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Immune responses can protect hosts from the fitness costs of parasitism; however, the strength and effectiveness of immune-mediated defense varies among individuals. Variability has been linked to factors including, but not limited to, host reproductive condition (Horak et al. 1999; Ilmonen et al. 2002), stress (Lacoste et al. 2002), evolutionary history of exposure (Lee and Klasing 2004; Matson 2006; Bonneaud et al. 2012), and genetic factors (Beadell et al. 2007). The ability to identify underlying causes of variation is limited by the context in which studies are performed (Graham et al. 2011). A major challenge in ecological immunology has been drawing causal relationships between host immune responses and actual parasites, under natural conditions (Owen and Clayton 2007; Owen et al. 2010; Boughton et al. 2011). Studies that experimentally manipulate parasite abundance and test for parasite-induced host immune responses have the potential to be very informative; unfortunately, such studies are few in number (Buechler et al. 2002; De Coster et al. 2010). In this article we report the results of one of the first such studies in a natural host–parasite system.
In the Galápagos Islands of Ecuador an introduced parasitic nest fly, Philornis downsi, has been implicated in recent population declines of several species of Darwin's finches (Dvorak et al. 2004, 2012; Wiedenfeld et al. 2007; O'Connor et al. 2010b,c). Adult flies, which are not parasitic, lay their eggs in the nests of finches (Couri and Carvalho 2003; Fessl et al. 2006), or in the nares (nostrils) of nestlings (Galligan and Kleindorfer 2009). Once the eggs hatch, the larvae live in the nest and feed on the blood of the nestling and adult female birds (Dudaniec et al. 2006; Huber et al. 2010). Philornis downsi is known to have a significant negative effect on the reproductive success of its hosts (reviewed in Koop et al. 2011).
A recent study by Huber et al. (2010) of medium ground finches (Geospiza fortis; Fig. 1) demonstrated increased levels of P. downsi-binding antibodies in birds during the nesting season, compared to birds sampled immediately prior to nesting. This increase in antibodies was observed in adult female birds, but not in adult males. Female finches incubate eggs and brood offspring, hypothetically increasing their exposure to P. downsi larvae in the nest. Male finches do not incubate eggs or brood nestlings. Although Huber et al. (2010) showed a correlation between nesting and increased antibody level, this correlation could be driven by other variables such as immune stimulation induced by breeding stress (Pruett 2003). An experimental manipulation of parasite abundance is needed to confirm the extent to which the immune response is actually caused by the parasite. To this end, we manipulated parasite abundance in nests to confirm that the observed changes in immune response are, in fact, induced by P. downsi, and are not the product of other temporal correlates.
We also monitored adult and nestling behavior with respect to P. downsi in the nest. Behavioral defense can be integrated with immune responses against ectoparasites (Lehane 2005). For example, host antibodies produced against salivary proteins of ectoparasites are known to promote pruritus (itching), alerting the host to the presence of parasites (Wikel 1996; Owen et al. 2009). Hosts that respond to the presence of biting insects with defensive behaviors, such as preening, are far more likely to injure, kill, or reduce the feeding time of the parasite (Dusbabek and Skarkovaspakova 1988; O'Connor et al. 2010a).
Yet another goal of this study was to investigate the role of immune responses in mitigating the fitness effects of P. downsi. Antibodies produced by hosts have the potential to act defensively against ectoparasites, like P. downsi, by facilitating the speed and intensity of inflammatory responses (Owen et al. 2010). Inflammation of the skin inhibits blood feeding by preventing parasites from reaching host blood vessels with their mouthparts. Ectoparasites feeding on inflamed tissues may also ingest defensive peptides, or lytic molecules produced by the host that impair parasite feeding and digestion (Owen et al. 2009). These components of the immune response can lead to dramatic reductions in the survival, development, and reproduction of parasites (Owen et al. 2009). Thus, we compared the level of immune response by finches to the abundance of P. downsi larvae in their nests.
