Sex-dependent differences in avian malaria prevalence and consequences of infections on nestling growth and adult condition in the Tawny pipit, Anthus campestris

Theoretical and empirical work on a variety of host-parasite systems has demonstrated the central role that parasites play in many important aspects of host biology [1]. Prevalence of arthropod-borne parasites in wild animal populations reflects a balance between the exposure of hosts to the infective stages of a parasite, and the success of the parasite in infecting the hosts [2]. This balance between host exposure and parasite success is partially influenced by the immunological capability of the host, which is known to be sensitive to traits such as sex, age, physiology, body condition and reproductive effort [3‐7]. Avian malaria parasites of genera Plasmodium and Haemoproteus are blood parasites that occur regularly in wild populations and are one of the most popular model systems for studying host-parasite co-evolution [5, 8]. This type of parasites had attracted considerable attention as disease causes a variety of adverse effects on hosts [9‐17], including the extinction of naive host populations [18]. Although the infections caused by these parasites can also persist in hosts for years causing no direct effects or only mild effects on fitness [9, 12, 19‐21], recent studies have revealed that long-term chronic infections can produce important hidden costs for hosts [17].

The vast majority of studies exploring malaria impacts on wild avian hosts have been focused on adults, which inevitably restrict the ability to understand properly the consequences of infections for hosts. For example, during the acute phase of malaria infection many individuals were naturally removed from the population by dying [21], which makes really difficult to assess what type of parasites are involved and the real damage they caused at the population level. In this context, one interesting approach is to use nestlings as research focus. New-borns represent an attractive target for malaria parasites because of their low immunological and behavioural defences, which are relatively immature at hatching [22, 23]. Development of the immune system begins post-hatching and continues during the growth period, when birds are able to mount a significant immune response to blood parasites [24]. Therefore, age determines host cell maturation and the capacity to resist against parasites and support infections (reviewed in [25]). A naive immune system is known to increase susceptibility to parasitism and, therefore, those parasites that can take advantage of the immunologically naive hosts to increase its recruitment and survival rates should be favoured [25]. In birds, the immunological naive hosts (nestlings) are highly available to vectors only during early stages of development, whereas for much of the remaining year the host population consists of individuals with experience of prior infection. Moreover, infected nestlings may face at this stage a trade-off between allocating resources to growth or to keep them as immune enhancers for fighting parasites or diseases [26‐29]. Thus, adults and nestlings should be considered altogether in order to better understand how parasites (in terms of species composition and prevalence) impact globally on a population. Despite its importance, surprisingly little is known about the avian malaria infections during the crucial phase of nestling growth as compared to adult phase.

Furthermore, parasite prevalence in vertebrates is often different between males and females [30, 31], which has been mainly attributed to sex-specific host characteristics, such as the endocrine-immune interactions. Mounting evidence indicates that sex hormones influence the immune system [32‐34]. Androgens, primarily testosterone, can suppress cell-mediated and humoral immunity in males, whereas oestrogen can suppress cell-mediated immunity while boosting humoral immunity in females [32]. In birds, evidence suggests that an effective immune system is costly, and that there are trade-offs among investment in immune function and other physiological and ecological aspects during reproduction [35‐37]. This may result in differences in parasite prevalence between the sexes [4, 38]. There is, therefore, a need to assess what role, if any, sex-related trade-offs between investment in immunity and other aspects of reproduction play in mediating acquisition of parasites, and if costs of parasite infections are different for each sex.

In total, 92 out of the 200 birds screened were infected with malaria parasites, which represents an overall prevalence of 46 % (Table 1). Multiple infections (3.3 %) and unresolved (poor-quality) sequences (2.2 %) were excluded from further analyses. Sequences from 86 Plasmodium (99 %) and one Haemoproteus (1 %) species were recorded, yielding 16 unique Plasmodium and one Haemoproteus cytochrome b haplotypes. The most common parasite haplotype in Tawny pipits was BIC33 (31 %), followed by BIC39 (16.1 %), BIC8 (13.8 %), BIC29 (12.6 %) and BIC32 (11.5 %). Other haplotypes were detected at low rate (less than 3.0 % of all infections). A Blast search (GenBank’s basic alignment search tool) was performed for our cytochrome b haplotype dataset, identifying six Plasmodium haplotypes that had been previously recorded in other avian hosts: BIC29 (KF747759), BIC32 (KF747760), BIC33 (KF747761), BIC36 (KF747762), BIC37 (KF747763) and BIC39 (KF747764). The remaining 11 parasite haplotypes had not been previously described, and showed between 0.23 and 1.87 % of genetic divergence with respect to any other Plasmodium or Haemoproteus lineage recorded, to date, in avian hosts.

