Lifetime inclusive fitness effects of cooperative polygamy in the acorn woodpecker.

Although over 50 y have passed since W. D. Hamilton articulated kin selection and inclusive fitness as evolutionary explanations for altruistic behavior, quantifying inclusive fitness continues to be challenging. Here, using 30 y of data and two alternative methods, we outline an approach to measure lifetime inclusive fitness effects of cooperative polygamy (mate-sharing or cobreeding) in the cooperatively breeding acorn woodpecker Melanerpes formicivorus. For both sexes, the number of offspring (observed direct fitness) declined while the number of young parented by related cobreeders (observed indirect fitness effect) increased with cobreeding coalition size. Combining these two factors, the observed inclusive fitness effect of cobreeding was greater than breeding singly for males, while the pattern for females depended on whether fitness was age-weighted, as females breeding singly accrued greater fitness at younger ages than cobreeding females. Accounting for the fitness birds would have obtained by breeding singly, however, lifetime inclusive fitness effects declined with coalition size for males, but were greater for females breeding as duos compared to breeding singly, due largely to indirect fitness effects of kin. Our analyses provide a road map for, and demonstrate the importance of, quantifying indirect fitness as a powerful evolutionary force contributing to the costs and benefits of social behaviors.

Longitudinal gut microbiome dynamics in relation to age and senescence in a wild animal population.

In humans, gut microbiome (GM) differences are often correlated with, and sometimes causally implicated in, ageing. However, it is unclear how these findings translate in wild animal populations. Studies that investigate how GM dynamics change within individuals, and with declines in physiological condition, are needed to fully understand links between chronological age, senescence and the GM, but have rarely been done. Here, we use longitudinal data collected from a closed population of Seychelles warblers (Acrocephalus sechellensis) to investigate how bacterial GM alpha diversity, composition and stability are associated with host senescence. We hypothesised that GM diversity and composition will differ, and become more variable, in older adults, particularly in the terminal year prior to death, as the GM becomes increasingly dysregulated due to senescence. However, GM alpha diversity and composition remained largely invariable with respect to adult age and did not differ in an individual's terminal year. Furthermore, there was no evidence that the GM became more heterogenous in senescent age groups (individuals older than 6 years), or in the terminal year. Instead, environmental variables such as season, territory quality and time of day, were the strongest predictors of GM variation in adult Seychelles warblers. These results contrast with studies on humans, captive animal populations and some (but not all) studies on non-human primates, suggesting that GM deterioration may not be a universal hallmark of senescence in wild animal species. Further work is needed to disentangle the factors driving variation in GM-senescence relationships across different host taxa.

Adult telomere length is positively correlated with survival and lifetime reproductive success in a wild passerine.

Explaining variation in individual fitness is a key goal in evolutionary biology. Recently, telomeres, repeating DNA sequences capping chromosome ends, have gained attention as a biomarker for body state, physiological costs, and senescence. Existing research has provided mixed evidence for whether telomere length correlates with fitness, including survival and reproductive output. Moreover, few studies have examined how the rate of change in telomere length correlates with fitness in wild populations. Here, we intensively monitored an insular population of house sparrows, and collected longitudinal telomere and life history data (16 years, 1225 individuals). We tested whether telomere length and its rate of change predict fitness measures, namely survival, lifespan and annual and lifetime reproductive effort and success. Telomere length positively predicted short-term survival, independent of age, but did not predict lifespan, suggesting either a diminishing telomere length-survival correlation with age or other extrinsic factors of mortality. The positive association of telomere length with survival translated into reproductive benefits, as birds with longer telomeres produced more genetic recruits, hatchlings and reared more fledglings over their lifetime. In contrast, there was no association between telomere dynamics and annual reproductive output, suggesting telomere dynamics might not reflect the costs of reproduction in this population, potentially masked by variation in individual quality. The rate of change of telomere length did not correlate with neither lifespan nor lifetime reproductive success. Our results provide further evidence that telomere length correlates with fitness, and contribute to our understanding of the selection on, and evolution of, telomere dynamics.