Finally, we quantified host reproductive success to investigate potential fitness consequences of host immune responses. Immune responses, even those associated with negative consequences for parasites, do not necessarily lead to increases in host fitness (Sheldon and Verhulst 1996; Norris and Evans 2000). Mounting an immune response is energetically expensive and may involve trade-offs with other fitness components, such as parental care or reproductive effort (Raberg et al. 2000). Thus, hosts mounting strong immune responses against a parasite may have reduced fitness if they are less able to care for their offspring. Conversely, the benefit of reducing parasite abundance may outweigh the costs of an immune response and lead to a net increase in host fitness. Host immune response and behavior, parasite abundance, and host fitness must be measured simultaneously to rigorously interpret the influence of host immune defense on host fitness (Graham et al. 2011).
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This study experimentally demonstrates a parasite-induced immune response in a wild bird population. Our results show a causal link between a biologically relevant host immune response and an actual parasite, under natural conditions. Adult female, but not male, medium ground finches produced a significant immunological (antibody-mediated) response to P. downsi. Our results show experimentally that P. downsi does, in fact, stimulate an immune response in adult females, consistent with the correlation reported by Huber et al. (2010). Furthermore, we show that females mounting stronger parasite-induced immune responses tend to have fewer parasites in their nests.
This study is one of the first demonstrations of an apparent effect of a parasite-induced immune response on parasite abundance in a wild bird. Work with other bird parasites has shown that antibody-mediated immune responses can increase the speed and intensity of the inflammatory response, preventing successful feeding of parasites and reducing parasite survival (Owen et al. 2009). Alternatively, the mechanism could be indirect; for example, female antibody responses may promote itching that alerts the host to biting insects (Wikel 1996; Owen et al. 2009). Females that respond with defensive behaviors, such as preening, could kill, injure, or remove parasites (Dusbabek and Skarkovaspakova 1988; O'Connor et al. 2010a). Further work is needed to explore additional variables that may be co-correlated with female immune response and parasite abundance.
A reduction in parasite burden is expected to benefit nestlings and thereby improve host reproductive success. However, within the parameters of this study, the observed decrease in parasite abundance did not help nestlings, as no nestlings fledged from any of the sham-fumigated nests. This result was surprising, given the results of a previous study performed in 2008 which showed that eradicating some, but not all, P. downsi from medium ground finch nests leads to increased fledging success (Koop et al. 2011). Koop et al. (2011) significantly reduced mean P. downsi abundance to ~21 parasites in nests treated with nest liners, compared to untreated nests, which had ~38 parasites per nest. This reduction was sufficient to increase fledgling success in lined nests, where 33% of nests fledged at least one offspring, compared to unlined nests, where only 4% of nests fledged any offspring. In this study, parasite abundance in sham-fumigated nests ranged from 5 to 79 P. downsi per nest (Fig. 2), yet no nestlings survived from nests in this treatment. The young age at which nestlings in this study died and the complete failure of nests even with low parasite abundance suggests that the impact of P. downsi on finches was unusually severe at our study site in 2010. One possible reason is that 2009 was a very dry year; annual rainfall in 2009 was 219 mm, compared to 503 mm in 2010 (Charles Darwin Foundation 2012, Meteorological Database). Dry years reduce overall seed availability, meaning that the seed bank in 2010 may have been depleted (Schluter 1982). Limited food resources are expected to negatively affect adult condition (Boag and Grant 1984), which may have placed additional stress on nestlings.
Furthermore, annual differences in rainfall may have contributed to changes in P. downsi virulence. Multiple P. downsi females can infest a single finch nest and female flies can mate with multiple males (Dudaniec et al. 2010). Thus, the relatedness between P. downsi larvae in a single finch nest has a relatively high degree of variability. Models of kin selection predict that when genetic relatedness of parasites is low, competition for within-host resources increases, leading to greater costs to the host (Frank 1994, 1996). While we did not collect data to directly test this idea, annual variation in climatic conditions may have altered the egg laying strategy of female flies, causing variation in parasite virulence between years. However, variation in parasite virulence could also be due to a number of other factors, such as host bird traits. Further investigation is needed to determine the role of biotic and abiotic factors on P. downsi virulence.
