Association between telomere length and Plasmodium falciparum malaria endemicity in sub-Saharan Africans.

Leukocyte telomere length (LTL) varies significantly across human populations, with individuals of African ancestry having longer LTL than non-Africans. However, the genetic and environmental drivers of LTL variation in Africans remain largely unknown. We report here on the relationship between LTL, genetics, and a variety of environmental and climatic factors in ethnically diverse African adults (n = 1,818) originating from Botswana, Tanzania, Ethiopia, and Cameroon. We observe significant variation in LTL among populations, finding that the San hunter-gatherers from Botswana have the longest leukocyte telomeres and that the Fulani pastoralists from Cameroon have the shortest telomeres. Genetic factors explain ∼50% of LTL variation among individuals. Moreover, we observe a significant negative association between Plasmodium falciparum malaria endemicity and LTL while adjusting for age, sex, and genetics. Within Africa, adults from populations indigenous to areas with high malaria exposure have shorter LTL than those in populations indigenous to areas with low malaria exposure. Finally, we explore to what degree the genetic architecture underlying LTL in Africa covaries with malaria exposure.

High-throughput single telomere analysis using DNA microarray and fluorescent in situ hybridization.

The human telomere system is highly dynamic. Both short and long leucocyte average telomere lengths (aTL) are associated with an increased risk of cancer and early death, illustrating the complex relationship between TL and human health and the importance of assessing TL distributions with single TL analysis. A DNA microarray and telomere fluorescent in situ hybridization (DNA-array-FISH) approach was developed to measure the base-pair (bp) lengths of single telomeres. On average 32000 telomeres were measured per DNA sample with one microarray chip assaying 96 test DNA samples. Various telomere parameters, i.e. aTL and the frequency of short/long telomeres, were computed to delineate TL distribution. The intra-assay and inter-assay coefficient of variations of aTL ranged from 1.37% to 3.98%. The correlation coefficient (r) of aTL in repeated measurements ranged from 0.91 to 1.00, demonstrating high measurement precision. aTLs measured by DNA-array-FISH predicted aTLs measured by terminal restriction fragment (TRF) analysis with r ranging 0.87-0.99. A new accurate and high-throughput method has been developed to measure the bp lengths of single telomeres. The large number of single TL data provides an opportunity for an in-depth analysis of telomere dynamics and the complex relationship between telomere and age-related diseases.

Hot and dry conditions predict shorter nestling telomeres in an endangered songbird: Implications for population persistence.

Climate warming is increasingly exposing wildlife to sublethal high temperatures, which may lead to chronic impacts and reduced fitness. Telomere length (TL) may link heat exposure to fitness, particularly at early-life stages, because developing organisms are especially vulnerable to adverse conditions, adversity can shorten telomeres, and TL predicts fitness. Here, we quantify how climatic and environmental conditions during early life are associated with TL in nestlings of wild purple-crowned fairy-wrens (Malurus coronatus), endangered songbirds of the monsoonal tropics. We found that higher average maximum air temperature (range 31 to 45 °C) during the nestling period was associated with shorter early-life TL. This effect was mitigated by water availability (i.e., during the wet season, with rainfall), but independent of other pertinent environmental conditions, implicating a direct effect of heat exposure. Models incorporating existing information that shorter early-life TL predicts shorter lifespan and reduced fitness showed that shorter TL under projected warming scenarios could lead to population decline across plausible future water availability scenarios. However, if TL is assumed to be an adaptive trait, population viability could be maintained through evolution. These results are concerning because the capacity to change breeding phenology to coincide with increased water availability appears limited, and the evolutionary potential of TL is unknown. Thus, sublethal climate warming effects early in life may have repercussions beyond individual fitness, extending to population persistence. Incorporating the delayed reproductive costs associated with sublethal heat exposure early in life is necessary for understanding future population dynamics with climate change.

Ectoparasite presence and brood size manipulation interact to accelerate telomere shortening in nestling jackdaws.