The long-lasting legacy of reproduction: lifetime reproductive success shapes expected genetic contributions of humans after 10 generations.

An individual's lifetime reproductive success (LRS) measures its realized genetic contributions to the next generation, but how well does it predict this over longer periods? Here we use human genealogical data to estimate expected individual genetic contributions (IGC) and quantify the degree to which LRS, relative to other fitness proxies, predicts IGC over longer periods. This allows an identification of the life-history stages that are most important in shaping variation in IGC. We use historical genealogical data from two non-isolated local populations in Switzerland to estimate the stabilized IGC for 2230 individuals approximately 10 generations after they were born. We find that LRS explains 30% less variation in IGC than the best predictor of IGC, the number of grandoffspring. However, albeit less precise than the number of grandoffspring, we show that LRS does provide an unbiased prediction of IGC. Furthermore, it predicts IGC better than lifespan, and accounting for offspring survival to adulthood does not improve the explanatory power. Overall, our findings demonstrate the value of human genealogical data to evolutionary biology and suggest that reproduction-more than lifespan or offspring survival-impacts the long-term genetic contributions of historic humans, even in a population with appreciable migration.

DNA methylation markers of age(ing) in non-model animals.

Inferring the chronological and biological age of individuals is fundamental to population ecology and our understanding of ageing itself, its evolution, and the biological processes that affect or even cause ageing. Epigenetic clocks based on DNA methylation (DNAm) at specific CpG sites show a strong correlation with chronological age in humans, and discrepancies between inferred and actual chronological age predict morbidity and mortality. Recently, a growing number of epigenetic clocks have been developed in non-model animals and we here review these studies. We also conduct a meta-analysis to assess the effects of different aspects of experimental protocol on the performance of epigenetic clocks for non-model animals. Two measures of performance are usually reported, the R2 of the association between the predicted and chronological age, and the mean/median absolute deviation (MAD) of estimated age from chronological age, and we argue that only the MAD reflects accuracy. R2 for epigenetic clocks based on the HorvathMammalMethylChip4 was higher and the MAD scaled to age range lower, compared with other DNAm quantification approaches. Scaled MAD tended to be lower among individuals in captive populations, and decreased with an increasing number of CpG sites. We conclude that epigenetic clocks can predict chronological age with relatively high accuracy, suggesting great potential in ecological epigenetics. We discuss general aspects of epigenetic clocks in the hope of stimulating further DNAm-based research on ageing, and perhaps more importantly, other key traits.

Causes and consequences of telomere lengthening in a wild vertebrate population.

Telomeres have been advocated to be important markers of biological age in evolutionary and ecological studies. Telomeres usually shorten with age and shortening is frequently associated with environmental stressors and increased subsequent mortality. Telomere lengthening - an apparent increase in telomere length between repeated samples from the same individual - also occurs. However, the exact circumstances, and consequences, of telomere lengthening are poorly understood. Using longitudinal data from the Seychelles warbler (Acrocephalus sechellensis), we tested whether telomere lengthening - which occurs in adults of this species - is associated with specific stressors (reproductive effort, food availability, malarial infection and cooperative breeding) and predicts subsequent survival. In females, telomere shortening was observed under greater stress (i.e., low food availability, malaria infection), while telomere lengthening was observed in females experiencing lower stress (i.e., high food availability, assisted by helpers, without malaria). The telomere dynamics of males were not associated with the key stressors tested. These results indicate that, at least for females, telomere lengthening occurs in circumstances more conducive to self-maintenance. Importantly, both females and males with lengthened telomeres had improved subsequent survival relative to individuals that displayed unchanged, or shortened, telomeres - indicating that telomere lengthening is associated with individual fitness. These results demonstrate that telomere dynamics are bidirectionally responsive to the level of stress that an individual faces, but may poorly reflect the accumulation of stress over an individuals lifetime.

Telomere heritability and parental age at conception effects in a wild avian population.

