Parental food provisioning and nestling growth under Philornis downsi parasitism in the Galapagos Green Warbler-Finch, classified as 'vulnerable' by the IUCN.

In the Galapagos Islands, many endemic landbird populations are declining due to habitat degradation, food availability, introduced species and other factors. Given nestlings typically lack efficient defense mechanisms against parasites, hematophagous ectoparasites such as the larvae of the introduced Avian Vampire Fly, Philornis downsi, can impose high brood mortality and cause threatening population declines in Darwin finches and other landbirds. Here, we assess whether the food compensation hypothesis (i.e., the parents' potential to compensate for deleterious parasite effects via increased food provisioning) applies to the Green Warbler-Finch. We differentiated nests with low or high infestation levels by P. downsi and quantified food provisioning rates of male and female parents, time females spent brooding nestlings, and nestling growth. Male provisioning rates, total provisioning rates and female brooding time did not significantly vary in relation to infestation levels, nor by the number of nestlings. Opposed to the predictions of the food compensation hypothesis, females showed significantly reduced provisioning rates at high infestation levels. Nestling body mass was significantly lower and there was a reduction of skeletal growth, although not significantly, in highly infested nests. The females' response to high infestation may be due to parasites directly attacking and weakening brooding females, or else that females actively reduce current reproductive effort in favor of future reproduction. This life-history trade-off may be typical for Darwin finches and many tropical birds with long lifespans and therefore high residual reproductive value. Conservation strategies may not build on the potential for parental food compensation by this species.

Female in-nest attendance predicts the number of ectoparasites in Darwin's finch species.

Selection should act on parental care and favour parental investment decisions that optimize the number of offspring produced. Such predictions have been robustly tested in predation risk contexts, but less is known about alternative functions of parental care under conditions of parasitism. The avian vampire fly (Philornis downsi) is a myasis-causing ectoparasite accidentally introduced to the Galápagos Islands, and one of the major mortality causes in Darwin's finch nests. With an 11-year dataset spanning 21 years, we examine the relationship between parental care behaviours and number of fly larvae and pupae in Darwin's finch nests. We do so across three host species (Camarhynchus parvulus, C. pauper, Geospiza fuliginosa) and one hybrid Camarhynchus group. Nests with longer female brooding duration (minutes per hour spent sitting on hatchlings to provide warmth) had fewer parasites, and this effect depended on male food delivery to chicks. Neither male age nor number of nest provisioning visits were directly associated with number of parasites. While the causal mechanisms remain unknown, we provide the first empirical study showing that female brooding duration is negatively related to the number of ectoparasites in nests. We predict selection for coordinated host male and female behaviour to reduce gaps in nest attendance, especially under conditions of novel and introduced ectoparasites.

Epigenetic effects of parasites and pesticides on captive and wild nestling birds.

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ectoparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free-living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced-representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we conducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identified a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontarget effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost-effective way to characterize genome-wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects.

Parental care in the Small Tree Finch Camarhynchus parvulus in relation to parasitism and environmental factors.

The parental food compensation hypothesis suggests that parents may compensate for the negative effects of parasites on chicks by increased food provisioning. However, this ability differs widely among host species and may also depend on ecological factors such as adverse weather conditions and habitat quality. Although weed management can improve habitat quality, management measures can bring about a temporary decrease in food availability and thus may reduce parents' ability to provide their nestlings with enough energy. In our study we investigated the interaction of parasitism and weed management, and the influence of climate on feeding rates in a Darwin's tree finch species, which is negatively impacted by two invasive species. The larvae of the invasive parasitic fly Philornis downsi ingest the blood and body tissues of tree finch nestlings, and the invasive Blackberry Rubus niveus affects one of the main habitats of Darwin's tree finches. We compared parental food provisioning of the Small Tree Finch Camarhynchus parvulus in parasitized and parasite-free nests in three different areas, which differed in invasive weed management (no management, short-term and long-term management). In a parasite reduction experiment, we investigated whether the Small Tree Finch increases food provisioning rates to nestlings when parasitized and whether this ability depends on weed management conditions and precipitation. Our results provide no evidence that Small Tree Finches can compensate with additional food provisioning when parasitized with P. downsi. However, we found an increase in male effort in the short-term management area, which might indicate that males compensate for lower food quality with increased provisioning effort. Furthermore, parental food provisioning was lower during rainfall, which provides an explanation for the negative influence of rain on breeding success found in earlier studies. Like other Darwin's finches, the Small Tree Finch seems to lack the ability to compensate for the negative effects of P. downsi parasitism, which is one explanation for why this invasive parasite has such a devastating effect on this host species.

Timing of infestation influences virulence and parasite success in a dynamic multi-host-parasite interaction between the invasive parasite, Philornis downsi, and Darwin's finches.