Early-life conditions impact fitness, but whether the combined effect of extrinsic stressors is additive or synergistic is not well known. This is a major knowledge gap because exposure to multiple stressors is frequent. Telomere dynamics may be instrumental when testing how stressors interact because many factors affect telomere shortening, and telomere shortening predicts survival. We evaluated the effect of manipulated brood size and natural infestation by the carnid fly Carnus hemapterus on nestling growth and telomere shortening of wild jackdaws (Corvus monedula). Telomere length, measured in blood using TRF, shortened on average by 264 bp, and on average, Carnus infection induced more telomere shortening. Further analyses showed that in enlarged broods, nestlings' telomeres shortened more when parasitized, while in reduced broods there was no effect of infection on telomere shortening. We conclude that there is a synergistic effect of number of siblings and Carnus infection on telomere shortening rate: blood-sucking parasites may negatively impact telomeres by increasing cell proliferation and/or physiological stress, and coping with infection may be less successful in enlarged broods with increased sibling competition. Larger nestlings had shorter telomeres independent of age, brood manipulation or infection. Growth was independent of infestation but in enlarged broods, nestlings were lighter at fledging. Our findings indicate that (i) evaluating consequences of early-life environmental conditions in isolation may not yield a full picture due to synergistic effects, and (ii) effects of environmental conditions may be cryptic, for example, on telomeres, with fitness consequences expressed beyond the temporal framework of the study.

When does early-life telomere length predict survival? A case study and meta-analysis.

Suboptimal conditions during development can shorten telomeres, the protective DNA caps on the end of chromosomes. Shorter early-life telomere length (TL) can indicate reduced somatic maintenance, leading to lower survival and shorter lifespan. However, despite some clear evidence, not all studies show a relationship between early-life TL and survival or lifespan, which may be due to differences in biology or study design (e.g., survival period measured). In superb fairy-wrens (Malurus cyaneus), we assessed whether early-life TL predicts mortality across different life-history stages (fledgling, juvenile, adult). However, in contrast to a similar study on a congener, early-life TL did not predict mortality across any life stage in this species. We then performed a meta-analysis including 32 effect sizes from 23 studies (15 birds and three mammals) to quantify the effect of early-life TL on mortality whilst taking into consideration potential sources of biological and methodological variation. Overall, the effect of early-life TL on mortality was significant, corresponding to a 15% reduction in mortality risk with each standard deviation increase in TL. However, the effect became weaker when correcting for publication bias. Contrary to our predictions, there was no evidence that effects of early-life TL on mortality varied with species lifespan or the period over which survival was measured. However, negative effects of early-life TL on mortality risk were pervasive throughout life. These results imply that effects of early-life TL on mortality are more likely to be context-dependent than age-dependent, although substantial power and publication bias issues highlight the need for more research.

Cool, dry nights and short heatwaves during growth result in longer telomeres in temperate songbird nestlings.

Exposure to rising sublethal temperatures can affect development and somatic condition, and thereby Darwinian fitness. In the context of climate warming, these changes could have implications for population viability, but they can be subtle and consequently difficult to quantify. Using telomere length (TL) as a known biomarker of somatic condition in early life, we investigated the impact of pre-hatching and nestling climate on six cohorts of wild nestling superb fairy wrens (Malurus cyaneus) in temperate south-eastern Australia. Models incorporating only climate information from the nestling phase were best supported compared to those including the (pre-)laying to incubation phase (previously shown to affect mass) or both phases combined. This implies that nestling TL is most sensitive to ambient climate in the nestling phase. The top model showed a negative relationship between early-life TL and nestling mean daily minimum temperature when rainfall was low which gradually became positive with increasing rainfall. In addition, there was a positive relationship between TL and the frequency of hot days (daily maximum temperature ≥35°C), although these temperatures were rare and short-term. Including other pre-hatching and nestling period, climate variables (e.g., mean daily maximum temperature and mean diurnal temperature variability) did not improve the prediction of nestling TL. Overall, our results suggest that cooler nights when conditions are dry and short-term temperature spikes above 35°C during development are conducive for somatic maintenance. While these findings indicate a potential pathway for climate warming to impact wildlife fitness, they emphasize the need to elucidate the mechanisms underlying these complex associations.

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.

Short- and long-term effects of nutritional state on IGF-1 levels in nestlings of a wild passerine.