Individual variation in telomere length is predictive of health and mortality risk across a range of species. However, the relative influence of environmental and genetic variation on individual telomere length in wild populations remains poorly understood. Heritability of telomere length has primarily been calculated using parent-offspring regression which can be confounded by shared environments. To control for confounding variables, quantitative genetic "animal models" can be used, but few studies have applied animal models in wild populations. Furthermore, parental age at conception may also influence offspring telomere length, but most studies have been cross-sectional. We investigated within- and between-parental age at conception effects and heritability of telomere length in the Seychelles warbler using measures from birds caught over 20 years and a multigenerational pedigree. We found a weak negative within-paternal age at conception effect (as fathers aged, their offspring had shorter telomeres) and a weak positive between-maternal age at conception effect (females that survived to older ages had offspring with longer telomeres). Animal models provided evidence that heritability and evolvability of telomere length were low in this population, and that variation in telomere length was not driven by early-life effects of hatch period or parental identities. Quantitative polymerase chain reaction plate had a large influence on telomere length variation and not accounting for it in the models would have underestimated heritability. Our study illustrates the need to include and account for technical variation in order to accurately estimate heritability, as well as other environmental effects, on telomere length in natural populations.

Early-life seasonal, weather and social effects on telomere length in a wild mammal.

Early-life environmental conditions can provide a source of individual variation in life-history strategies and senescence patterns. Conditions experienced in early life can be quantified by measuring telomere length, which can act as a biomarker of survival probability in some species. Here, we investigate whether seasonal changes, weather conditions and group size are associated with early-life and/or early-adulthood telomere length in a wild population of European badgers (Meles meles). We found substantial intra-annual changes in telomere length during the first 3 years of life, where within-individual effects showed shorter telomere lengths in the winter following the first spring and a trend for longer telomere lengths in the second spring compared to the first winter. In terms of weather conditions, cubs born in warmer, wetter springs with low rainfall variability had longer early-life (3-12 months old) telomeres. Additionally, cubs born in groups with more cubs had marginally longer early-life telomeres, providing no evidence of resource constraint from cub competition. We also found that the positive association between early-life telomere length and cub survival probability remained when social and weather variables were included. Finally, after sexual maturity, in early adulthood (i.e., 12-36 months) we found no significant association between same-sex adult group size and telomere length (i.e., no effect of intrasexual competition). Overall, we show that controlling for seasonal effects, which are linked to food availability, is important in telomere length analyses, and that variation in telomere length in badgers reflects early-life conditions and also predicts first year cub survival.

A meta-analysis on the heritability of vertebrate telomere length.

Telomere dynamics are linked with both cellular and organismal senescence, and life history, individual quality and health. Telomere dynamics, particularly telomere length, have therefore garnered much research interest in evolutionary biology. To examine the evolution of telomere length, it is important to quantify its heritability, the proportion of total variation explained by additive genetic effects. Many studies have quantified telomere length heritability, but estimates are varied, and no general conclusion has been drawn. Additionally, it is unclear whether biological and methodological factors influence telomere length heritability estimates. We present the first meta-analysis of telomere length heritability, using 104 estimates from 43 studies over 18 vertebrate species. We calculated an overall mean heritability and examined how estimates varied by study, phylogeny, species-specific ecology, environmental setting, age at sampling, laboratory methods, statistical methods, sex and repeated measurements. Overall heritability was moderate (44.9%, 95% CI: 25.2-64.7%), and there was considerable heterogeneity in heritability estimates, in particular among studies and estimates. Laboratory method influenced heritability estimates, with in-gel hybridization TRF yielding higher heritabilities than qPCR and Southern blot TRF. There was also an effect from statistical method, with twin-based and SNP-based estimates lower than correlation-based or pedigree-based estimates. Our results highlight an overall heritable basis of telomere length, and we recommend future research on a wider range of taxa, and the use of variance-partitioning methods with relatedness or SNP data over correlation methods to minimize heritability estimation bias.

Population level consequences of facultatively cooperative behaviour in a stochastic environment.

