Testosterone treatment is immunosuppressive in superb fairy-wrens, yet free-living males with high testosterone are more immunocompetent

The immunocompetence handicap hypothesis proposes that the immunosuppressive effect of testosterone enforces honesty of sexual signalling via a physiological trade-off between signal intensity and immunocompetence. However, evidence that testosterone is immunosuppressive is scant, particularly in birds. I studied the correlation between immunocompetence and testosterone in superb fairy-wrens (Malurus cyaneus), a species with intense intersexual selection. Males are seasonally dichromatic and testosterone increases during the moult from dull brown eclipse plumage into bright nuptial plumage. I determined the primary antibody response to immunization with sheep red blood cells (SRBCs) in (i) control and testosterone-implanted males in captivity, and (ii) a cross-section of free-living males with basal and elevated testosterone (in eclipse plumage, moulting and in nuptial plumage). Experimental treatment with testosterone decreased the likelihood of an antibody response to SRBCs in captive birds. In contrast, free-living males which had acquired the nuptial plumage and had naturally elevated testosterone were more likely to respond to SRBCs than males in eclipse plumage with basal testosterone levels. The association between higher immunocompetence and higher immunosuppressive testosterone could arise if both are positively correlated with male phenotypic quality In addition, the association could result if males compensate for potential immunosuppression by enhancing their humoral immune responses, particularly since high testosterone is linked to other demanding activities such as moulting and courtship displays.     

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.     

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 role of testosterone in bib size determination in the male house sparrow Passer domesticus,is age dependent

Secondary sexual signals are thought to indicate individual quality. In order to understand the evolutionary pressures that give rise to such traits it is important to understand the physiological mechanisms underlying their production. The black bib of the house sparrow Passer domesticus is known to function as a badge of social status in males. Past studies have found that the size of the bib in older males is determined, at least partly, by the androgen testosterone. The immunocompetence handicap hypothesis suggests that testosterone has a key role in maintaining honest signalling – it is both involved in the development or expression of sexual signals and is immunosuppressive. In this paper we test experimentally two hypo theses relating to bib size development, whether 1) testosterone is only immunosuppressive in conditions where the natural feedback loop from the testes has been removed, and 2) testosterone is, in addition to influencing the bib size of older males, responsible for the size of the bib in juvenile sparrows. In the first experiment we found that exogenous testosterone administered to intact males during the winter (when LH and FSH levels are very low and were not artificially increased by castration) caused significant immunosuppression, albeit in interaction with the stress hormone corticosterone. Second, we found that exogenous testosterone administration in castrated fledgling male house sparrows had no effect on subsequent post‐juvenile moult bib size relative to controls. Our results suggest that in some circumstances testosterone can be immunosuppressive, but that its role in bib size determination is age‐dependent.     

The role of juvenile hormone in immune function and pheromone production trade-offs: a test of the immunocompetence handicap principle

The immunocompetence handicap hypothesis postulates that secondary sexual traits are honest signals of mate quality because the hormones (e.g. testosterone) needed to develop secondary sexual traits have immunosuppressive effects. The best support for predictions arising from the immunocompetence handicap hypothesis so far comes from studies of insects, although they lack male-specific hormones such as testosterone. In our previous studies, we found that female mealworm beetles prefer pheromones of immunocompetent males. Here, we tested how juvenile hormone (JH) affects male investment in secondary sexual characteristics and immune functions in the mealworm beetle, Tenebrio molitor. We injected male mealworm beetles with JH (type III) and found that injection increased the attractiveness of male pheromones but simultaneously suppressed immune functions (phenoloxidase activity and encapsulation). Our results suggest that JH, which is involved in the control of reproduction and morphogenesis, also plays a central role in the regulation of a trade-off between the immune system and sexual advertisement in insects. Thus, the results reflect a general mechanism by which the immunocompetence handicap hypothesis may work in insects.     

