Carlos Alonso-Alvarez

An Experimental Test of the Dose-Dependent Effect of Carotenoids and Immune Activation on Sexual Signals and Antioxidant Activity

Carotenoid‐based sexual traits are thought to be reliable indicators of male quality because they might be scarce and therefore might indicate the ability of males to gather high‐quality food and because they are involved in important physiological functions (as immune enhancers and antioxidants). We performed an experiment where male and female zebra finches (Taeniopygia guttata) were provided with increasing carotenoid doses in the drinking water during 4 weeks (bill color of this species is a carotenoid‐based sexual signal). Simultaneously, birds were split into two groups: one receiving weekly injections of Escherichia coli lipopolysaccharide in order to activate the immune system, the other being injected with the same volume of phosphate buffered saline. We assessed how carotenoid availability and immune activation affected the amount of circulating plasma carotenoids, the beak color, and the antioxidant defenses (assessed as the resistance of red blood cells to a controlled free radical attack). Carotenoid availability affected the amount of circulating carotenoids and beak color; both variables reached a plateau at the highest carotenoid doses. Immune activation diverted carotenoids from plasma, and this in turn affected the expression of the sexual trait. Finally, we found a positive correlation between the change in circulating carotenoids and antioxidant defenses. These results support the idea that carotenoids have important physiological properties that ensure the honesty of carotenoid‐based sexual traits.

Testosterone and oxidative stress: the oxidation handicap hypothesis

Secondary sexual traits (SST) are usually thought to have evolved as honest signals of individual quality during mate choice. Honesty of SST is guaranteed by the cost of producing/maintaining them. In males, the expression of many SST is testosterone-dependent. The immunocompetence handicap hypothesis has been proposed as a possible mechanism ensuring honesty of SST on the basis that testosterone, in addition to its effect on sexual signals, also has an immunosuppressive effect. The immunocompetence handicap hypothesis has received mixed support. However, the cost of testosterone-based signalling is not limited to immunosuppression and might involve other physiological functions such as the antioxidant machinery. Here, we tested the hypothesis that testosterone depresses resistance to oxidative stress in a species with a testosterone-dependent sexual signal, the zebra finch. Male zebra finches received subcutaneous implants filled with flutamide (an anti-androgen) or testosterone, or kept empty (control). In agreement with the prediction, we found that red blood cell resistance to a free radical attack was the highest in males implanted with flutamide and the lowest in males implanted with testosterone. We also found that cell-mediated immune response was depressed in testosterone-treated birds, supporting the immunocompetence handicap hypothesis. The recent finding that red blood cell resistance to free radicals is negatively associated with mortality in this species suggests that benefits of sexual signalling might trade against the costs derived from oxidation.

AN EXPERIMENTAL MANIPULATION OF LIFE-HISTORY TRAJECTORIES AND RESISTANCE TO OXIDATIVE STRESS

Abstract Optimal investment into life‐history traits depends on the environmental conditions that organisms are likely to experience during their life. Evolutionary theory tells us that optimal investment in reproduction versus maintenance is likely to shape the pattern of age‐associated decline in performance, also known as aging. The currency that is traded against different vital functions is, however, still debated. Here, we took advantage of a phenotypic manipulation of individual quality in early life to explore (1) long‐term consequences on life‐history trajectories, and (2) the possible physiological mechanism underlying the life‐history adjustments. We manipulated phenotypic quality of a cohort of captive zebra finches (Taeniopygia guttata) by assigning breeding pairs to either an enlarged or a reduced brood. Nestlings raised in enlarged broods were in poorer condition than nestlings raised in reduced broods. Interestingly, the effect of environmental conditions experienced during early life extended to the age at first reproduction. Birds from enlarged broods delayed reproduction. Birds that delayed reproduction produced less offspring but lived longer, although neither fecundity nor longevity were directly affected by the experimental brood size. Using the framework of the life‐table response experiment modeling, we also explored the effect of early environmental condition on population growth rate and aging. Birds raised in reduced broods tended to have a higher population growth rate, and a steeper decrease of reproductive value with age than birds reared in enlarged broods. Metabolic resources necessary to fight off the damaging effect of reactive oxygen species (ROS) could be the mechanism underlying the observed results, as (1) birds that engaged in a higher number of breeding events had a weaker red blood cell resistance to oxidative stress, (2) red blood cell resistance to oxidative stress predicted short‐term mortality (but not longevity), and (3) was related with a parabolic function to age. Overall, these results highlight that early condition can have long‐term effects on life‐history trajectories by affecting key life‐history traits such as age at first reproduction, and suggest that the trade‐off between reproduction and self‐maintenance might be mediated by the cumulative deleterious effect of ROS.