Growth trajectories of young animals are intimately connected to their fitness prospects, but we have little knowledge of growth regulation mechanisms, particularly in the wild. Insulin-like growth factor 1 (IGF-1) is a central hormone in regulating resource allocation, with higher IGF-1 levels resulting in more growth. IGF-1 levels generally increase in conjunction with nutritional state, but whether IGF-1 levels are adjusted in response to current nutrient availability or to the nutrient availability integrated over a longer term is not well known. We tested for such effects by supplementary feeding the jackdaw (Corvus monedula) nestlings in experimentally reduced or enlarged broods with either water (control) or a food solution; these manipulations have long- and short-term effects on the nutritional state, respectively. Baseline plasma IGF-1 levels were higher in reduced broods. Food supplementation induced an increase in plasma IGF-1 levels measured one hour later, and this effect was significantly more substantial in nestlings in reduced broods. Changes in plasma IGF-1 levels increased with increased retention of the supplementary food, which was higher in reduced broods, explaining the stronger IGF-1 response. Thus, IGF-1 levels respond to short-term variations in the nutritional state, but this effect is amplified by longer-term variations in the nutritional state. We discuss our findings using a graphical model that integrates the results of the two treatments.

IGF-1 receptor inhibitor OSI-906 reduces growth in nestlings of a wild passerine.

Young animals need to grow to a large body size fast to maximise their survival prospects until sexual maturity. However, body size varies substantially in wild populations, and neither the selection pressures maintaining this variation, nor the regulatory mechanisms are well understood. IGF-1 administration has been shown to accelerate growth, but this does not necessarily imply that natural variation in growth rate is IGF-1 dependent. To test the latter we administered OSI-906 to pied flycatcher Ficedula hypoleuca nestlings, which has an inhibitory effect on IGF-1 receptor activity. We performed the experiment in two breeding seasons to test the prediction that blocking the IGF-1 receptor downregulates growth. As predicted, OSI-906 treated nestlings had lower body mass and reached a smaller structural size than siblings receiving a vehicle only, with the mass difference being most profound at the age preceding the highest body mass growth rate. The IGF-1 receptor inhibition effect on growth varied with age and year of study, and we discuss possible explanations. The OSI-906 administration results indicate that natural variation in growth rate is regulated by IGF-1, and constitutes a novel tool to study causes and consequences of growth variation, but details of the underlying mechanism still need to be resolved.

Genetics and geography of leukocyte telomere length in sub-Saharan Africans.

Leukocyte telomere length (LTL) might be causal in cardiovascular disease and major cancers. To elucidate the roles of genetics and geography in LTL variability across humans, we compared LTL measured in 1295 sub-Saharan Africans (SSAs) with 559 African-Americans (AAms) and 2464 European-Americans (EAms). LTL differed significantly across SSAs (P = 0.003), with the San from Botswana (with the oldest genomic ancestry) having the longest LTL and populations from Ethiopia having the shortest LTL. SSAs had significantly longer LTL than AAms [P = 6.5(e-16)] whose LTL was significantly longer than EAms [P = 2.5(e-7)]. Genetic variation in SSAs explained 52% of LTL variance versus 27% in AAms and 34% in EAms. Adjustment for genetic variation removed the LTL differences among SSAs. LTL genetic variation among SSAs, with the longest LTL in the San, supports the hypothesis that longer LTL was ancestral in humans. Identifying factors driving LTL variation in Africa may have important ramifications for LTL-associated diseases.

Telomere dynamics in relation to experimentally increased locomotion costs and fitness in great tits.

Evidence that telomere length (TL) and dynamics can be interpreted as proxy for 'life stress' experienced by individuals stems largely from correlational studies. We tested for effects of an experimental increase of workload on telomere dynamics by equipping male great tits (Parus major) with a 0.9 g backpack for a full year. In addition, we analysed associations between natural life-history variation, TL and TL dynamics. Carrying 5% extra weight for a year did not significantly accelerate telomere attrition. This agrees with our earlier finding that this experiment did not affect survival or future reproduction. Apparently, great tit males were able to compensate behaviourally or physiologically for the increase in locomotion costs we imposed. We found no cross-sectional association between reproductive success and TL, but individuals with higher reproductive success (number of recruits) lost fewer telomere base pairs in the subsequent year. We used the TRF method to measure TL, which method yields a TL distribution for each sample, and the association between reproductive success and telomere loss was more pronounced in the higher percentiles of the telomere distribution, in agreement with the higher impact of ageing on longer telomeres within individuals. Individuals with longer telomeres and less telomere shortening were more likely to survive to the next breeding season, but these patterns did not reach statistical significance. Whether successful individuals are characterized by losing fewer or more base pairs from their telomeres varies between species, and we discuss aspects of ecology and social organisation that may explain this variation.