Testing the immunocompetence handicap hypothesis: a review of the evidence

The immunocompetence handicap hypothesis was formulated 12 years ago in an attempt to offer a proximate mechanism by which female choice of males could be explained by endocrine control of honest signalling. The hypothesis suggested that testosterone has a dual effect in males of controlling the development of sexual signals while causing immunosuppression. Our purpose in this review is to examine the empirical evidence to date that has attempted to test the hypothesis, and to conduct a meta-analysis on two of the assumptions of the hypothesis, that testosterone reduces immunocompetence and increases parasitism, to ascertain any statistical trend in the data. There is some evidence to suggest that testosterone is responsible for the magnitude of trait expression or development of sexual traits, but this is by no means conclusive. The results of many studies attempting to find evidence for the supposed immunosuppressive qualities of testosterone are difficult to interpret since they are observational rather than experimental. Of the experimental studies, the data obtained are ambiguous, and this is reflected in the result of the meta-analysis. Overall, the meta-analysis found a significant suppressive effect of testosterone on immunity, in support of the hypothesis, but this effect disappeared when we controlled for multiple studies on the same species. There was no effect of testosterone on direct measures of immunity, but it did increase ectoparasite abundance in several studies, in particular in reptiles. A funnel analysis indicated that the results were robust to a publication bias. Alternative substances that interact with testosterone, such as glucocorticoids, may be important. Ultimately, a greater understanding is required of the complex relationships that exist both within and between the endocrine and immune systems and their consequences for mate choice decision making.     

Immune activation suppresses plasma testosterone level: a meta-analysis

Females often select mates on the basis of sexual signals, which can be reliable indicators of male quality when the costliness of these signals prevents cheating. The immunocompetence handicap hypothesis (ICHH) provides a mechanistic explanation of these costs, by proposing a trade-off between immune function and sexual displays. This trade-off arises because testosterone enhances sexual signals, but suppresses immune function. Many studies have investigated the ICHH by administrating testosterone, and a recent meta-analysis found little evidence that testosterone suppressed immune function. However, another component of the ICHH, which has received less empirical interest, suggests that there may also be an interaction in the other direction, with immune activation suppressing testosterone levels. We present a meta-analysis to test for this effect. Overall, there was a strong suppressive effect of experimental immune activation on testosterone levels (r=-0.52), regardless of whether live pathogens or non-pathogenic antigens were used to challenge the immune system. The latter is important because it shows that immune activation per se suppresses testosterone levels. Thus, a trade-off between immunocompetence and sexual displays may primarily be generated by the effect of immune activation on testosterone, rather than the opposite effect that has received most attention.     

Energetic reserves, leptin and testosterone: a refinement of the immunocompetence handicap hypothesis

The immunocompetence handicap hypothesis (ICHH) assumes that testosterone (T), required for the expression of sexual traits, can also incur a cost due to its immunosuppressive properties. However, T-dependent immunosuppression could also arise as an indirect consequence of energy reallocation from the immune system to other metabolic demands. Leptin is mostly produced in lipogenic tissues and its circulating level is positively correlated with the amount of lipid reserves. Leptin also has an important role as immunoenhancer and we suggest that this hormone could play a role as a mediator of the immunosuppressive effect of testosterone. In particular, we propose that only the individuals able to maintain large lipid reserves (with high leptin levels), while sustaining high testosterone levels, might be able to develop sexual displays without an impairment of their immune defences. Here, we tested one of the assumptions underlying this extension of the ICHH: leptin administration should attenuate testosterone-induced immunosuppression. T-implanted and control male zebra finches (Taeniopygia guttata) received daily injections of leptin or phosphate buffered saline. T-implants initially depressed the phytohaemagglutinin-induced immune response. However, T-birds injected with leptin enhanced their immune response to the level of control birds. These results open a new perspective on the study of the ICHH.     

Sexual dimorphism in immunocompetence: what does life-history theory predict?

Sexual dimorphism in immunocompetence, usually in the directionof inferior male immunocompetence, has historically been explainedas the result of proximate physiological mechanisms such asthe immunosuppressive effects of the male hormone testosterone.More recently, it has been argued that this pattern is bestunderstood as a result of resource-based trade-offs betweenmale mating effort and immune defense, a trade-off that femalesdo not make. The central prediction of this hypothesis is thatas the strength of sexual selection on males increases, themagnitude of the sex differences in immunocompetence will increase.Two implicit assumptions of this argument are that 1) longevityis of more importance for female than for male fitness and 2)that the primary benefit of immunocompetence is increased longevity.However, both of these assumptions may not be as broadly applicableas has been argued. We have modeled the optimal allocation toimmunocompetence for males and females without making theseassumptions. We find that the optimal allocation to immune defensefor males decreases as the strength of sexual selection increases,as predicted. However, males may still invest more, relativeto females, into immunocompetence if the impact of parasiteson condition differs for the sexes and/or if the relationshipbetween condition and reproduction differs for the sexes. Weargue that these previously overlooked assumptions may be criticalfor predicting sex-specific patterns of immunocompetence.     

