Abstract

Elevated breeding effort is known to increase an individual’s rate of senescence, although the underlying mechanisms are not well understood. One possibility is that the ability to resist senescence is limited by the availability of antioxidants, which are necessary to mitigate the deleterious effects of oxidative stress thought to underlie the aging process. Susceptibility to oxidative stress is likely to be particularly high during reproduction, and so for a given level of reproductive effort, the rate of senescence should be fastest in those individuals with the poorest antioxidant capacity. We tested this hypothesis in an experimental study of breeding male three-spined sticklebacks (Gasterosteus aculeatus) held under a high or low reproduction regime and fed on either high or low levels of carotenoids (potentially limiting dietary compounds with antioxidant properties). Fish on the high reproduction regime and those on the low-carotenoid diet both showed an accelerated decline in sustained swimming performance (an indicator of locomotor senescence) compared with males on the low reproduction regime and high-carotenoid diet, respectively. The swimming performance of fish subjected to the high rate of reproduction, but fed a low-carotenoid diet, appeared to decline most rapidly, perhaps because of the additive effects of increased levels of reproduction-induced oxidative stress and lower antioxidant availability. These findings show that both dietary carotenoid intake and breeding effort can impact on the age-related decline in swimming performance and have important implications for female choice and the capacity of males with insufficient antioxidant defenses to adequately perform paternal care.

A key assumption of most life-history evolution models is that increased allocation of resources to one function precludes the allocation of these resources to other functions (Stearns 1992). A classic example of this is the cost of reproduction, where elevated breeding effort leads to lowered subsequent fecundity and an increase in the rate of senescence (Stearns 1992). However, the proximate mechanisms underlying this trade-off are poorly understood, and in particular, the currency that is actually traded off is still under debate (Zera and Harshman 2001; Barnes and Partridge 2003). Models of life-history evolution have tended to assume that energy is the primary limitation, although recent studies have suggested that increased susceptibility to oxidative stress might represent a general cost of reproduction, independent of energy allocation (von Schantz et al. 1999; Monaghan et al. 2009).

Conditions of oxidative stress occur when there is an imbalance between the production of reactive oxygen species (ROS) and the capacity of the antioxidant system to neutralize them (Finkel and Holbrook 2000). ROS are by-products of normal metabolic activities (Fang et al. 2002) but can have a damaging effect on biomolecules, such as DNA, proteins, and lipids (Finkel and Holbrook 2000), the cumulative effects of which are thought to underlie the age-associated decline in performance (the free radical theory of ageing: Harman 1956; Finkel and Holbrook 2000; Kirkwood and Austad 2000). Antioxidant molecules and enzymes scavenge ROS and limit their detrimental effects (Surai 2002), but the antioxidant defense system may be unable to cope during times when ROS production is high. This may be particularly pronounced during energetically expensive activities because higher metabolic activity may result in elevated ROS production (Pamplona and Barja 2003; Monaghan et al. 2009). In particular, it is well established that reproduction is energetically costly (Zera and Harshman 2001) and usually results in increases in energy expenditure by elevating metabolic rate (Angilletta and Sears 2000; Nilsson 2002). Given that reproduction also requires resources that may have to be diverted away from other needs, such as somatic maintenance, it may increase the rate of ROS-induced damage and so accelerate the rate of senescence. However, individuals with access to sufficient antioxidants to limit the damaging effects of ROS would be predicted to show slower age-related declines in performance than those for which antioxidants are limiting.