High heritability of telomere length and low heritability of telomere shortening in wild birds.

Telomere length and telomere shortening predict survival in many organisms. This raises the question of the contribution of genetic and environmental effects to variation in these traits, which is still poorly known, particularly for telomere shortening. We used experimental (cross-fostering) and statistical (quantitative genetic "animal models") means to disentangle and estimate genetic and environmental contributions to telomere length variation in pedigreed free-living jackdaws (Corvus monedula). Telomere length was measured twice in nestlings, at ages 4 (n = 715) and 29 days (n = 474), using telomere restriction fragment (TRF) analysis, adapted to exclude interstitial telomeric sequences. Telomere length shortened significantly over the nestling period (10.4 ± 0.3 bp day-1 ) and was highly phenotypically (rP  = 0.95 ± 0.01) and genetically (rG  > 0.99 ± 0.01) correlated within individuals. Additive genetic effects explained a major part of telomere length variation among individuals, with its heritability estimated at h2  = 0.74 on average. We note that TRF-based studies reported higher heritabilities than qPCR-based studies, and we discuss possible explanations. Parent-offspring regressions yielded similar heritability estimates for mothers and fathers when accounting for changes in paternal telomere length over life. Year effects explained a small but significant part of telomere length variation. Heritable variation for telomere shortening was low (h2  = 0.09 ± 0.11). The difference in heritability between telomere length (high) and telomere shortening (low) agrees with evolutionary theory, in that telomere shortening has stronger fitness consequences in this population. Despite the high heritability of telomere length, its evolvability, which scales the additive genetic variance by mean telomere length, was on average 0.48%. Hence, evolutionary change of telomere length due to selection is likely to be slow.

Telomere length is highly repeatable and shorter in individuals with more elaborate sexual ornamentation in a short-lived passerine.

Quantifying an individual's state as a fitness proxy has proven challenging, but accumulating evidence suggests that telomere length and attrition may indicate individual somatic state and success at self-maintenance, respectively. Sexual ornamentation is also thought to signal phenotypic quality, but links between telomeres and sexual ornamentation have been little explored. To address this issue, we examined whether telomere length and dynamics are predicted by the expression of a sexually selected ornament, the length of the outermost tail feathers (streamers), using longitudinal data from a population of European barn swallows (Hirundo rustica). In 139 adult individuals, each measured twice, we further assessed associations of telomere length with age, sex, breeding status and survival. Telomere length showed high individual repeatability (R = .97) across years while shortening with age in both sexes. Telomere length and dynamics were not significantly associated with survival to the next year, remaining lifespan or reproduction status (comparing breeding and nonbreeding yearlings). Tail streamer length, a sexually selected trait in barn swallows, was negatively associated with telomere length, independent of sex. Thus, telomere length may reflect the costs of carrying an elaborated sexual ornament, although ornament size did not significantly predict telomere shortening. In conclusion, telomere length in adult barn swallows is a highly consistent trait that shows a negative relationship with sexual ornamentation, suggesting a trade-off between sexual ornamentation and telomere length.

Telomere length is highly heritable and independent of growth rate manipulated by temperature in field crickets.

Many organisms are capable of growing faster than they do. Restrained growth rate has functionally been explained by negative effects on lifespan of accelerated growth. However, the underlying mechanisms remain elusive. Telomere attrition has been proposed as a causal agent and has been mostly studied in endothermic vertebrates. We established that telomeres exist as chromosomal-ends in a model insect, the field cricket Gryllus campestris, using terminal restriction fragment and Bal 31 methods. Telomeres comprised TTAGGn repeats of 38 kb on average, more than four times longer than the telomeres of human infants. Bal 31 assays confirmed that telomeric repeats were located at the chromosome-ends. We tested whether rapid growth between day 1, day 65, day 85, and day 125 is achieved at the expense of telomere length by comparing nymphs reared at 23°C with their siblings reared at 28°C, which grew three times faster in the initial 65 days. Surprisingly, neither temperature treatment nor age affected average telomere length. Concomitantly, the broad sense heritability of telomere length was remarkably high at ~100%. Despite high heritability, the evolvability (a mean-standardized measure of genetic variance) was low relative to that of body mass. We discuss our findings in the context of telomere evolution. Some important features of vertebrate telomere biology are evident in an insect species dating back to the Triassic. The apparent lack of an effect of growth rate on telomere length is puzzling, suggesting strong telomere length maintenance during the growth phase. Whether such maintenance of telomere length is adaptive remains elusive and requires further study investigating the links with fitness in the wild.