Trade-offs between immune investment and sexual signaling in male mallards

Allocation trade-offs between the immune system and sexual traits are central to current sexual selection hypotheses but remain contentious. Such trade-offs could be brought about by the dual action of testosterone that stimulates sexual signals but also suppresses immune functions and/or by competition for carotenoids that can be deposited in ornaments or used as antioxidants in support of immune functions. We investigated the trade-off between investment in immunity and maintenance of testosterone, carotenoids, and sexually selected, carotenoid-based bill color in male mallards. Following a nonpathogenic immune challenge, facultative immune investment resulted in a syndrome of changes in allocation. Plasma carotenoids disappeared from circulation proportional to antibody production. In addition, the reflectance spectrum of the bill was affected; greater antibody production was associated with an increase in relative UV reflectance. Although changes in bill reflectance and plasma carotenoids were related, the relationship appeared more complex than direct competition with immunity. Finally, maintenance of testosterone was affected by immune investment: testosterone levels declined substantially when males produced more antibodies. Because males with high testosterone are preferred by females, the decline in testosterone, in addition to carotenoid depletion and effects on bill reflectance, could constitute a significant cost of immune investment.     

Adiposity,compared with masculinity,serves as a more valid cue to immunocompetence in human mate choice

According to the ‘good genes’ hypothesis, females choose males based on traits that indicate the male''s genetic quality in terms of disease resistance. The ‘immunocompetence handicap hypothesis’ proposed that secondary sexual traits serve as indicators of male genetic quality, because they indicate that males can contend with the immunosuppressive effects of testosterone. Masculinity is commonly assumed to serve as such a secondary sexual trait. Yet, women do not consistently prefer masculine looking men, nor is masculinity consistently related to health across studies. Here, we show that adiposity, but not masculinity, significantly mediates the relationship between a direct measure of immune response (hepatitis B antibody response) and attractiveness for both body and facial measurements. In addition, we show that circulating testosterone is more closely associated with adiposity than masculinity. These findings indicate that adiposity, compared with masculinity, serves as a more important cue to immunocompetence in female mate choice.     

Enhanced male coloration after immune challenge increases reproductive potential

In many animal species, females select a mate on the basis of the expression of secondary sexual traits. A prevalent theory suggests that male ornaments are reliable indicators of immunocompetence, because the cost of immune function prevents cheating. However, sexual signalling is a component of male reproductive effort, and an immune challenge may also alter his perceived future prospects and hence signalling effort. In this study, blue‐footed booby males (Sula nebouxii) were inoculated with a diphtheria–tetanus vaccine during courtship to investigate the consequences of mounting an immune response on signalling effort. We found that, after this immune challenge, on average, males increased their signalling effort but lost more body mass compared with control males. Importantly, vaccination affected the partner's reproductive decisions: compared with control females, females paired with vaccinated males laid eggs earlier and increased clutch volume in pairs that laid early. Overall, our results suggest that blue‐footed booby males invest more in sexual signals when future breeding opportunities are at risk, eliciting a greater reproductive investment by their partners. Increased signalling effort by infected individuals may contrast the idea of sexual ornaments as signals of infection status.     

Carotenoid supplementation positively affects the expression of a non-visual sexual signal

Carotenoids are a class of pigments which are widely used by animals for the expression of yellow-to-red colour signals, such as bill or plumage colour. Since they also have been shown to promote immunocompetence and to function as antioxidants, many studies have investigated a potential allocation trade-off with respect to carotenoid-based signals within the context of sexual selection. Although an effect of carotenoids on non-visual (e.g. acoustic) signals involved in sexual selection has been hypothesized, this has to date not been investigated. First, we examined a potential effect of dietary carotenoid supplementation on overall song rate during the non-breeding season in captive male European starlings (Sturnus vulgaris). After only 3-7 days, we found a significant (body-mass independent) positive effect of carotenoid availability on overall song rate. Secondly, as a number of studies suggest that carotenoids could affect the modulation of sexual signals by plasma levels of the steroid hormone testosterone (T), we used the same birds to subsequently investigate whether carotenoid availability affects the increase in (nestbox-oriented) song rate induced by experimentally elevated plasma T levels. Our results suggest that carotenoids may enhance the positive effect of elevated plasma T levels on nestbox-oriented song rate. Moreover, while non-supplemented starlings responded to T-implantation with an increase in both overall song rate and nestbox-oriented song, carotenoid-supplemented starlings instead shifted song production towards (reproductively relevant) nestbox-oriented song, without increasing overall song rate. Given that song rate is an acoustic signal rather than a visual signal, our findings therefore indicate that the role of carotenoids in (sexual) signalling need not be dependent on their function as pigments.     