An Intracellular Antioxidant Determines the Expression of a Melanin-Based Signal in a Bird

To understand how traits used in animal communication evolved and are maintained as honest signals, we need to understand the mechanisms that prevent cheating. It has been proposed that honest signaling is guaranteed by the costs associated with the signal expression. However, the nature of these costs is still under debate. Melanin-based signals are intriguing because their expression seems to be tightly controlled by genes and the resource involved (i.e. melanin) seems to be not limited. However, in vertebrates, low levels of a key intracellular antioxidant (i.e. glutathione) are needed to promote melanogenesis. We propose that melanin-based ornaments can signal the ability to cope with oxidative stress because those individuals with low enough levels of glutathione, such as those required for melanin production, should manage well the whole of the antioxidant machinery in order to maintain a certain oxidative status. We analysed the expression of a melanin-based signal: the well-known black stripe of the great tit (Parus major). Great tit nestlings were injected with a specific inhibitor of glutathione production (DL-buthionine-S,R-sulfoximine; BSO) throughout their development. BSO effectively decreased intracellular glutathione levels without apparent side effects on growth or body condition. Instead, treated nestlings developed black breast stripes 70–100% larger than controls. Moreover, treated nestlings also compensated the decrease in glutathione levels by increasing the levels of circulating antioxidants. Results indicate that melanin-based signals can be at least partially permeable to environmental influences such as those associated to oxidative stress. They also reveal a potential handicap associated to the expression of this kind of signals. Finally, although other contributing factors could have been present, our findings emphasize the role of oxidative stress in shaping the evolution of animal signals in general and, in particular, those produced by pigments.

Carotenoids modulate the trade-off between egg production and resistance to oxidative stress in zebra finches

The allocation of resources to reproduction and survival is a central question of studies of life history evolution. Usually, increased allocation to current reproduction is paid in terms of reduced future reproduction and/or decreased survival. However, the proximal mechanisms underlying the cost of reproduction are poorly understood. Recently, it has been shown that increased susceptibility to oxidative stress might be one of such proximate links between reproduction and self-maintenance. Organisms possess a range of antioxidant defenses, including endogenously produced molecules (e.g., enzymes) and compounds ingested with food (e.g., carotenoids). If reproductive effort increases the production of reactive oxygen species, the availability of antioxidant defenses may partly or fully counteract the free-radical damages. One could, therefore, expect that the trade-off between reproduction and oxidative stress is modulated by the availability of antioxidant defenses. We tested this hypothesis in zebra finches. We manipulated reproductive effort by either allowing or preventing pairs to breed. Within each breeding or non-breeding group, the availability of antioxidant compounds was manipulated by supplementing or not supplementing the drinking water with carotenoids. We found that although birds in the breeding and non-breeding groups did not differ in their resistance to oxidative stress (the breakdown of red blood cells submitted to a controlled free-radical attack), one aspect of breeding effort (i.e., the number of eggs laid by birds in both breeding and non-breeding groups) was negatively correlated with resistance to oxidative stress only in birds that did not benefit from a carotenoid-supplemented diet. This result therefore suggests that carotenoid availability can modulate the trade-off between reproduction and resistance to oxidative stress.

The oxidation handicap hypothesis and the carotenoid allocation trade‐off

The oxidation handicap hypothesis proposes that testosterone mediates the trade‐off between the expression of secondary sexual traits and the fight against free radicals. Coloured traits controlled by testosterone can be produced by carotenoid pigments (yellow–orange–red traits), but carotenoids also help to quench free radicals. Recently, it has been shown that testosterone increases the amount of circulating carotenoids in birds. Here, a testosterone‐mediated trade‐off in the carotenoid allocation between colour expression and the fight against oxidative stress is proposed. Male red‐legged partridges were treated with testosterone, anti‐androgens or manipulated as controls. Testosterone‐treated males maintained the highest circulating carotenoid levels, but showed the palest red traits and no evidence of oxidative damage. Increased levels of a key intracellular antioxidant (i.e. glutathione) indicated that an oxidative challenge was in fact induced but controlled. The trade‐off was apparently solved by reducing redness, allowing increased carotenoid availability, which could have contributed to buffer oxidative stress.

The expression of melanin-based plumage is separately modulated by exogenous oxidative stress and a melanocortin.