The present study adds data on host-parasite relationships for a so far non-investigated passerine species, the Tawny pipit. Host sex-related differences affecting prevalence of avian malaria, but no evidence of age-related differences, were reported here. Tawny pipit males were more likely to be infected by malaria parasites compared to females, and this sex-bias parasitism was evident in both adults and nestlings. Avian malaria parasites were also prevalent in 7–11 day-old nestlings, consistent with other reports [54]. Most importantly, infected males showed lower daily mass gain during nestling growth than infected females, whereas infections at adulthood were associated with lower condition in females than in males.

The effect of host age on parasite prevalence is a controversial topic in avian ecology. On one hand, the number of host-parasite encounters and the accumulation of infections over time means that we can expect a pattern of higher parasite prevalence in adults than at early stages of life [55, 56]. On the other hand, the low immunological and behavioural defences against parasites of nestlings compared to adults suggest the opposite pattern, with higher expected prevalence in nestlings than in adults [57, 58]. Few studies have explicitly compared avian malaria infections between nestlings and adults in the wild, since the prepatent period (when the parasite is developing in the host tissues and absent from blood) is often longer than the nestling period of most bird species (see [59]). Results from Tawny pipit supported the hypothesis that the naïve immune system makes nestlings highly susceptible to developing infections. The age-pattern of malaria infection found in this study indicated a rapid accumulation of parasites at the age at which an individual is first susceptible to infection. Considering the age of nestlings studied (7–11 days) and that prepatent periods for avian species of Plasmodium and Haemoproteus range from 2 days to several months [60], it is likely that some of the nestlings considered as uninfected in this study might be truly infected, but the disease has not yet reached the blood (prepatent stage) at the time of sampling. If so, the real parasite prevalence in nestlings can be underestimated, and the age-prevalence curve would reflect a decreasing incidence of malaria with age [58] typical of a parasite-mediated viability selection pattern [61, 62]. A reduction of prevalence with age should merely indicate the loss (death) of infected individuals from the population [58, 61], or that parasites were cleared from hosts or entered a latent stage (remaining usually in host tissues) later in life [63, 64]. Unfortunately discrimination among these possibilities is not possible in the present study, but the topic merits further research as they can reveal patterns that may allow quantification of rates of parasite transmission, parasite-induced host mortality, and development of host resistance.

Regarding the processes whereby infections occur, age-specific differences in physiological trade-offs would explain, at least in part, the observed pattern [58]. Nestlings hatch when parasite prevalence peaks in vector populations [65, 66], so they are expected to rapidly acquire numerous parasite lineages present in the breeding area. Maturation of the immune system in birds may take several weeks after hatching and, therefore, investment in immune defence may come at the expense of investment in somatic growth (review in [67]), which may be especially patent for species with determinate growth (i.e. growth ceases before chicks leave the nest) like passerines. Moreover, in species or populations at higher risk of nest predation the trade-off between investment in immune defence and investment in growth may be particularly important, as predation is one ecological factor that exerts strong selection on growth rates in passerines [68, 69]. Ground-nesting species living in open environments in the Mediterranean region are under strong predation rates [40, 70], and previous work conducted on the same Tawny pipit population has shown that nest predation is a key factor driving the species’ reproduction [40]. As a consequence, nestlings of altricial or semi-altricial species, such as Tawny pipits, should reach adult size faster than precocial species [71], indicating that growth is compressed into a shorter period fuelled by a strong evolutionary pressure to leave the nest as soon as possible in order to avoid or minimize predation [72]. Therefore, nestlings may prioritize growth over development of immune function, causing high disease incidence in this age group despite the short time of exposure to parasites. The high prevalence found in nestlings, together with the fact that most parasite haplotypes were found infecting both nestlings and adults points towards transmission of these parasites in the European breeding grounds of Tawny pipits. The only exception was the Haemoproteus haplotype, which was detected only in adults. This suggest that parasites of this clade could have been transmitted in winter quarters (i.e. Africa), as proposed in the case of many other Haemoproteus and Plasmodium haplotypes for which the occurrence of suitable vectors in Europe could be currently absent [73]. Alternatively, perhaps, the prepatent period for Haemoproteus spp. described in the literature (between 11 days and 3 weeks) diminishes the probability of detection in the nestling age cohort.