Carotenoids are one class of compound which, through their role as antioxidants (Krinsky and Yeum 2003; El-Agamey et al. 2004), may protect against the damage caused by ROS and hence the costs of reproduction. Carotenoids cannot be synthesized de novo by vertebrates and so must be obtained through the diet. Carotenoid supply may therefore be limiting, and indeed, there is evidence from a variety of species that individuals with greater dietary access to carotenoids may have reduced levels of oxidative stress (Alonso-Álvarez et al. 2004; Bertrand et al. 2006; Pike, Blount, Bjerkeng, et al. 2007; Thomson et al. 2007). These effects have been suggested to underlie the enhanced ability of individuals with high access to carotenoids to engage in energetically expensive activities, such as parental care (Pike, Blount, Lindström, et al. 2007) and escape flight (Blount and Matheson 2006), and may explain the observed link between carotenoid supply and adult survival (Hill 1991; Hõrak et al. 2001; Pike, Blount, Bjerkeng, et al. 2007). However, experimental evidence for a link between the availability of carotenoids and an individual’s capacity to counteract the costs of reproduction is lacking. The three-spined stickleback (Gasterosteus aculeatus) is an ideal species in which to investigate this question. Many populations (including our study population) are annual and so are limited to a single breeding season during which individuals of both sexes invest heavily in reproduction. For males in particular, which must bear the costs of courtship followed by obligate paternal care (Wootton 1984), reproductive activities are energetically expensive (Chellappa et al. 1989; Fitzgerald et al. 1989; Smith and Wootton 1999), and individuals involved in paternal care show elevated metabolic rates compared with noncaring males (Smith and Wootton 1999). These costs mean that in annual populations, many breeding adults die before the end of the breeding season (Chellappa et al. 1989; Poizat et al. 1999). We have demonstrated previously that male sticklebacks fed a diet low in carotenoids suffered from an increased susceptibility to oxidative stress compared with those fed higher levels of carotenoids, leading to a reduced life span (Pike, Blount, Bjerkeng, et al. 2007). However, in this experiment, reproductive investment was not controlled for, and so the precise interaction between reproductive effort and carotenoid antioxidants is unclear. In this paper, we explicitly test the effect of this interaction on the rate of decline in swimming performance, which we use as an indicator of locomotor senescence as in other studies of small fish (Reznick et al. 2004; Valenzano et al. 2006).

METHODS

Source of fish and dietary manipulation

Juvenile three-spined sticklebacks were captured with dip nets from the River Endrick, Scotland (lat 56°04′N, long 4°23′W), during November 2005. After capture, individual fish were allocated randomly to 1 of 12 holding aquaria (80 × 50 × 30 cm, at a density of ∼30 fish per tank), each containing only a water filter and several artificial plants (to provide refuges and reduce stress), and held until the start of the natural breeding season under a simulated natural temperature and photoperiod regime that was updated each week to match the mean for that time of the year at the source river (e.g., mean for December: 4 °C, 8.25-h photoperiod; mean for May: 15 °C, 17.25-h photoperiod).

Throughout the experiment, fish were fed to satiation daily on a customized diet based on anchovy meal-based fish feed pellets, manufactured to lack supplemental antioxidants, including carotenoids. The experimental diets were prepared by mixing the fish pellets with equal quantities of astaxanthin (Carophyll Pink; DSM, Basel, Switzerland) and lutein (FloraGLO; Kemin Health, Des Moines, IA, which also contains approximately 4.2% zeaxanthin) at a total concentration of either 200 mg carotenoids kg−1 pellets (high-carotenoid diet) or 10 mg kg−1 (low-carotenoid diet) and grinding to a fine powder. This was then combined with carotenoid-free vegetable fat and red food coloring (10 ml kg−1; SuperCook, Leeds, UK). The vegetable fat prevented the feed from breaking up in the water, and the red coloring ensured that food was eaten promptly, as sticklebacks exhibit a strong preference for red food items (Smith et al. 2004). The 2 diets did not differ in nutritional content other than carotenoid levels and had a similar visual appearance. It was unlikely that total food intake differed between fish on the different diets because if presented with a simultaneous binary choice between high- and low-carotenoid feed prepared as described, there was no evidence that they preferred one over the other (mean ± standard deviation weight of food consumed in an 24-h binary choice trial, high-carotenoid food: 49.2 ± 18.4 mg and low-carotenoid food: 47.4 ± 18.9 mg; paired t-test: t = 1.21, N = 20, P = 0.24). The concentration and composition of carotenoids in both the high- and the low-carotenoid diets are known to allow male sticklebacks to develop normal sexual coloration and successfully engage in reproductive activities (Pike, Blount, Bjerkeng, et al. 2007). Algal growth was suppressed using 2-chloro-4, 6-bis-(ethylamino)-s-triazine (Algae Destroyer; Aquarium Pharmaceuticals, Chalfont, PA), and so we were confident that the only source of carotenoids available to these fish was through the diet.

Experimental design

We used 80 males for this experiment, 40 from the high-carotenoid diet treatment and 40 from the low-carotenoid diet. When males began to develop blue eye coloration (an indicator of sexual maturation), they were transferred to individual experimental aquaria (33 × 18 × 19 cm), where they were maintained on the same experimental diets. The experimental aquaria contained only a filter and an artificial plant and had the same photoperiod, temperature, and water conditions as the holding aquaria. Individual aquaria were separated by opaque partitions, so males were not in visual or olfactory contact with each other. Each male was provided with a nesting dish and nesting material (following Pike, Blount, Bjerkeng, et al. 2007) and shown a gravid female enclosed in a Plexiglas container for 5 min twice daily for 10 days, after which most males had developed red nuptial coloration and entered the courtship phase. In total, 67 males successfully completed nest building. The remaining 13 males (4 on the high-carotenoid diet and 9 on the low-carotenoid diet; binomial test comparing proportion of the total on each of the 2 diets: P = 0.267) failed to build nests and so were excluded from the experiment.

