Introduction

Sexual selection imposes different selective pressures on males and females resulting in a dichotomy in fitness strategies [1]. In efforts to maximize their number of progeny, females typically invest more in processes affecting longevity [2], [3]. Male investment is dependent on short term mating success, which can be maximized by increased mating rate often at the expense of immune defenses [3]. Differences in susceptibility to infection may therefore reflect differential selection on males and females as well as sex-biased life-history traits relating to mating and reproduction. Previous research across multiple taxa found tradeoffs between reproduction and immunity that can occur pre- and post-mating, however there is little consistency in the patterns between these two processes when looking at natural populations [1], [4]. This inconsistency may be attributed to the many behavioral and physiological processes an individual utilizes in an effort to counter parasitic infections [5].

Male vertebrates typically show increased susceptibility to disease and higher rates of parasitic infections compared to females [6]–[8]. These differences may be a result of males being in worse body condition [9] but often are attributed to the negative effects of androgens and the positive effects of estrogens[5], [10]–[14]. An individual’s ability to respond to foreign antigens in order to reduce costs associated with infection is often referred to in the literature as ‘immunocompetence’ [15]. Life history trade-offs are such that reproductive strategies often reflect immunological strategies [15], as demonstrated by the robust association between sexual selection and male-biased parasitism [7]. For example, in monogamous species, where pressures to compete for females are less intense, males and females are less likely to differ in disease susceptibility [1], [14]. As competition for mates increases, however, males invest more energy into courtship displays, intrasexual competition, and sperm competition. Such competitive investment is energetically expensive and requires high levels of testosterone to express secondary sex characters [10]. Testosterone is hypothesized to have a dualistic effect, stimulating sexual character development while simultaneously reducing immune response. Folstad & Karter [10] formalized the ‘immunocompetence handicap hypothesis’ (ICHH) based on the assumption that testosterone suppresses immune function during spermatogenesis because sperm are recognized as foreign bodies. Testosterone-dependent characters therefore represent honest indicators of quality because only highly immunocompetent males are capable of trading-off between reproduction and immunity [10], [11]. However, types of parasites may be impacted differently by androgens suggesting this relationship is much more complex than originally thought [16], [17].

The ICHH predicts species with high investment in spermatogenesis are more likely to demonstrate a trade-off between reproductive and immunological investment [10], [18]. This relationship is not always found, possibly because immune response is also dependent on social and environmental circumstances. Likewise, the pattern of lower male immunocompetence compared to females is not always explicit as multiple ecological and behavioral factors play a role in the evolution of immune defenses [3]. Patterns of increased parasitic infection could be a response to differences in habitat quality, food preferences, body size, or sexual differences in life history tactics that directly or indirectly influence parasite burdens [2], [8], [12], [14], [19], [20]. For example, exposure rates due to different mating behaviors or increased home range sizes may cause the sex with greater exposure, often males, to have increased parasitism [7], [14]. Characteristics such as increased sociality and promiscuity increase the likelihood of infection and transmission of pathogens altering immune response [20] and may be different between males and females resulting in variation in parasite loads. In promiscuous systems, individuals have increased contact with others, which increases both the probability of exchanging parasites, particularly ectoparasites, and the risk of acquiring sexually transmitted infections [21], [22].

We addressed sex differences in immunity in the Cape ground squirrel (Xerus inauris), a species that is both highly social and promiscuous [23], [24]. Such a high amount of promiscuity results in one of the largest investments in male reproductive anatomy compared to other sciurids [25]. This investment is attributed to males outcompeting others via sperm competition [23], [25]. Although females breed year round [26], estrous opportunities are limited [23], suggesting males are under greater selection to increase short term mating success and therefore invest more in reproductive morphology compared to females who invest in processes affecting longevity and offspring survival. The result of these differing investments is predicted to be lower male immunocompetence compared to females. Previous research supports this prediction with males carry higher ectoparasite loads compared to females, a factor attributed to increased testosterone [27]. Because increases in testosterone are important for sperm production, we predict males that are able to invest more in sperm competition do so at a cost to their immune system resulting in a trade-off between immune and reproductive investment.

Results

We euthanized 26 males and 11 females and found no significant differences between body mass (t_13.0 = −0.26, P = 0.797) or spleen size (t₃₅ = −0.26, P = 0.800) after controlling for body size. Despite finding no significant difference between body condition of males and females (t_10.8 = 0.26, P = 0.798), males did have significantly more ectoparasites per individual (t₁₀ = 3.19, P = 0.005) averaging 71.3±18.3 compared to females (10.8±5.1 per individual). The majority of those ectoparasites for both sexes were lice (males: 63.0±16.1; females: 9.3±5.0). We took blood from live animals in addition to those we euthanized such that we had 7 additional male samples and 22 additional females, although red blood cell counts were not performed on all additional animals. We found the percentage of red blood cells to be significantly lower in males than females (t₆₂ = 2.97, P = 0.004; Table 1). We also found significant differences in white blood cell percentages, with males having higher basophils (t_40.2 = −5.65, P<0.001) and lymphocytes (t_28.2 = −2.82, P = 0.009) and females having higher percentages of neutrophils (t_16.7 = 2.58, P = 0.020) (Table 1). Testes size for one subadult male fit our outlier criterion and was removed. We found a significant negative correlation between the residuals of spleen mass and testes mass (Figure 1). Despite the small sample size, we did find a significant positive correlation between testes mass and lice, after controlling for body mass, but not fleas (Figure 2).

