Lorenzo Pérez-Rodríguez

Testosterone increases bioavailability of carotenoids: Insights into the honesty of sexual signaling

Androgens and carotenoids play a fundamental role in the expression of secondary sex traits in animals that communicate information on individual quality. In birds, androgens regulate song, aggression, and a variety of sexual ornaments and displays, whereas carotenoids are responsible for the red, yellow, and orange colors of the integument. Parallel, but independent, research lines suggest that the evolutionary stability of each signaling system stems from tradeoffs with immune function: androgens can be immunosuppressive, and carotenoids diverted to coloration prevent their use as immunostimulants. Despite strong similarities in the patterns of sex, age and seasonal variation, social function, and proximate control, there has been little success at integrating potential links between the two signaling systems. These parallel patterns led us to hypothesize that testosterone increases the bioavailability of circulating carotenoids. To test this hypothesis, we manipulated testosterone levels of red-legged partridges Alectoris rufa while monitoring carotenoids, color, and immune function. Testosterone treatment increased the concentration of carotenoids in plasma and liver by >20%. Plasma carotenoids were in turn responsible for individual differences in coloration and immune response. Our results provide experimental evidence for a link between testosterone levels and immunoenhancing carotenoids that (i) reconciles conflicting evidence for the immunosuppressive nature of androgens, (ii) provides physiological grounds for a connection between two of the main signaling systems in animals, (iii) explains how these signaling systems can be evolutionary stable and honest, and (iv) may explain the high prevalence of sexual dimorphism in carotenoid-based coloration in animals.

Carotenoids in evolutionary ecology: re-evaluating the antioxidant role

The antioxidant role of carotenoids in the living organism was proposed as a possible basis for the honesty of carotenoid‐based signals. However, recent studies have questioned the relevance of carotenoids as powerful antioxidants in vivo. Current evidence does not seem to support the “antioxidant role” hypothesis, but it does not allow us to reject it either. This paper proposes some steps to solve this controversy, such as taking a dynamic approach to antioxidant responses, designing protocols that expose individuals to oxidative challenges, analyzing tissues other than blood, and obtaining measures of antioxidant capacity and oxidative damage simultaneously. However, it should be considered that, irrespective of their antioxidant potential, carotenoids might still give information on oxidative stress levels if they are particularly sensitive to free radicals. Finally, lumping together the immunostimulatory and antioxidant roles of carotenoids should be avoided as these functions are not necessarily associated.

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.

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.

Nematode parasites reduce carotenoid-based signalling in male red grouse

Carotenoids determine the yellow–red colours of many ornaments, which often function as signals of quality. Carotenoid-based signalling may reliably advertise health and should be particularly sensitive to parasite infections. Nematodes are among the commonest parasites of vertebrates, with well-documented negative effects on their hosts. However, to date, little is known about the effects that these parasites may have on carotenoid-based signalling. Tetraonid birds (grouse) exhibit supra-orbital combs, which are bright integumentary ornaments pigmented by carotenoids. We tested the effect of the nematode parasite Trichostrongylus tenuis on signalling in free-living male red grouse Lagopus lagopus scoticus. We show that experimentally reduced nematode infection increases plasma carotenoid concentration and comb redness, demonstrating for the first time that nematodes can influence carotenoid-based signals.

Condition and androgen levels: are condition-dependent and testosterone-mediated traits two sides of the same coin?

The immunocompetence handicap hypothesis posits that androgen-mediated signals honestly indicate the responsiveness of the immune system because of the immunosuppressive effect of androgens. However, androgen levels may also be related to nutritional status, and differences in body condition could be a more parsimonious explanation for variation in the expression of the signal. We maintained captive male red-legged partridges, Alectoris rufa, under regulated food shortage until they reached 85% of their initial body mass. Controls were provided with food ad libitum. After food shortage, experimental birds had lower androgen and higher corticosterone levels than controls. The condition dependence in androgen levels suggests that androgen-mediated signals could be indicating general nutritional state rather than immune function specifically. We propose that androgen-dependent signals may act as indicators of body condition or indicators of immune system quality, depending on the nutritional status of the individual.