When nest building was complete, we quantified each breeding male’s sustained swimming performance as the amount of time a fish could swim against a current of water. This technique has been used successfully to measure swimming stamina in juvenile brown trout, Salmo trutta (Ojanguren and Braña 2000, 2003); green swordtails, Xiphophorus helleri (Royle et al. 2006); and sticklebacks (Álvarez and Metcalfe 2005), and full details of this particular experimental setup are given in Álvarez and Metcalfe (2005) and Royle et al. (2006). The experiments were conducted inside a temperature-controlled room that maintained the temperature at 14 ± 1 °C. One fish at a time was placed into a cylindrical swimming chamber (50-cm long and 20-cm diameter) through which could be pumped a steady but controllable flow of water. We initially set the flow rate at 4 cm s−1 (ca. 1 body length s−1) to allow the test fish to orient toward the water inflow and to acclimatize to the swimming chamber for 5 min. After this initial period, the velocity of the water was increased in one step to 20 cm s−1 (selected after preliminary trials) and maintained until fatigue. Fish were deemed to be exhausted when they fell back against the fine mesh grid at the downstream end of the compartment for more than 5 s (Ryan 1998) and were no longer able to continue swimming, despite tapping of the side of the chamber (Ojanguren and Braña 2000). Once exhausted, the pump was immediately turned off, and the subject was allowed 5-min recuperation time before being placed back in its nesting tank. Endurance is defined as the amount of time (s) that a fish swam at the higher flow rate.

At least 2 h after measuring sustained swimming performance, a gravid female was placed temporarily in each male’s tank and allowed to spawn. The males were then allowed to care for the eggs until the end of the incubation period (day 8, just prior to hatching) when the nests and nesting dishes were removed. After this first breeding attempt, reproductive rate was manipulated within each dietary treatment as follows. A random sample of 40 males (20 from each diet treatment) were allocated to the high reproduction group. These males were induced to build another nest and were presented with a new female every 20 days so that the cycle of breeding continued (exactly as described above) for a total of 3 breeding rounds. The remaining fish (14 on the high-carotenoid diet and 13 on the low-carotenoid diet) were allocated to the low reproduction group; their nesting dish was removed after the first breeding round, and they did not see another female. The sample size discrepancy between reproduction treatments was in order to maximize the number of males in the high reproduction group. Sustained swimming performance was measured in all fish from the high reproduction group that successfully built a new nest and all surviving fish from the low reproduction group every 20 days corresponding to the start of each new breeding cycle for high reproduction males. All nest-building males courted females and mated successfully, and therefore, the probability of neither courting nor mating was affected by the level of carotenoids in the diet. We also measured male standard length (±0.01 mm) and body weight (±0.1 g) immediately after each assessment of sustained swimming ability.

Statistical analysis

The seasonal change in sustained swimming performance was analyzed using a general linear mixed-effects model in R (R Core Development Team, version 2.8.0), with diet treatment (high or low carotenoid), reproduction group (high or low reproduction), and breeding round (first, second, or third) as fixed factors; a random effects term of breeding round given male identity; and body length (which is known to correlate with swimming performance in fish, e.g., Videler 1993) as a covariate. This allowed changes in an individual fish’s performance to be assessed over the breeding season.

RESULTS

A total of 6 males died during the experiment, but mortality did not differ between diet treatments or reproduction groups (binary logistic regression, diet: z = 0.87, P = 0.39 and group: z = 1.16, P = 0.25; Table 1). Furthermore, the number of high reproduction group males successfully building nests decreased over the season (Table 1), although there was no evidence that the number of rounds completed differed between high- and low-carotenoid diet males (Mann–Whitney test: W = 383.5, N1 = N2 = 20, P = 0.48).