The social environment in which individuals live affects their fitness and in turn population dynamics as a whole. Birds with facultative cooperative breeding can live in social groups with dominants, subordinate helpers that assist with the breeding of others, and subordinate non-helpers. Helping behaviour benefits dominants through increased reproductive rates and reduced extrinsic mortality, such that cooperative breeding might have evolved in response to unpredictable, harsh conditions affecting reproduction and/or survival of the dominants. Additionally, there may be different costs and benefits to both helpers and non-helpers, depending on the time-scale. For example, early-life costs might be compensated by later-life benefits. These differential effects are rarely analysed in the same study. We examined whether helping behaviour affects population persistence in a stochastic environment and whether there are direct fitness consequences of different life-history tactics adopted by helpers and non-helpers. We parameterised a matrix population model describing the population dynamics of female Seychelles warblers Acrocephalus sechellensis, birds that display facultative cooperative breeding. The stochastic density-dependent model is defined by a (st)age structure that includes life-history differences between helpers and non-helpers and thus can estimate the demographic mechanisms of direct benefits of helping behaviour. We found that population dynamics are strongly influenced by stochastic variation in the reproductive rates of the dominants, that helping behaviour promotes population persistence and that there are only early-life differences in the direct fitness of helpers and non-helpers. Through a matrix population model, we captured multiple demographic rates simultaneously and analysed their relative importance in determining population dynamics of these cooperative breeders. Disentangling early-life versus lifetime effects of individual tactics sheds new light on the costs and benefits of helping behaviour. For example, the finding that helpers and non-helpers have similar lifetime reproductive outputs and that differences in reproductive values between the two life-history tactics arise only in early life suggests that overall, helpers and non-helpers have a similar balance of costs and benefits when analysing direct benefits. We recommend analysing the consequence of different life-history tactics, during both early life and over the lifetime, as analyses of these different time frames may produce conflicting results.

Lifetime reproductive benefits of cooperative polygamy vary for males and females in the acorn woodpecker (Melanerpes formicivorus).

Cooperative breeding strategies lead to short-term direct fitness losses when individuals forfeit or share reproduction. The direct fitness benefits of cooperative strategies are often delayed and difficult to quantify, requiring data on lifetime reproduction. Here, we use a longitudinal dataset to examine the lifetime reproductive success of cooperative polygamy in acorn woodpeckers (Melanerpes formicivorus), which nest as lone pairs or share reproduction with same-sex cobreeders. We found that males and females produced fewer young per successful nesting attempt when sharing reproduction. However, males nesting in duos and trios had longer reproductive lifespans, more lifetime nesting attempts and higher lifetime reproductive success than those breeding alone. For females, cobreeding in duos increased reproductive lifespan so the lifetime reproductive success of females nesting in duos was comparable to those nesting alone and higher than those nesting in trios. These results suggest that for male duos and trios, reproductive success alone may provide sufficient fitness benefits to explain the presence of cooperative polygamy, and the benefits of cobreeding as a duo in females are higher than previously assumed. Lifetime individual fitness data are crucial to reveal the full costs and benefits of cooperative polygamy.

Contemporary evolution of the innate immune receptor gene TLR3 in an isolated vertebrate population.

Understanding where genetic variation exists, and how it influences fitness within populations is important from an evolutionary and conservation perspective. Signatures of past selection suggest that pathogen-mediated balancing selection is a key driver of immunogenetic variation, but studies tracking contemporary evolution are needed to help resolve the evolutionary forces and mechanism at play. Previous work in a bottlenecked population of Seychelles warblers (Acrocephalus sechellensis) show that functional variation has been maintained at the viral-sensing Toll-like receptor 3 (TLR3) gene, including one nonsynonymous SNP, resulting in two alleles. Here, we characterise evolution at this TLR3 locus over a 25-year period within the original remnant population of the Seychelles warbler, and in four other derived, populations. Results show a significant and consistent temporal decline in the frequency of the TLR3C allele in the original population, and that similar declines in the TLR3C allele frequency occurred in all the derived populations. Individuals (of both sexes) with the TLR3CC genotype had lower survival, and males - but not females - that carry the TLR3C allele had significantly lower lifetime reproductive success than those with only the TLR3A allele. These results indicate that positive selection on the TLR3A allele, caused by an as yet unknown agent, is driving TLR3 evolution in the Seychelles warbler. No evidence of heterozygote advantage was detected. However, whether the positive selection observed is part of a longer-term pattern of balancing selection (through fluctuating selection or rare-allele advantage) cannot be resolved without tracking the TLR3C allele over an extended time period.