Sexual selection and immune function in Drosophila melanogaster

The evolution of immune function depends not only on variation in genes contributing directly to the immune response, but also on genetic variation in other traits indirectly affecting immunocompetence. In particular, sexual selection is predicted to trade-off with immunocompetence because the extra investment of resources needed to increase sexual competitiveness reduces investment in immune function. Additional possible immunological consequences of intensifying sexual selection include an exaggeration of immunological sexual dimorphism, and the reduction of condition-dependent immunological costs due to selection of 'good genes' (the immunocompetence handicap hypothesis, ICHH). We tested for these evolutionary possibilities by increasing sexual selection in laboratory populations of Drosophila melanogaster for 58 generations by reestablishing a male-biased sex ratio at the start of each generation. Sexually selected flies were larger, took longer to develop, and the males were more sexually competitive than males from control (equal sex ratio) lines. We found support for the trade-off hypothesis: sexually selected males were found to have reduced immune function compared to control males. However, we found no evidence that sexual selection promoted immunological sexual dimorphism because females showed a similar reduction in immune function. We found no evidence of evolutionary changes in the condition-dependent expression of immunocompetence contrary to the expectations of the ICHH. Lastly, we compared males from the unselected base population that were either successful (IS) or unsuccessful (IU) in a competitive mating experiment. IS males showed reduced immune function relative to IU males, suggesting that patterns of phenotypic correlation largely mirror patterns of genetic correlation revealed by the selection experiment. Our results suggest increased disease susceptibility could be an important cost limiting increases in sexual competitiveness in populations experiencing intense sexual selection. Such costs may be particularly important given the high intersex correlation, because this represents an apparent genetic conflict, preventing males from reaching their sexually selected optimum.     

Honest signals and sexual conflict: Female lizards carry undesirable indicators of quality

Sex differences in animal coloration often result from sex‐dependent regulatory mechanisms. Still, some species exhibit incomplete sexual dimorphism as females carry a rudimentary version of a costly male trait, leading to intralocus sexual conflict. The underlying physiology and condition dependence of these traits can inform why such conflicts remain unresolved. In eastern fence lizards (Sceloporus undulatus), blue iridophore badges are found in males and females, but melanin pigmentation underneath and surrounding badges is male‐exclusive. We track color saturation and area of badges across sexual maturity, and their relationship to individual quality (body condition and immunocompetence) and relevant hormones (testosterone and corticosterone). Saturation and testosterone were positively correlated in both sexes, but hormone and trait had little overlap between males and females. Saturation was correlated with body condition and immunocompetence in males but not in females. Co‐regulation by androgens may have released females from resource allocation costs of color saturation, even when in high condition. Badge area was independent of testosterone, but associated with low corticosterone in females, indicating that a nonsex hormone underlies incomplete sexual dimorphism. Given the evidence in this species for female reproductive costs associated with ornamentation, this sex‐nonspecific regulation of an honest signal may underlie intralocus sexual conflict.     

Dressed to impress: breeding plumage as a reliable signal of innate immunity

Animal signals involved in sexual selection are often indicators of individual quality. The assumption that sexual characters such as breeding plumage may indicate immune state has rarely been tested in free‐living migratory birds, particularly in relation to innate immunity. If sexual characters indeed reflect immune condition, then these could be used to evaluate individual quality. Melanin is a common pigment used in animal communication that mitigates the effects of oxidative stress and has positive effects on energy homeostasis, important functions during the strenuous activity of long‐distance flights. However, melanin is also immunosuppressive, and the melanised patches of breeding plumage may to some extent compromise immune responsiveness. We studied melanin‐based secondary sexual characters (SSC) in a long‐distance migratory wader, the black‐tailed godwit Limosa limosa, and found that breeding plumage features of male and female godwits are linked to components of innate immunity. Males with a larger colour extension had higher circulating levels of haptoglobin and hemolysis activity, while they also presented a lower body condition; whereas females presented a negative trend between colour and bar extension and hemolysis activity, and a positive trend for natural antibodies. The association between signal, immune state and physical condition in males suggests a cost for signal production and immune condition during prenuptial migration. Sex differences in how signals relate with immune capacity are a likely consequence of sex‐specific signalling roles and energy demands. Our results indicate that male godwit breeding plumage reflects innate immunity state, and is therefore a likely signal for females to use during mate choice as an honest indicator of male's capacity to allocate energy/resources to both expensive traits during periods of energetic constraint.     

