitted splines suggests lower early-life fecundity of LD evolution flies tested on LD when compared with SD and HD evolution diet flies (Supplementary Figure S1), lower early-life fecundity of SD evolution flies on SD assay diet when compared with LD and HD evolution diet, and no difference due to evolution diet when tested on HD. Analysis of age classes (week 1, weeks 2 and 3, older than 3 weeks [week 4 up]; see Methods) showed that evolution diet affected age-classed fecundity in females tested on LD diet (age class × evolution diet: F4,73.3 = 2.92, p = .027), but not on SD (age class × evolution diet: F4,32.7 = 2.39, p = .071) and HD assay diet (age class × evolution diet: F4,31.1 = 2.35, p = .077). The effect on LD assay diet was driven by lower initial fecundity of flies evolved on LD (Supplementary Figure S1), compared with flies evolved on SD (week 1: t23 = −3.16, p = .004) and HD (week 1: t24.3 = −2.35, p = .027). This supports the visual difference in spline shapes on low evolution diet, but not on standard evolution diet. Body Mass Female body mass did not differ between evolution diet regimes (F2,2.53 = 5.77, p = .114). Discussion The life span extending effect of DR is often explained as an adaptive plastic response, which reallocates energy from reproduction to somatic maintenance to survive temporary periods of food shortage (16). When DR becomes chronic, such strategy becomes maladaptive, and selection is predicted to favor reproduction over somatic maintenance and longevity. In accordance with this prediction, we found decreased life span of females that evolved on LD, compared with females evolved on SD, when populations from both evolutionary regimes were tested on low assay diet. However, the evolution of shorter life span under LD was not accompanied by the evolution of increased reproduction, as predicted by the disposable soma hypothesis. On the contrary, early fecundity was reduced in lines that evolved on the LD and were tested on the LD, compared with the SD. We previously tested this prediction in males, using the same experimental evolution lines as in the present study (24). In contrast to females, male reproduction increased when evolving on low-protein diet. However, we did not observe a simultaneous decrease in survival, as would be expected from a negative correlation between reproduction and survival. Together, our results from this long-term DR experiment show that although both sexes evolved in response to different dietary regimes, there was no detectable correlated response between reproduction and survival in either sex. The evolutionary response of the sexes to dietary regimes differed considerably, but the lack of genetic correlation between survival and reproduction across populations was, perhaps, one unifying feature. A previous experimental evolution study that manipulated larval diet, instead of adult diet as in the present study, found a negative effect of low nutrient food (restricted in protein and carbohydrates) on adult body mass (35). However, there is no indication that our results were affected by differences in female body mass because we observed no evolutionary response of body mass in either of our dietary regimes. Although empirical studies often support a trade-off between reproduction and survival—the so-called cost of reproduction (6,36,37)—including in D. melanogaster females (5,15,36), there are many studies in which no trade-off has been detected (reviewed for example in refs (36,37)). For example, recent studies show that ratios of dietary amino acids can be manipulated to produce the standard DR life span extension, without any reduction in reproductive output (38,39). This reveals that survival and reproduction can be uncoupled to a substantial extent. In the study by Grandison and colleagues (38), the level of only one amino acid, methionine, was increased in a DR diet to result in the apparent resolution of a potential trade-off between reproduction and life span. Another line of evidence for a substantially decoupled effect of DR on reproduction and survival comes from studies that show a DR-induced increase in life span when reproduction is experimentally inhibited (40,41). It is important to recognize that if no trade-off is detected, there is still a possibility that trade-offs are manifest only with other fitness components, such as immune response, which can have a weak undetectable correlation with fitness under the specific experimental conditions and might not even be measured. Discussing our previous results in males, we invoked insulin/insulin-like signaling/target of rapamycin signaling–dependent autophagy (42). This process is upregulated in low dietary resource environments (43) and could be a potential mechanism to explain higher reproduction without lowered survival in males, which has been previously suggested as a general explanation for DR effects on life span (44). We hypothesized that a sexually antagonistic effect, for example, through the p53 pathway (45) that is involved in regulating autophagy, might explain the positive effect on reproduction in males, trading off with fitness effects in females. If this would be the case, evolving under chronic DR would be expected to have negative effects in females, presumably in reproductive traits, as a more efficient reuse of internal resources through increased autophagy (organelles and long-lived proteins) (46) might also negatively affect processes related to egg production under DR. A certain level of autophagy and apoptosis, targeted at somatic nurse cells and germline follicle cells that are essential during oocyte development, is part of the normal process of oogenesis (47). Although extreme nutrient depletion increases the level of autophagy in ovaries (48,49), it is not clear at this stage whether restricted nutrient regimes have a less pronounced but similar effect on egg production. We did not find a strong effect of multigenerational chronic DR on female reproduction: Evolving on LD decreased early female fecundity, with no significant effect on total reproduction. Females evolved under DR had lower survival compared with females evolved on SD. Together, these responses can be cautiously interpreted as negative effects of multigenerational chronic DR on females, compared with positive effect on male fitness, and thus putatively support a role for sexual antagonistic genetic variation in the observed qualitative sex differences in response to chronic DR. Genetically based metabolic and physiological constraints that are genotype (female/male) and environment (protein rich/protein poor) specific might also constrain the evolution of similar phenotypes in females, compared with males. When tested on LD, flies evolved on SD had a lower baseline hazard rate and therefore lived longer than flies evolved on LD or HD, as observed in other studies (50–52). Flies evolved on LD and tested on LD showed slower actuarial aging, compared with flies evolved on SD. It, therefore, seems that evolution under DR not only removes any life span extension observed in female flies evolved on SD, but is also characterized by an earlier onset of aging. Evolution in a rich resource environment (HD) resulted in low life span not only when tested on DR, but also when assayed on SD. The fact that females evolved on HD and tested on DR had very low survival, but did not show a simultaneous increase in reproduction also does not support a direct reallocation between reproduction and survival. However, the disposable soma theory is generally not very suitable to explain phenotypes in resource-rich environments, as one of its fundamental assumptions is that resources are limited. The negative effect on life span caused by evolving on high-protein diet points to a specific loss of plasticity in the ability to adjust life span to nutrition and to survive longer when assayed in nutritionally less rich environments. Measuring trade-offs is always a difficult endeavor, even in the established model species such as D. melanogaster. We used female fecundity, measured as the number of eggs laid, as our fitness measure. Negative fitness effects could potentially manifest in the quality of the offspring, for example through egg viability, hatching success, and condition of eclosed offspring, which we did not capture in our assay. Another caveat that concerns all experimental evolution and artificial selection studies is the possibility of parental effects through nongenetic transgenerational inheritance. To lower these effects, we allowed one generation of relaxed selection on SD, before assessing treatment effects. In summary, our findings do not support the leading hypothesis that life span extension under DR results from the strategic reallocation of resources from reproduction to survival to survive a temporary famine. It is possible that DR is reducing superfluous nutrient-sensing signaling in late life, as suggested by the hyperfunction theory of aging (53,54). Future studies should aim to test the whole range of new theoretical approaches to solve the paradox of cost-free life span extension. Supplementary Material Supplementary data is available at The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online. Funding This study was supported by a Wenner-Gren Postdoctoral Fellowship to F.Z., a Swedish Research Council grant to U.F., and a European Research Council Starting Grant (AGINGSEXDIFF) and Consolidator Grant (GermlineAgeingSoma 724909) to A.A.M. Conflict of Interest None reported. 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The Journals of Gerontology Series A: Biomedical Sciences and Medical SciencesOxford University Press

Published: Apr 28, 2018

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