Genetic, social and maternal contributions to Mycobacterium bovis infection status in European badgers (Meles meles).

Within host populations, individuals can vary in their susceptibility to infections and in the severity and progression of disease once infected. Though mediated through differences in behaviour, resistance or tolerance, variation in disease outcomes ultimately stems from genetic and environmental (including social) factors. Despite obvious implications for the evolutionary, ecological and epidemiological dynamics of disease traits, the relative importance of these factors has rarely been quantified in naturally infected wild animal hosts. Here, we use a long-term capture-mark-recapture study of group-living European badgers (Meles meles) to characterize genetic and environmental sources of variation in host infection status by Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB). We find that genetic factors contribute to M. bovis infection status, whether measured over a lifetime or across repeated captures. In the latter case, the heritability (h2 ) of infection status is close to zero in cubs and yearlings but increases in adulthood. Overall, environmental influences arising from a combination of social group membership (defined in time and space) and maternal effects appear to be more important than genetic factors. Thus, while genes do contribute to among-individual variation, they play a comparatively minor role, meaning that rapid evolution of host defences under parasite-mediated selection is unlikely (especially if selection is on young animals where h2 is lowest). Conversely, our results lend further support to the view that social and early-life environments are important drivers of the dynamics of bTB infection in badger populations specifically, and of disease traits in wild hosts more generally.

Estimation of environmental, genetic and parental age at conception effects on telomere length in a wild mammal.

Understanding individual variation in fitness-related traits requires separating the environmental and genetic determinants. Telomeres are protective caps at the ends of chromosomes that are thought to be a biomarker of senescence as their length predicts mortality risk and reflect the physiological consequences of environmental conditions. The relative contribution of genetic and environmental factors to individual variation in telomere length is, however, unclear, yet important for understanding its evolutionary dynamics. In particular, the evidence for transgenerational effects, in terms of parental age at conception, on telomere length is mixed. Here, we investigate the heritability of telomere length, using the 'animal model', and parental age at conception effects on offspring telomere length in a wild population of European badgers (Meles meles). Although we found no heritability of telomere length and low evolvability (<0.001), our power to detect heritability was low and a repeatability of 2% across individual lifetimes provides a low upper limit to ordinary narrow-sense heritability. However, year (32%) and cohort (3%) explained greater proportions of the phenotypic variance in telomere length, excluding qPCR plate and row variances. There was no support for cross-sectional or within-individual parental age at conception effects on offspring telomere length. Our results indicate a lack of transgenerational effects through parental age at conception and a low potential for evolutionary change in telomere length in this population. Instead, we provide evidence that individual variation in telomere length is largely driven by environmental variation in this wild mammal.

Age-dependent changes in infidelity in Seychelles warblers.

Extra-pair paternity (EPP) is often linked to male age in socially monogamous vertebrates; that is, older males are more likely to gain EPP and less likely to be cuckolded. However, whether this occurs because males improve at gaining paternity as they grow older, or because "higher quality" males that live longer are preferred by females, has rarely been tested, despite being central to our understanding of the evolutionary drivers of female infidelity. Moreover, how extra-pair reproduction changes with age within females has received even less attention. Using 18 years of longitudinal data from an individually marked population of Seychelles warblers (Acrocephalus sechellensis), we found considerable within-individual changes in extra-pair reproduction in both sexes: an early-life increase and a late-life decline. Furthermore, males were cuckolded less as they aged. Our results indicate that in this species age-related patterns of extra-pair reproduction are determined by within-individual changes with age, rather than differences among individuals in longevity. These results challenge the hypothesis-based on longevity reflecting intrinsic quality-that the association between male age and EPP is due to females seeking high-quality paternal genes for offspring. Importantly, EPP accounted for up to half of male reproductive success, emphasizing the male fitness benefits of this reproductive strategy. Finally, the occurrence of post-peak declines in extra-pair reproduction provides explicit evidence of senescence in infidelity in both males and females.