Differences between males and females in prevalence and/or intensity of infection, in particular male-biased parasitic infections, are often observed in nature [6, 31, 35, 74‐76]. However, sex-bias in parasitism is not universal and consistent, and often varies between and within host-parasite systems [3, 58, 77]. In Tawny pipits, males had higher parasite prevalence than females in both age cohorts, a pattern compatible with gender differences in susceptibility to parasites according to biological differences between host sexes [78]. In the avian malaria system, host sex is considered as a potentially important source of variation in both prevalence and cost of parasites for hosts [78]. The proximate explanations for sex-bias in parasitism may be caused by many different factors that revolve around two hypotheses, which are not mutually exclusive: 1) certain hormones have an immunosuppressive effect upon the host defence against pathogens, and 2) because of morphological and/or behavioural differences, one sex has a greater likelihood of being parasitized by differential exposure to vectors [35, 79]. In Tawny pipits, males and females do not differ substantially in mass or colour [80] and their home ranges overlap within territories, which were probably not large enough (3.5–12.1 ha; [81]) to explain differences in vector exposure between males and females. Although behavioural differences cannot be excluded as a potential explanation, there is overwhelming evidence that sex-associated hormones can directly influence the susceptibility of each sex to infections [35]. For example, testosterone has immunosuppressive effects in some species, leading to increased susceptibility of males to parasite infections [82]. However, while the immunosuppressive effect of sex-linked hormones is a well-recognized phenomenon in adulthood [83, 84], that could explain the results found in adults, its rationale in the case of nestlings is not clear, as differences in susceptibility to parasite infection might not be attributable directly to sexual differences in circulating testosterone levels [85]. Firm conclusions cannot be drawn about why males were more infected than female nestlings, but the results found on differential growth between sexes (discussed below) should shed some light on this issue.

Male and female nestlings leave the nest at the same time; therefore, for a species where males are slighter larger in wing size than females [80], males may need to grow faster than their smaller sisters, as supported by the higher wing growth rate of male nestling compared to females found in this study. The growth strategy of small breeding-ground passerines under strong nest-predation rates may be due to the need to leave the nest as soon as possible, which depends on their ability to fly and, thus, the development of the wings. If male nestlings need to allocate more resources to growth and less to prevent infections than females this could result in increased parasite prevalence in males as compared to females. In a comparative study with amphibians, Johnson et al. [86] showed that amphibian species with rapid growth were more susceptible to infections and pathology than species with slow growth. Within birds, selection for growth compromises the immune function in lines of commercial poultry (meta-analysed by van der Most et al. [87]). Although the increment in wing length was higher for male than for female nestlings, wing-growth strategies in both sexes seem to be unaffected by parasite infections. As a faster development of wings can facilitate both escape from predators and survival outside the nest, growth of body components that reduces the risk of predation may be relatively prioritized over mass in species at higher risk of nestling predation [71]. In contrast, there is a clear sex-biased growth strategy in relation to the rate at which individuals gain body mass under malaria infestation. The advantage of ‘being a male’—attaining higher weight and larger biometric size than sisters—, and the competitive strength related to this [88] becomes a handicap under parasite infestation, when the selective pressure for a faster growth is strong. In our study, the impact of parasites in term of mass gain is particularly important in the fastest growing sex [89], which support this idea. Unfortunately, the potential incidence of a reduced condition during growth on posterior mortality rates cannot be evaluated here, but several authors have demonstrated this kind of relationships [90, 91]. Therefore, in species/populations that evolved under strong (nest) predation pressures, such as Tawny pipits [40], the impact of malaria parasites might ultimately influence offspring mortality rates.

Once adulthood is reached, selection pressures changed, and the trade-offs between immune system and investment in reproduction makes females more susceptible to the effect of malaria parasites than males. In our species model, females make greater investment than males in terms of time and energy during breeding, since females are solely responsible for incubation and chick rearing, which included the search for food and feeding chicks [40]. As a result, Tawny pipit females show a continuous decline in body condition during the breeding season of around 27 % of initial mass [92]. Here, infection was associated to a reduced host condition for females but not for males, supporting the view that infections by malaria parasites cause sex-related differences in host body condition, presumably as a result of increased investment in reproduction by females compared to males. However, it is also possible that host with reduced body mass may be less likely to fight off parasites or their vectors. Further work is clearly needed to conclusively evaluate the link between reproductive effort in both sexes and the effect of parasites acquired by Tawny pipits.

Most studies only consider adults when investigating avian malaria parasites and as a result infection in nestlings is under-represented in the literature. This study has focussed not only in adults (in terms of parasite prevalence and association with body condition), but also considered infections of these parasites in nestlings (prevalence and effects on nestling growth). This study has demonstrated high prevalence of avian malaria parasites in nestlings (7–11 day-old), comparable to adult prevalence, and sex-related differences in both age groups. Both the acquisition of these parasites as well as the consequences of parasites for individuals depends on host sex and age. Trade-offs between investment in immune system and investment in other tasks (reproduction in adults and growth in nestlings) may explain the results found, and some ecological factors (nest predation) could exacerbate the effects of malaria parasites for avian hosts at the population level.