Table 1

Data on the change in body weight, sustained swimming performance, number of fish successfully breeding, and the number of fish surviving over successive breeding rounds in relation to diet treatment (low or high levels of dietary carotenoids) and reproduction group (low or high levels of reproductive effort)

Breeding round Diet treatment Reproduction group Mean ± SD body weight (g) Mean ± SD sustained swimming performance (s) Number breeding (sample size) Number surviving 
Low Low 1.02 ± 0.30 131.9 ± 45.6 13 13 
Low High 0.94 ± 0.28 133.6 ± 32.7 20 20 
High Low 0.98 ± 0.31 131.0 ± 39.8 14 14 
High High 0.94 ± 0.26 126.9 ± 36.5 20 20 
Low Low 1.01 ± 0.29 117.3 ± 42.5 13 13 
Low High 0.94 ± 0.28 110.2 ± 34.6 14 19 
High Low 0.97 ± 0.32 119.6 ± 28.1 14 14 
High High 0.93 ± 0.26 123.8 ± 33.8 17 19 
Low Low 1.02 ± 0.29 107.1 ± 26.7 12 12 
Low High 0.94 ± 0.29 58.7 ± 22.2 17 
High Low 0.95 ± 0.31 118.9 ± 38.9 14 14 
High High 0.93 ± 0.27 100.1 ± 24.8 18 
Breeding round Diet treatment Reproduction group Mean ± SD body weight (g) Mean ± SD sustained swimming performance (s) Number breeding (sample size) Number surviving 
Low Low 1.02 ± 0.30 131.9 ± 45.6 13 13 
Low High 0.94 ± 0.28 133.6 ± 32.7 20 20 
High Low 0.98 ± 0.31 131.0 ± 39.8 14 14 
High High 0.94 ± 0.26 126.9 ± 36.5 20 20 
Low Low 1.01 ± 0.29 117.3 ± 42.5 13 13 
Low High 0.94 ± 0.28 110.2 ± 34.6 14 19 
High Low 0.97 ± 0.32 119.6 ± 28.1 14 14 
High High 0.93 ± 0.26 123.8 ± 33.8 17 19 
Low Low 1.02 ± 0.29 107.1 ± 26.7 12 12 
Low High 0.94 ± 0.29 58.7 ± 22.2 17 
High Low 0.95 ± 0.31 118.9 ± 38.9 14 14 
High High 0.93 ± 0.27 100.1 ± 24.8 18 

SD, standard deviation.

Male body weight declined significantly over the breeding season, but this did not differ between diet treatments or reproduction groups or their interaction with breeding round (Tables 1 and 2). There were, however, significant interactions between breeding round and both diet treatment and reproduction group on sustained swimming performance (Tables 1 and 2; Figure 1) such that fish on the high-carotenoid diet treatment had a slower rate of decline in swimming endurance than those on the low-carotenoid diet, and high reproduction group fish showed a faster rate of decline than those in the low reproduction treatment. The lack of significant interaction between diet treatment and reproduction group (Table 2) suggests that these 2 factors had an additive effect on swimming performance, explaining why the fastest rate of decline in swimming performance was observed in males on the low-carotenoid diet and in the high reproduction group (Figure 1). Swimming performance was also significantly predicted by body length (Table 2), with larger individuals performing better than smaller fish. The 3-way interaction between diet treatment, reproduction group, and breeding round on sustained swimming performance was not significant (Table 2). There was no evidence that swimming performance in one breeding round predicted the probability of successfully nest building in the subsequent round (generalized linear mixed model: F1,67 = 0.01, P = 0.93) and no interaction between breeding round and swimming ability (F1,67 = 0.14, P = 0.71).

Table 2

Output of general linear mixed models assessing the effects of the experimental manipulations (and their interactions) on body weight and sustained swimming performance

Variable Body weight Sustained swimming performance 
Diet treatment F1,63 = 0.09, P = 0.769 F1,63 = 1.92, P = 0.171 
Reproduction group F1,63 = 0.84, P = 0.361 F1,63 = 0.35, P = 0.554 
Breeding round F2,90 = 127.75, P < 0.001F2,90 = 15.88, P < 0.001 
Body length F1,90 = 0.32, P = 0.573 F1,90 = 7.61, P = 0.007 
Diet × reproduction F1,63 = 0.06, P = 0.802 F1,63 = 0.03, P = 0.861 
Diet × round F2,90 = 0.15, P = 0.864 F2,90 = 3.34, P = 0.039 
Reproduction × round F2,90 = 0.97, P = 0.381 F2,90 = 4.43, P = 0.015 
Diet × reproduction × round F2,90 = 1.71, P = 0.187 F2,90 = 0.69, P = 0.501 
Variable Body weight Sustained swimming performance 
Diet treatment F1,63 = 0.09, P = 0.769 F1,63 = 1.92, P = 0.171 
Reproduction group F1,63 = 0.84, P = 0.361 F1,63 = 0.35, P = 0.554 
Breeding round F2,90 = 127.75, P < 0.001F2,90 = 15.88, P < 0.001 
Body length F1,90 = 0.32, P = 0.573 F1,90 = 7.61, P = 0.007 
Diet × reproduction F1,63 = 0.06, P = 0.802 F1,63 = 0.03, P = 0.861 
Diet × round F2,90 = 0.15, P = 0.864 F2,90 = 3.34, P = 0.039 
Reproduction × round F2,90 = 0.97, P = 0.381 F2,90 = 4.43, P = 0.015 
Diet × reproduction × round F2,90 = 1.71, P = 0.187 F2,90 = 0.69, P = 0.501 
a