Social effects on age-related and sex-specific immune cell profiles in a wild mammal.

Evidence for age-related changes in innate and adaptive immune responses is increasing in wild populations. Such changes have been linked to fitness, and knowledge of the factors driving immune response variation is important for understanding the evolution of immunity. Age-related changes in immune profiles may be owing to factors such as immune system development, sex-specific behaviour and responses to environmental conditions. Social environments may also contribute to variation in immunological responses, for example, through transmission of pathogens and stress arising from resource and mate competition. Yet, the impact of the social environment on age-related changes in immune cell profiles is currently understudied in the wild. Here, we tested the relationship between leukocyte cell composition (proportion of neutrophils and lymphocytes [innate and adaptive immunity, respectively] that were lymphocytes) and age, sex and group size in a wild population of European badgers (Meles meles). We found that the proportion of lymphocytes in early life was greater in males in smaller groups compared to larger groups, but with a faster age-related decline in smaller groups. By contrast, the proportion of lymphocytes in females was not significantly related to age or group size. Our results provide evidence of sex-specific age-related changes in immune cell profiles in a wild mammal, which are influenced by the social environment.

Kinship and familiarity mitigate costs of social conflict between Seychelles warbler neighbors.

Because virtually all organisms compete with others in their social environment, mechanisms that reduce conflict between interacting individuals are crucial for the evolution of stable families, groups, and societies. Here, we tested whether costs of social conflict over territorial space between Seychelles warblers (Acrocephalus sechellensis) are mitigated by kin-selected (genetic relatedness) or mutualistic (social familiarity) mechanisms. By measuring longitudinal changes in individuals' body mass and telomere length, we demonstrated that the fitness costs of territoriality are driven by a complex interplay between relatedness, familiarity, local density, and sex. Physical fights were less common at territory boundaries shared between related or familiar males. In line with this, male territory owners gained mass when living next to related or familiar males and also showed less telomere attrition when living next to male kin. Importantly, these relationships were strongest in high-density areas of the population. Males also had more rapid telomere attrition when living next to unfamiliar male neighbors, but mainly when relatedness to those neighbors was also low. In contrast, neither kinship nor familiarity was linked to body mass or telomere loss in female territory owners. Our results indicate that resolving conflict over territorial space through kin-selected or mutualistic pathways can reduce both immediate energetic costs and permanent somatic damage, thus providing an important mechanism to explain fine-scale population structure and cooperation between different social units across a broad range of taxa.

Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

Individual variation in early-life telomere length and survival in a wild mammal.

Individual variation in survival probability due to differential responses to early-life environmental conditions is important in the evolution of life histories and senescence. A biomarker allowing quantification of such individual variation, and which links early-life environmental conditions with survival by providing a measure of conditions experienced, is telomere length. Here, we examined telomere dynamics among 24 cohorts of European badgers (Meles meles). We found a complex cross-sectional relationship between telomere length and age, with no apparent loss over the first 29 months, but with both decreases and increases in telomere length at older ages. Overall, we found low within-individual consistency in telomere length across individual lifetimes. Importantly, we also observed increases in telomere length within individuals, which could not be explained by measurement error alone. We found no significant sex differences in telomere length, and provide evidence that early-life telomere length predicts lifespan. However, while early-life telomere length predicted survival to adulthood (≥1 year old), early-life telomere length did not predict adult survival probability. Furthermore, adult telomere length did not predict survival to the subsequent year. These results show that the relationship between early-life telomere length and lifespan was driven by conditions in early-life, where early-life telomere length varied strongly among cohorts. Our data provide evidence for associations between early-life telomere length and individual life history, and highlight the dynamics of telomere length across individual lifetimes due to individuals experiencing different early-life environments.

The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction.

Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12 myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast culture. Resveratrol (10 µM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the 'slow' type I MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for 'intermediate' and 'faster' IIx, IIa and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48-72 h. Overall, resveratrol evoked myotube hypertrophy in DM conditions while favouring 'slower' Myhc gene expression and acutely ameliorated impaired myotube growth observed during glucose restriction.