Differential effects of testosterone,dihydrotestosterone and estradiol on carotenoid deposition in an avian sexually selected signal

Recent studies have demonstrated that carotenoid-based traits are under the control of testosterone (T) by up-regulation of carotenoid carriers (lipoproteins) and/or tissue-specific uptake of carotenoids. T can be converted to dihydrotestosterone (DHT) and estradiol (E2), and variation in conversion rate may partly explain some contradictory findings in the literature. Moreover, most studies on the effect of T on sexual signals have focused on the male sex only, while in many species females show the same signal, albeit to a lesser extent. We studied the effects of T, DHT, and E2 treatment in male and female diamond doves Geopelia cuneata in which both sexes have an enlarged red eye ring, which is more pronounced in males. We first showed that this periorbital ring contains very high concentration of carotenoids, of which most are lutein esters. Both T and DHT were effective in enhancing hue, UV-chroma and size in both sexes, while E2 was ineffective. However, E2 dramatically increased the concentration of circulating lipoproteins. We conclude that in both sexes both color and size of the secondary sexual trait are androgen dependent. The action of androgens is independent of lipoproteins regulation. Potential mechanisms and their consequences for trade-off are discussed.     

Trade-off between warning signal efficacy and mating success in the wood tiger moth

The coloration of species can have multiple functions, such as predator avoidance and sexual signalling, that directly affect fitness. As selection should favour traits that positively affect fitness, the genes underlying the trait should reach fixation, thereby preventing the evolution of polymorphisms. This is particularly true for aposematic species that rely on coloration as a warning signal to advertise their unprofitability to predators. Nonetheless, there are numerous examples of aposematic species showing remarkable colour polymorphisms. We examined whether colour polymorphism in the wood tiger moth is maintained by trade-offs between different functions of coloration. In Finland, males of this species have two distinct colour morphs: white and yellow. The efficacy of the warning signal of these morphs was tested by offering them to blue tits in the laboratory. Birds hesitated significantly longer to attack yellow than white males. In a field experiment, the survival of the yellow males was also higher than white males. However, mating experiments in the laboratory revealed that yellow males had lower mating success than white males. Our results offer an explanation for the maintenance of polymorphism via trade-off between survival selection and mating success.     

Tight hormonal phenotypic integration ensures honesty of the electric signal of male and female Brachyhypopomus gauderio

Hormones mediate sexually selected traits including advertisement signals. Hormonal co-regulation links the signal to other hormonally-mediated traits such that the tighter the integration, the more reliable the signal is as a predictor of those other traits. Androgen administration increases the duration of the communication signal pulse in both sexes of the electric fish Brachyhypopomus gauderio. To determine whether the duration of the signal pulse could function as an honest indicator of androgen levels and other androgen-mediated traits, we measured the variation in sex steroids, signal pulse duration, and sexual development throughout the breeding season of B. gauderio in marshes in Uruguay. Although the sexes had different hormone titres and signal characteristics, in both sexes circulating levels of the androgens testosterone (T) and 11-ketotestosterone (11-KT) were strongly related to signal pulse duration. Consequently, signal pulse duration can serve as an honest indicator of circulating androgens in males and females alike. Additionally, through phenotypic integration, signal pulse duration also predicts other sexual traits directly related to androgen production: gonad size in males and estradiol (E2) levels in females. Our findings show that tight hormonal phenotypic integration between advertisement signal and other sex steroid-mediated traits renders the advertisement signal an honest indicator of a suite of reproductive traits.     

Reproductive ageing and sexual selection on male body size in a wild population of antler flies (Protopiophila litigata)

Little is known about the importance of trade-offs between ageing and other life history traits, or the effects of ageing on sexual selection, particularly in wild populations suffering high extrinsic mortality rates. Life history theory suggests that trade-offs between reproduction and somatic maintenance may constrain individuals with higher initial reproductive rates to deteriorate more rapidly, resulting in reduced sexual selection strength. However, this trade-off may be masked by increased condition dependence of reproductive effort in older individuals. We tested for this trade-off in males in a wild population of antler flies (Protopiophila litigata). High mating rate was associated with reduced longevity, as a result of increased short-term mortality risk or accelerated ageing in traits affecting viability. In contrast, large body size was associated with accelerated ageing in traits affecting mating success, resulting in reduced sexual selection for large body size. Thus, ageing can affect sexual selection and evolution in wild populations.