Melanin-based traits involved in animal communication have been traditionally viewed as occurring under strict genetic control. However, it is generally accepted that both genetic and environmental factors influence melanin production. Medical studies suggest that, among environmental factors influencing melanization, oxidative stress could play a relevant role. On the other hand, genetic control would be exerted by the melanocortin system, and particularly by the alpha-melanocyte-stimulating hormone (α-MSH), which triggers the production of eumelanins (black pigments). To determine how the melanocortin system and an exogenous source of oxidative stress interact in the expression of melanin-based plumage, developing red-legged partridges (Alectoris rufa) were manipulated. Some partridges were injected with α-MSH, while other birds received a pro-oxidant molecule (diquat) in drinking water. Controls and birds receiving both treatments were also studied. Both α-MSH- and diquat-treated individuals presented larger eumelanin-based traits than controls, but α-MSH+diquat-treated birds showed the largest traits, suggesting that oxidative stress and melanocortins promote additive but independent effects. Diquat also induced a decline in the level of a key intracellular antioxidant (glutathione), which is associated with high expression of eumelanin-based signals in other bird species. Some scenarios for the evolution of melanin-based traits in relation to oxidative stress are proposed.

Cell-mediated immune activation rapidly decreases plasma carotenoids but does not affect oxidative stress in red-legged partridges (Alectoris rufa).

SUMMARY In animals yellow-orange-red sexual traits pigmented by carotenoids have been suggested to act as signals of current health. Because carotenoids have important physiological functions, individuals might trade-off allocating these pigments to self-maintenance versus coloration. Carotenoids may act as scavengers of free radicals that are released during an immune response. Here, we experimentally assessed whether a local cell-mediated immune response affects circulating carotenoids, antioxidant status, oxidative damage and the expression of a carotenoid-based trait. Male red-legged partridges (Alectoris rufa) were subcutaneously injected with phytohaemagglutinin (PHA) or with phosphate buffer solution (controls). The effect of the treatment on circulating carotenoids, total plasma antioxidant status (TAS), lipid oxidative damage in erythrocytes (TBARS) and ornamentation was assessed. Immune challenge induced a 13% decrease in circulating carotenoids within 24 h. However, this treatment did not affect TAS, TBARS or coloration. Coloration, circulating carotenoids and cell-mediated immune response were positively correlated, but these were not related to TAS or TBARS. Carotenoids were only weakly related to TAS after controlling for the effect of uric acid levels. These results suggest that carotenoid-based ornaments may honestly indicate immunocompetence but probably not antioxidant capacity in this species, and that carotenoids might be relatively weak antioxidants in the plasma. Furthermore, even a relatively harmless and locally elicited immune challenge had important effects on circulating carotenoids, but this effect did not appear to be associated with oxidative stress. Alternative mechanisms linking carotenoids to immunity (not necessarily relying on the use of these pigments as antioxidants) should be considered in future studies on birds.

Avoiding bad genes: oxidatively damaged DNA in germ line and mate choice

August Weismann proposed that genetic changes in somatic cells cannot pass to germ cells and hence to next generations. Nevertheless, evidence is accumulating that some environmental effects can promote heritable changes in the DNA of germ cells, which implies that some somatic influence on germ line is possible. This influence is mostly detrimental and related to the presence of oxidative stress, which induces mutations and epigenetic changes. This effect should be stronger in males due to the particular characteristics of sperm. Here, we propose the hypothesis that females are able to avoid males with oxidatively damaged DNA in the germ line by using oxidative-dependent (pre- and post-mating) signals. This new hypothesis may shed light on unsolved questions in evolutionary biology, such as the benefits of polyandry, the lek paradox, or the role of sexual selection on the evolution of aging.

A Biochemical Study of Fasting, Subfeeding, and Recovery Processes in Yellow‐Legged Gulls

An investigation of the effects of fasting, subfeeding, and refeeding on plasma biochemistry was carried out on 22 captive yellow‐legged gulls Larus cachinnans Pallas. These birds showed the same fasting endurance model described in other species, but with an important decrease in glucose plasma concentration and very great differences between individuals when reaching the deterioration limit, suggesting a moderate physiological adaptation to long periods of fasting. A different model was proposed in subfed gulls in relation to fasted gulls, based on lipid and protein use, which could be reflected by changes in nitrogen wastes and triglyceride levels in this experiment. Thus, the subfed gulls might use protein directly from the diet as an energy source, thereby reducing the use of fat stores. The gulls quickly recovered body mass during the refeeding period, but while some plasma substances quickly reached their initial values, others showed many changes before the end of the experiment, which could reflect a process of metabolic restabilization. These results contribute to a better knowledge of fasting, subfeeding, and refeeding processes in birds and can be added to a recent study about fasting in gulls.