Age and Breeding Effort as Sources of Individual Variability in Oxidative Stress Markers in a Bird Species

Oxidative stress is the imbalance between the production of pro‐oxidant substances and the level of antioxidant defenses, which leads to oxidative damage. It has been proposed that senescence is the result of accumulated oxidative damage throughout life. In birds, the sources of individual variability in oxidative stress are still poorly understood. Among these sources, age, as related to senescence, should be particularly relevant. Furthermore, recent findings suggest that breeding effort may also deeply influence susceptibility to oxidative stress. However, there is still no evidence of a link between breeding effort and oxidative damage in any vertebrate. Here we analyzed 288 captive red‐legged partridges (Alectoris rufa) across a wide age range (i.e., 1–8 yr old), thus including potentially senescent birds. In spite of limitations due to the cross‐sectional approach, results revealed that old birds produced less offspring and endured higher levels of oxidized glutathione and peroxidized lipids in erythrocytes than did middle‐aged individuals. Old birds also showed higher plasma total antioxidant status and uric acid levels than did younger birds, but lower amounts of circulating carotenoids. Furthermore, hatching success was negatively correlated to lipid peroxidation in females but not in males, supporting the hypothesis that breeding effort promotes oxidative damage.

Avoiding bias in parasite excretion estimates: the effect of sampling time and type of faeces

The study of host-parasite relationships usually requires reliable estimates of parasite intensity, which is often estimated from parasite propagule concentration in faeces. However, parasite excretion in faeces may be subject to variation due to endogenous or exogenous factors that must be identified to obtain reliable results. We analysed the effect of the hour of sample collection on propagule counts of 2 intestinal parasites infecting the red-legged partridge: the capillarid nematode Aonchoteca caudinflata and coccidia of the genus Eimeria (Protozoa). Also, we test whether there are differences in propagule counts between caecal and intestinal faeces. Individual faecal samples from infected birds were collected daily at 4 different hours during several days. The hour of the day exerted a very strong effect on propagule counts, excretion of both types of parasites showing a clear and constant increase from dawn to dusk. Also, capillarid eggs were more abundant in intestinal than in caecal faeces, whereas the inverse pattern was found for coccidian oocysts. Standardization of the hour of sample collection or statistical control of this variable is recommendable to prevent bias. Similarly, in bird species with long caeca, consistent collection of one type of faeces may avoid significant errors in parasite burden estimates.

Oxidative stress and the effect of parasites on a carotenoid-based ornament.

SUMMARY Oxidative stress, the physiological condition whereby the production of reactive oxygen and nitrogen species overwhelms the capacity of antioxidant defences, causes damage to key bio-molecules. It has been implicated in many diseases, and is proposed as a reliable currency in the trade-off between individual health and ornamentation. Whether oxidative stress mediates the expression of carotenoid-based signals, which are among the commonest signals of many birds, fish and reptiles, remains controversial. In the present study, we explored interactions between parasites, oxidative stress and the carotenoid-based ornamentation of red grouse Lagopus lagopus scoticus. We tested whether removing nematode parasites influenced both oxidative balance (levels of oxidative damage and circulating antioxidant defences) and carotenoid-based ornamentation. At the treatment group level, parasite purging enhanced the size and colouration of ornaments but did not significantly affect circulating carotenoids, antioxidant defences or oxidative damage. However, relative changes in these traits among individuals indicated that males with a greater number of parasites prior to treatment (parasite purging) showed a greater increase in the levels of circulating carotenoids and antioxidants, and a greater decrease in oxidative damage, than those with initially fewer parasites. At the individual level, a greater increase in carotenoid pigmentation was associated with a greater reduction in oxidative damage. Therefore, an individuals ability to express a carotenoid-based ornament appeared to be linked to its current oxidative balance and susceptibility to oxidative stress. Our experimental results suggest that oxidative stress can mediate the impact of parasites on carotenoid-based signals, and we discuss possible mechanisms linking carotenoid-based ornaments to oxidative stress.

Testosterone-mediated trade-offs in the old age: a new approach to the immunocompetence handicap and carotenoid-based sexual signalling

The immunocompetence handicap hypothesis proposes that testosterone mediates a trade-off between sexual signalling and immunocompetence in males. Such a trade-off could favour the reliability of sexual signals on the basis that testosterone required for signal expression also promotes immunosuppression. However, the immunosuppressive activity of testosterone has not been convincingly demonstrated. We propose that the optimal solution to the testosterone-mediated trade-off should change with age, explaining ambiguous results in the past. Testosterone and ageing would promote two simultaneous immunosuppressive challenges unaffordable for low-quality males. Oxidative stress, as intimately related to ageing and immunosenescence, could contribute to enhance signal reliability. In this context, traits coloured by carotenoids (yellow–red traits) could play a crucial role due to the immunostimulatory and antioxidant properties of these pigments. Here, old and middle-aged male red-legged partridges were treated with testosterone or manipulated as controls. In the presence of high-testosterone levels, middle-aged males increased both circulating carotenoid levels and colour expression, whereas their cell-mediated immunity was not significantly altered. However, in old males, neither circulating carotenoids nor sexual signalling increased when treated with testosterone, but immunosuppression was detected. The link between testosterone and carotenoids could favour the reliability of sexual signals throughout the life.