Telomere length is heritable and genetically correlated with lifespan in a wild bird.

Telomeres are protective caps at the end of eukaryotic chromosomes that shorten with age and in response to stressful or resource-demanding conditions. Their length predicts individual health and lifespan across a wide range of animals, but whether the observed positive association between telomere length and lifespan is environmentally induced, or set at conception due to a shared genetic basis, has not been tested in wild animals. We applied quantitative genetic "animal models" to longitudinal telomere measurements collected over a 10-year period from individuals of a wild seabird (common tern; Sterna hirundo) with known pedigree. We found no variation in telomere shortening with age among individuals at the phenotypic and genetic level, and only a small permanent environmental effect on adult telomere length. Instead, we found telomere length to be highly heritable and strongly positively genetically correlated with lifespan. Such heritable differences between individuals that are set at conception may present a hitherto underappreciated component of variation in somatic state.

Telomere dynamics in the first year of life, but not later in life, predict lifespan in a wild bird.

Telomeres are protective, nucleoprotein structures at the end of chromosomes that have been associated with lifespan across taxa. However, the extent to which these associations can be attributed to absolute length vs. the rate of telomere shortening prior to sampling remains unresolved. In a longitudinal study, we examined the relationship between lifespan, telomere length and the rate of telomere shortening in wild, purple-crowned fairy-wrens (Malurus coronatus coronatus). To this end, we measured telomere length using quantitative polymerase chain reaction in the blood of 59 individuals sampled as nestlings and 4-14 months thereafter, and in 141 known-age individuals sampled on average three times across adulthood. We applied within-subject centring analyses to simultaneously test for associations between lifespan and average telomere length and telomere shortening. We reveal that the rate of telomere shortening and to a lesser extent telomere length in the first year of life independently predicted lifespan, with individuals with faster shortening rates and/or shorter telomeres living less long. In contrast, in adulthood neither telomere shortening nor telomere length predicted lifespan, despite a considerably larger data set. Our results suggest that telomere length measured very early in life (during development) and longitudinal assessments of telomere shortening during the first year of life constitute more useful biomarkers of total life expectancy than either telomere length measured after development, or telomere shortening later in adulthood.

Glucose tolerance predicts survival in old zebra finches.

The capacity to deal with external and internal challenges is thought to affect fitness, and the age-linked impairment of this capacity defines the ageing process. Using a recently developed intra-peritoneal glucose tolerance test (GTT), we tested for a link between the capacity to regulate glucose levels and survival in zebra finches. We also investigated for the effects of ambient factors, age, sex, and manipulated developmental and adult conditions (i.e. natal brood size and foraging cost, in a full factorial design) on glucose tolerance. Glucose tolerance was quantified using the incremental 'area under the curve' (AUC), with lower values indicating higher tolerance. Glucose tolerance predicted survival probability in old birds, above the median age, with individuals with higher glucose tolerance showing better survival than individuals with low or intermediate glucose tolerance. In young birds there was no association between glucose tolerance and survival. Experimentally induced adverse developmental conditions did not affect glucose tolerance, but low ambient temperature at sampling and hard foraging conditions during adulthood induced a fast return to baseline levels (i.e. high glucose tolerance). These findings can be interpreted as an efficient return to baseline glucose levels when energy requirements are high, with glucose presumably being used for energy metabolism or storage. Glucose tolerance was independent of sex. Our main finding that old birds with higher glucose tolerance had better survival supports the hypothesis that the capacity to efficiently cope with a physiological challenge predicts lifespan, at least in old birds.

A comparison of continuous and intermittent EEG recordings in geese: How much data are needed to reliably estimate sleep-wake patterns?