Recently commenced host-parasite interactions provide an excellent opportunity to study co-evolutionary processes. Multi-host systems are especially informative because variation in virulence between hosts and temporal changes provides insight into evolutionary dynamics. However, empirical data under natural conditions are scarce. In the present study, we investigated the interaction between Darwin's finches and the invasive fly Philornis downsi whose larvae feed on the blood of nestlings. Recently, however, the fly has changed its behavior and now also attacks incubating females. Two sympatric hosts are affected differently by the parasite and parasite load has changed over time. Our study observed a reversal of trends described two decades ago: while, currently, small tree finches (Camarhynchus parvulus) experience significantly higher parasite load than warbler finches (Certhidea olivacea), this was the opposite two decades ago. Currently, fledging success is higher in warbler finches compared to small tree finches. Our data indicate that not only intensity but also timing of infestation influences hosts' reproductive success and parasite fitness. During incubation, prevalence was higher in warbler finches, but once chicks had hatched, prevalence was 100% in both species and parasite load was higher in small tree finches. Furthermore, our results suggest faster development and higher reproductive success of P. downsi in small tree finch nests. A change in host preference driven by larvae competition could have led to the reversal in parasite load.

Parasitism by an invasive nest fly reduces future reproduction in Galápagos mockingbirds.

Organisms allocate limited resources to competing activities such as reproduction, growth, and defense against parasites and predators. The introduction of a novel parasite may create new life history trade-offs. As hosts increase their investment in self-maintenance or defense, the cost of parasitism may carry over to other aspects of host biology. Here, in an experimental field study, we document delayed effects of an introduced nest parasite, Philornis downsi, on reproduction of Galápagos mockingbirds (Mimus parvulus). Parasitism of first nests reduced both the number and size of chicks that parents hatched when they re-nested several weeks later. The delayed effect of P. downsi on future reproduction may have been mediated by behavioral shifts by the parents to avoid or resist parasitism. Our results demonstrate that effects of parasitism can persist even after immediate exposure ends. We draw attention to the potential implications that introduced parasites have for host reproductive strategies.

Genetic admixture predicts parasite intensity: evidence for increased hybrid performance in Darwin's tree finches.

Hybridization can increase adaptive potential when enhanced genetic diversity or novel genetic combinations confer a fitness advantage, such as in the evolution of anti-parasitic mechanisms. Island systems are especially susceptible to invasive parasites due to the lack of defence mechanisms that usually coevolve in long-standing host-parasite relationships. We test if host genetic admixture affects parasite numbers in a novel host-parasite association on the Galápagos Islands. Specifically, we compare the number of Philornis downsi in nests with offspring sired by Darwin's small tree finch (Camarhynchus parvulus), Darwin's medium tree finch (C. pauper) and hybrids of these two species. The number of P. downsi decreased with an increasing genetic admixture of the attending male, and nests of hybrid males had approximately 50% fewer parasites than C. parvulus nests, and approximately 60% fewer parasites than C. pauper nests. This finding indicates that hybridization in this system could be favoured by selection and reveal a mechanism to combat an invasive parasite.

Annual environmental variation influences host tolerance to parasites.

When confronted with a parasite or pathogen, hosts can defend themselves by resisting or tolerating the attack. While resistance can be diminished when resources are limited, it is unclear how robust tolerance is to changes in environmental conditions. Here, we investigate the sensitivity of tolerance in a single host population living in a highly variable environment. We manipulated the abundance of an invasive parasitic fly, Philornis downsi, in nests of Galápagos mockingbirds ( Mimus parvulus) over four field seasons and measured host fitness in response to parasitism. Mockingbird tolerance to P. downsi varied significantly among years and decreased when rainfall was limited. Video observations indicate that parental provisioning of nestlings appears key to tolerance: in drought years, mockingbirds likely do not have sufficient resources to compensate for the effects of P. downsi. These results indicate that host tolerance is a labile trait and suggest that environmental variation plays a major role in mediating the consequences of host-parasite interactions.

Host-specific associations affect the microbiome of Philornis downsi, an introduced parasite to the Galápagos Islands.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.

Identification and Optimization of Microbial Attractants for Philornis downsi, an Invasive Fly Parasitic on Galapagos Birds.