Significant terms (P < 0.05) are shown in bold.

Figure 1

Mean ± standard error change in sustained swimming endurance (a measure of locomotor senescence) over 3 successive breeding rounds for males on high- (black data points) and low- (white data points) carotenoid diets in the high (solid lines) and low (dashed lines) reproduction groups.

Figure 1

Mean ± standard error change in sustained swimming endurance (a measure of locomotor senescence) over 3 successive breeding rounds for males on high- (black data points) and low- (white data points) carotenoid diets in the high (solid lines) and low (dashed lines) reproduction groups.

DISCUSSION

This study tested the hypothesis that a high dietary availability of antioxidant carotenoids can offset the increased rate of senescence induced by enhanced reproductive effort. Our results show that in breeding male sticklebacks, both dietary carotenoid intake and breeding effort impact on the rate of decline in sustained swimming performance, which we used as an indicator of locomotor senescence. Fish on the high reproduction regime and those on the low-carotenoid diet both showed an accelerated decline in sustained swimming performance compared with males on the low reproduction regime and high-carotenoid diet, respectively. We predicted, a priori, a 3-way interaction between diet treatment, reproduction group, and breeding round on swimming performance, although found instead an additive effect of a diet low in carotenoids and increased reproductive effort. This may explain why males in the high reproduction and low-carotenoid condition showed the fastest rate of decline in swimming performance, whereas males in the other conditions (including those either receiving low levels of carotenoids or experiencing high levels of reproductive expenditure) showed similar slower rates of decline. Although the hypothesized 3-way interaction was not statistically significant, we note that the power of this analysis was likely to be fairly low due to the relatively small sample size in the later breeding rounds. It is therefore difficult to draw firm conclusions concerning this possible 3-way interaction from this result.

Sticklebacks on the low-carotenoid diet are known to have greater susceptibility to oxidative stress than high-carotenoid diet males (Pike, Blount, Bjerkeng, et al. 2007), probably because they can utilize carotenoids as antioxidants (Burton 1989; Kiokias and Gordon 2004) to inactivate ROS. Therefore, fish in this study receiving low levels of dietary carotenoids may have been unable to mitigate the accumulation of ROS-induced damage to biomolecules, resulting in accelerated decline in swimming performance (Harman 1956; Sohal et al. 1994; Beckman and Ames 1998; Finkel and Holbrook 2000). The faster decline in performance in males on the high reproduction group may similarly have been caused by energetically costly reproduction (Chellappa et al. 1989; Fitzgerald et al. 1989) elevating metabolic rate (Angilletta and Sears 2000; Nilsson 2002) and thereby increasing ROS production (Pamplona and Barja 2003; Monaghan et al. 2009). In addition, the slow rate of decline in swimming performance in low reproduction group males may have been facilitated by the fact that, unlike males in the high reproduction group, they were not regularly exposed to females and so may not have maintained a carotenoid-rich sexual signal. A greater pool of carotenoids may therefore have been available for combating ROS-induced somatic damage. We can hypothesize that those fish in the high reproduction group and on the low-carotenoid diet therefore experienced elevated levels of reproduction-induced oxidative stress but had inadequate levels of antioxidant carotenoids to combat this; the resulting levels of ROS-induced damage may underpin the apparently divergent increase in the decline in swimming performance in this group (Figure 1).