Recent technological advancements allow researchers to measure electrophysiological parameters of animals, such as sleep, in remote locations by using miniature dataloggers. Yet, continuous recording of sleep might be constrained by the memory and battery capacity of the recording devices. These limitations can be alleviated by recording intermittently instead of continuously, distributing the limited recording capacity over a longer period. We assessed how reduced sampling of sleep recordings affected measurement precision of NREM sleep, REM sleep, and Wake. We analysed a dataset on sleep in barnacle geese that we resampled following 12 different recording schemes, with data collected for 1 min per 5 min up to 1 min per 60 min in steps of 5 min. Recording 1 min in 5 min still yielded precise estimates of hourly sleep-wake values (correlations of 0.9) while potentially extending the total recording period by a factor of 5. The correlation strength gradually decreased to 0.5 when recording 1 min per 60 min. For hourly values of Wake and NREM sleep, the correlation strength in winter was higher compared with summer, reflecting more fragmented sleep in summer. Interestingly for hourly values of REM sleep, correlations were unaffected by season. Estimates of total 24 h sleep-wake values were similar for all intermittent recording schedules compared to the continuous recording. These data indicate that there is a large safe range in which researchers can periodically record sleep. Increasing the sample size while maintaining precision can substantially increase the statistical power, and is therefore recommended whenever the total recording time is limited.

Effects of manipulated food availability and seasonality on innate immune function in a passerine.

The innate immune system is essential for survival, yet many immune traits are highly variable between and within individuals. In recent years, attention has shifted to the role of environmental factors in modulating this variation. A key environmental factor is food availability, which plays a major role in shaping life histories, and may affect resource allocation to immune function through its effect on nutritional state. We developed a technique to permanently increase foraging costs in seed-eating birds, and leveraged this technique to study the effects of food availability on the innate immune system over a 3-year period in 230 zebra finches housed in outdoor aviaries. The immune components we studied were haptoglobin, ovotransferrin, nitric oxide, natural antibodies through agglutination, complement-mediated lysis, and killing capacity of Escherichia coli and Candida albicans, covering a broad spectrum of the innate immune system. We explored the effects of food availability in conjunction with other potentially important variables: season, age, sex and manipulated natal brood size. Increased foraging costs affected multiple components of the immune system, albeit in a variable way. Nitric oxide and agglutination levels were lower under harsh foraging conditions, while Escherichia coli killing capacity was increased. Agglutination levels also varied seasonally, but only at low foraging costs. C. albicans killing capacity was lower in winter, and even more so for animals in harsh foraging conditions that were raised in large broods. Effects of food availability on ovotransferrin were also seasonal, and only apparent in males. Haptoglobin levels were independent of foraging costs and season. Males had higher levels of immune function than females for three of the measured immune traits. Innate immune function was independent of age and manipulated natal brood size. Our finding that food availability affects innate immune function suggests that fitness effects of food availability may at least partially be mediated by effects on the immune system. However, food availability effects on innate immunity varied in direction between traits, illustrating the complexity of the immune system and precluding conclusions on the level of disease resistance.

Experimentally manipulated food availability affects offspring quality but not quantity in zebra finch meso-populations.

Food availability modulates survival, reproduction and thereby population size. In addition to direct effects, food availability has indirect effects through density of conspecifics and predators. We tested the prediction that food availability in isolation affects reproductive success by experimentally manipulating food availability continuously for 3 years in zebra finches (Taeniopygia guttata) housed in outdoor aviaries. To this end, we applied a technique that mimics natural variation in food availability: increasing the effort required per food reward without affecting diet. Lower food availability resulted in a slight delay of start of laying and fewer clutches per season, but did not affect clutch size or number of offspring reared per annum. However, increasing foraging costs substantially reduced offspring growth. Thus, food availability in isolation did not impact the quantity of offspring reared, at the expense of offspring quality. Growth declined strongly with brood size, and we interpret the lack of response with respect to offspring number as an adaptation to environments with low predictability, at the time of egg laying, of food availability during the period of peak food demand, typically weeks later. Manipulated natal brood size of the parents did not affect reproductive success. Individuals that were more successful reproducers were more likely to survive to the next breeding season, as frequently found in natural populations. We conclude that the causal mechanisms underlying associations between food availability and reproductive success in natural conditions may be more complex than usually assumed. Experiments in semi-natural meso-populations can contribute to further unravelling these mechanisms.