Independent of the effect on parasite abundance, female immune responses are thought to alter parental investment in current or future offspring (Raberg et al. 2000; Bonneaud et al. 2003). The ability of adult birds to perform parental behaviors can depend on the amount of energy invested (or not invested) in an immune response. Increases in nest sanitation and preening behaviors can serve to reduce parasite burden in the nest (Christe et al. 1996; Hurtrez-Bousses et al. 2000; Clayton et al. 2010). Parents can also alter the rate at which they feed nestlings in order to provide energetic compensation for the direct negative effects of parasitism (Tripet and Richner 1997; Hurtrez-Bousses et al. 1998). Alternatively, birds can abandon nests with parasites in favor of future reproductive efforts (Duffy 1983). O'Connor et al. (2010a) observed females of several finch species performing nest sanitation as well as allo-preening the feathers and nares of nestlings in nests with P. downsi. Interestingly, we observed almost no allo-preening; however, our observations were of younger nestlings (most of which were dead by 1 week of age). Our data show that while females did not abandon their parasitized nestlings or spend less time at the nest, they also did not significantly increase potentially beneficial behaviors, such as nest sanitation, or feeding nestlings. As in this study, O'Connor et al. (2010a) found no correlation between P. downsi intensity and parental feeding of nestlings.
Females in this study did, however, alter their brooding behavior; females in parasitized nests brooded significantly less and stood up more than females in fumigated nests. Whether this behavior was in response to agitated nestlings, or the parasites themselves, standing was probably an avoidance strategy for females (Hart 1990). Although this study shows that these responses were not sufficient to rescue current reproduction, further study is needed to determine whether female responses increase their ability to invest in future reproduction.
Young altricial nestlings are expected to serve as primary hosts for nest parasites because they lack the necessary motor skills to preen or stand. Furthermore, both the innate and acquired arms of the immune system are developing in nestlings, perhaps making them incapable of mounting a robust immune response to parasites (Palacios et al. 2009). We found no detectable difference in antibody levels of nestlings in fumigated and sham-fumigated nests. This result suggests that nestlings are not able to defend themselves immunologically against P. downsi in the nest. However, it should be noted that the rapid mortality of nestlings in sham-fumigated nests limited our sampling to young nestlings (~5 days of age). A recent study by King et al. (2010) found that nestlings of some species of birds can start producing parasite-induced antibodies endogenously within 3–6 days of age. Thus, quantification of antibodies from older nestlings (6–14 days old) may yield different results. Of course, the ability of nestlings to produce P. downsi-binding antibodies and immunologically defend themselves against nest parasites is dependent upon their survival to that time point.
Our results suggest that young nestlings are incapable of responding behaviorally to P. downsi. O'Connor et al. (2010a) observed medium ground finch nestlings preening themselves in a nest parasitized by P. downsi. The same nestlings were also observed trying to climb on top of one another, possibly to escape P. downsi larvae attempting to feed. Nestlings in this study were observed preening only rarely (<1% of time), and the behavior did not differ significantly between treatments. Nestlings from sham-fumigated nests tended to show more agitated behavior than those in fumigated nests. Periods of agitation included shaking and repositioning within the nest, but we did not observe nestlings standing on top of one another. Again, many of the nestlings observed by O'Connor et al. (2010a) were significantly older (>8 days of age), which may explain the differences in behavior between studies. Very young nestlings lack the necessary motor skills to preen themselves, or stand.
Studies that explore trade-offs between host immune response and life-history components often operate under the assumption that stronger immune responses are positively correlated with higher fitness (Norris and Evans 2000). This study demonstrates immunological activity of birds in response to a biologically relevant parasite. The data further suggest that stronger immune responses are defensive, because higher antibody levels are marginally correlated with lower parasite abundance. However, higher antibody levels did not result in higher reproductive success. This study provides a cautionary tale: even when stronger immune responses lead to lower parasite load, this does not necessarily result in higher host fitness. Our study underscores the importance of studying interactions between the host immune system, parasite load, and host fitness in order to derive robust conclusions regarding the functional significance of the immune system in an ecological context (Owen et al. 2010; Graham et al. 2011).