However, although both the current data and the previous findings on sticklebacks (e.g., Pike, Blount, Bjerkeng, et al. 2007) are consistent with a causal role of elevated oxidative stress in mediating the observed decline in swimming performance, alternative explanations also exist. In particular, reproductive activities may simply have reduced the available energy stores necessary for maintaining sustained swimming activity. We found that body weight declined slightly, although significantly, over successive breeding rounds in all treatment groups, but there was no difference between males on the different diet treatments or reproduction regimes. Because body weight is directly linked to available energy reserves in sticklebacks (Chellappa et al. 1989), this suggests that a reduction in energy reserves is unlikely to be the cause of the observed decline in swimming performance and that there was no difference in food intake between groups. We also cannot rule out the possibility that the decline in swimming performance was caused by an increased susceptibility to parasitic infection. Elevated stress levels, particularly in the high reproduction males, may have resulted in a decrease in immune function (e.g., Råberg et al. 1998), whereas low levels of carotenoids, which have been shown to have immunostimulatory properties (Blount et al. 2003; Faivre et al. 2003), may have had a negative effect on immune function in the low-carotenoid group. A significant 3-way interaction between the explanatory variables in which the high reproduction fish incurred a cost above and beyond the additive expectation for fish fed with the low-carotenoid diet would have been strong evidence for a single underlying mechanistic basis. Instead, therefore, these different mechanistic pathways may also have been acting independently, perhaps through the putative mechanisms described above. Our data cannot fully unravel these effects, and so, future work is needed to differentiate further between these alternative explanations.

Relatively few of the males actually died during the experiment, and the reduction in sample size over time was caused primarily by males ceasing to build nests. We found no differences in the mortality rate of males in the different treatment groups within the time frame of the experiment but suspect that if the subsequent fate of these males had been recorded, then such effects would have become apparent (as we have previously reported; Pike, Blount, Bjerkeng, et al. 2007). Furthermore, although there was no difference between groups in the number of breeding rounds completed, it is possible that a small number of males may have continued breeding beyond the 3 rounds of the current experiment or restarted breeding after one or more “skipped” rounds (Lindström et al. 2009).

These findings have important implications for female choice and the capacity of males with insufficient carotenoids or with a high reproductive burden to adequately perform paternal care. In sticklebacks, parental care is performed exclusively by the male and includes a number of energetically demanding components, such as changing nest structure with increasing egg development, removing dead and diseased eggs, and providing the eggs with oxygen and removing waste products by fanning movements of the pectoral fins (Wootton 1984; Chellappa et al. 1989). In particular, fanning can consume up to two-thirds of a male’s time budget (van Iersel 1953), and so, both fanning rate and bout length are likely to be severely affected by the current level of locomotor senescence, perhaps contributing to the reduced hatchability of eggs incubated by low-carotenoid diet males (Pike, Blount, Lindström, et al. 2007). It is likely that such males are also less able to perform energetically expensive aspects of courtship behavior, including the “zig-zag” courtship dance (Wootton 1984), and so may provide females with a direct means to assess a male’s current level of senescence and hence probability of successfully and adequately completing reproduction. Given that the differences in swimming endurance (and hence general fitness) became accentuated with successive breeding rounds, we should expect females to become more discriminating against these males as the breeding season progresses. This pattern has indeed been observed in fish from our study population in line with predictions from a theoretical model that predicts temporal changes in male condition, sexual attractiveness, and female choosiness (Lindström et al. 2009).

The relationships we observed between carotenoid intake, reproductive effort, and swimming performance are unlikely to be limited to male sticklebacks, and we would expect to find this relationship in any species of either sex where parental care is energetically expensive. However, such effects may be particularly evident where antioxidants are limiting in the diet or where there exist trade-offs between the allocation of antioxidants to somatic maintenance and their allocation to other functions, such as to sexual signaling (e.g., Alonso-Álvarez et al. 2004; Pike, Blount, Bjerkeng, et al. 2007; Monaghan et al. 2009) or egg production (e.g., Bertrand et al. 2006; Nordeide et al. 2006). Furthermore, there may be an effect of the type of antioxidant involved; for instance in many birds, where lutein and zeaxanthin are the predominant carotenoids, the in vivo antioxidant function of carotenoids has recently been questioned (e.g., Costantini and Møller 2007; Isaksson and Andersson 2008). In contrast, astaxanthin, which is commonly found in fish (including sticklebacks) and piscivorous birds, has a relatively higher antioxidant activity compared with other carotenoids (e.g., Di Mascio et al. 1991). The strength of relationships between carotenoid intake, reproductive effort, and senescence may therefore depend on the types of carotenoids predominating in the diet or the types that are preferentially absorbed by the species under study.

FUNDING

Natural Environment Research Council to N.B.M., J.D.B., and J.L.; Royal Society University Research Fellowship to J.D.B.

We thank Stirling University’s Institute of Aquaculture for providing the carotenoid-free food and J. Laurie and G. Adam for animal husbandry.

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