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Table 1. Average blood cell percentages for male and female Xerus inauris (significant differences indicated by bold text).

https://doi.org/10.1371/journal.pone.0038524.t001

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Figure 1. Relationship between residual masses of spleen and testes (r² = 0.21, F_1,23 = 6.07, P = 0.022) calculated from least squares regression against body mass in Xerus inauris.

Note: two points are overlapping at x = 1.1, y = −0.85.

https://doi.org/10.1371/journal.pone.0038524.g001

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Figure 2. Relationship between residual masses of testes and total flea and lice counts for male Xerus inauris (Lice: r² = 0.68, F_1,8 = 16.97, P = 0.003; Fleas: r² = 0.10, F_1,8 = 0.86, P = 0.381).

https://doi.org/10.1371/journal.pone.0038524.g002

Discussion

Sex biases in parasitism often are attributed to sex-specific life-history strategies that affect parasite susceptibility and exposure such that differences in reproductive strategies should determine male and female immunocompetence [14]. This rather simplistic view fails to incorporate the ecological and evolutionary dynamics, specifically host-parasite interactions, that may alter male and female immunological response [28]. X. inauris have been shown to carry high parasite loads with males having significantly higher ectoparasite loads and females having significantly higher endoparasite loads [27]. Endoparasite variation has been attributed to life history variations between male and female X. inauris while ectoparasites are suspected to be under hormonal control [27] suggesting different mechanisms may be employed for countering infections. Contrary to predictions based on these intersexual differences in parasite types and parasite loads, we found no differences in body condition or immunity as measured by spleen size. Spleen mass generally is indicative of overall immune strength [8], [9] and thus similarities in spleen size may be attributed to both sexes being highly infected, albeit with different parasite types [27].

The field of ecological immunity is becoming increasingly complex as variables such as parasite type [29] and host sexual reproduction [28] have been shown to elicit specific immunological responses. Endoparasites and ectoparasites are known to affect hosts differently [30] although rarely are accounted for separately in studies addressing sexual dichotomy of immune response. We found significant differences in percentages of both RBCs and leukocyte types, which may be attributed to variations in parasite type. Lower concentrations of RBCs in males are a likely response to higher numbers of lice and fleas, as they feed directly on host blood and fleas have been shown to induce anemia in rodents [31]. Within the WBC fraction of blood, males have significantly higher percentages of lymphocytes and basophils compared to females, possibly because of higher ectoparasite loads. Basophils are part of an allergic response to ectoparasites [32] while lymphocytes are involved in recognition of antigens and increase in response to inflammation caused by ectoparasites [29], [30]. Conversely, percent of neutrophils were higher in females and have been shown to increase in response to endoparasites [33]. While variation in parasite type has been demonstrated to elicit different immune responses in birds [29], further research is warranted in this species to explicitly link parasite exposure and type to immune response.

Previous studies of immunosuppression imply selection only imposes stress on males during the energetically expensive breeding season [11] and variations in spleen size has been shown to be influenced by sex and stress hormones [34]. X. inauris, however, are year-round, asynchronous breeders, requiring both sexes to continuously invest in reproduction [23], [26]. On average, 70% of estruses fail to produce offspring, which has been attributed to costs associated with parasites rather than scarce resources [26], [35], [36]. Parasitic infection often increases when animals reproduce, due to transmission through contact, endocrine changes, and reallocation of resources [37]. Removal of parasites from female X. inauris significantly increases reproductive success suggesting females also are immunologically challenged [35]. Given the costs associated with reproduction, females likely face a similar trade-off between investing in reproduction verses immune response. The lack of sexual differences in spleen size suggests immunological strategies in both sexes may be under intense selection in this species.

Significant increases in ectoparasite loads do not arise in males until they reach reproductive maturity [27] suggesting increases in testosterone that accompany reproductive maturity may alter immune response. The ICHH predicts testosterone decreases immunocompetence causing greater vulnerability to infection [10], [38]. Therefore, investing in sexual traits such as testis mass [18] or sperm viability [39] incurs a cost of decreased immune function and represents an evolutionary trade-off if decreased immune function reduces lifespan due to increased infection and mortality. This hypothesis was supported by our data which showed a negative association between testes size and immune function as well as a significant positive association between testes investment and number of lice, despite a low sample size. A similar relationship between testes size and flea and helminth species richness has been found in other rodents which was possibly attributed to testosterone-mediated immunosuppression [40]. However, as our data show, the relationship between testosterone and parasites also depends on the parasite [16], [40], [41]. Similarly, a recent study in spiny lizards (Sceloporus jarrovi) demonstrated a positive association between plasma testosterone levels and mites but a negative association with endoparasites [16]. Therefore, male immunity is not universally suppressed by testosterone suggesting these interactions are far more complex than originally thought.

Our findings support the ICHH and the male’s response to testosterone but also suggest a larger issue rarely addressed in the literature. Different parasite types trigger different immune responses. In this system, both males and females have high energetic constraints but significant differences in parasite loads [27]. Therefore, questions involving general immunological stategies measured by a single immune response (e.g. total white blood cells) may fail to detect differences relating to the ecological and evolutionary dynamics of host-parasite interactions and may contribute to mixed support for differences between sexes and the role of hormones in immunity [6]. We suggest future studies on this sexual dichotomy in natural populations should incorporate multiple types of parasites and leukocyte type not just overall numbers of WBCs.

Materials and Methods

Acknowledgments

The authors would like to thank Northwest Parks and Tourism Board and S.A. Lombard Nature Reserve as well as numerous colleagues at the University of Central Florida and the University of Illinois for comments and help with analyses. In addition, they thank M. Hillegass for blood analysis, L. Riato, P. Bateman, A. Roth, T. Roth, and J. Roth for necropsy and field assistance, and J. Osbourne for his never-ending encouragement and support.