We investigated the role of olfactory cues from actively fermenting yeast (Saccharomyces cerevisiae) in attraction of adult Philornis downsi and identified two synergistically attractive yeast volatiles. Larvae of this invasive fly parasitize the hatchlings of passerines and threaten the Galapagos avifauna. Gas chromatography coupled with electroantennographic detection (GC-EAD), coupled gas chromatography-mass spectrometry (GC-MS), and field trapping experiments were used to identify volatile compounds from a yeast-sugar solution. EAD responses were consistently elicited by 14 yeast volatiles. In a series of field trapping experiments, a mixture of the 14 EAD-active compounds was similarly attractive to P. downsi when compared to the yeast-sugar solution, and we found that acetic acid and ethanol were essential for attraction. A mixture of 0.03 % acetic acid and 3 % ethanol was as attractive as the 14-component blend, but was not as attractive as the yeast-sugar solution. Philornis downsi showed positive and negative dose-responses to acetic acid in the ranges of 0.01 ~ 0.3 % and 0.3 ~ 9 %, respectively. Further optimization showed that the mixture of 1 % acetic acid and 3 % ethanol was as attractive as the yeast-sugar solution. Both mixtures of acetic acid and ethanol were more selective than the yeast-sugar solution in terms of non-target moths and Polistes versicolor wasps captured. These results indicate that acetic acid and ethanol produced by yeasts are crucial for P. downsi attraction to fermented materials on which they feed as adults and can be used to manage this invasive fly in Galapagos.

An introduced parasitic fly may lead to local extinction of Darwin's finch populations.

Introduced pathogens and other parasites are often implicated in host population level declines and extinctions. However, such claims are rarely supported by rigorous real-time data. Indeed, the threat of introduced parasites often goes unnoticed until after host populations have declined severely. The recent introduction of the parasitic nest fly, Philornis downsi, to the Galápagos Islands provides an opportunity to monitor the current impact of an invasive parasite on endemic land bird populations, including Darwin's finches.In this paper we present a population viability model to explore the potential long-term effect of P. downsi on Darwin's finch populations. The goal of our study was to determine whether P. downsi has the potential to drive host populations to extinction and whether management efforts are likely to be effective.Our model is based on data from five years of experimental field work documenting the effect of P. downsi on the reproductive success of medium ground finch Geospiza fortis populations on Santa Cruz Island. Under two of the three scenarios tested, the model predicted medium ground finches are at risk of extinction within the next century.However, sensitivity analyses reveal that even a modest reduction in the prevalence of the parasite could improve the stability of finch populations. We discuss the practicality of several management options aimed at achieving this goal.Synthesis and applications. Our study demonstrates the predicted high risk of local extinction of an abundant host species, the medium ground finch Geospiza fortis due to an introduced parasite, Philornis downsi. However, our study further suggests that careful management practices aimed at reducing parasite prevalence have the potential to significantly lower the risk of host species extinction.

Galápagos mockingbirds tolerate introduced parasites that affect Darwin's finches.

Introduced parasites threaten native host species that lack effective defenses. Such parasites increase the risk of extinction, particularly in small host populations like those on islands. If some host species are tolerant to introduced parasites, this could amplify the risk of the parasite to vulnerable host species. Recently, the introduced parasitic nest fly Philornis downsi has been implicated in the decline of Darwin's finch populations in the Galápagos Islands. In some years, 100% of finch nests fail due to P. downsi; however, other common host species nesting near Darwin's finches, such as the endemic Galápagos mockingbird (Mimus parvulus), appear to be less affected by P. downsi. We compared effects of P. downsi on mockingbirds and medium ground finches (Geospiza fortis) on Santa Cruz Island in the Galápagos. We experimentally manipulated the abundance of P. downsi in nests of mockingbirds and finches to measure the direct effect of the parasite on the reproductive success of each species of host. We also compared immunological and behavioral responses by each species of host to the fly. Although nests of the two host species had similar parasite densities, flies decreased the fitness of finches but not mockingbirds. Neither host species had a significant antibody-mediated immune response to P. downsi. Moreover, finches showed no significant increase in begging, parental provisioning, or plasma glucose levels in response to the flies. In contrast, parasitized mockingbird nestlings begged more than nonparasitized mockingbird nestlings. Greater begging was correlated with increased parental provisioning behavior, which appeared to compensate for parasite damage. The results of our study suggest that finches are negatively affected by P. downsi because they do not have such behavioral mechanisms for energy compensation. In contrast, mockingbirds are capable of compensation, making them tolerant hosts, and a possible indirect threat to Darwin's finches.

Experimental test of the effect of introduced hematophagous flies on corticosterone levels of breeding Darwin's finches.

Parasites can negatively affect the evolutionary fitness of their hosts by eliciting physiological stress responses. Parasite-induced stress can be monitored by measuring changes in the adrenal steroid hormone corticosterone. We examined the effect of an invasive parasite on the corticosterone concentrations of a common species of Darwin's finch, the medium ground finch (Geospiza fortis). Philornis downsi (Diptera: Muscidae) is a parasitic nest fly recently introduced to the Galapagos Islands, where it feeds on the blood of nestlings and breeding adult female finches. Previous work shows that P. downsi significantly reduces the reproductive success of several species of finches. We predicted that the effect of P. downsi on host reproductive success is mediated by stress responses in breeding female finches. High stress levels could reduce the ability of females to invest in offspring, thus decreasing their reproductive success. To test this hypothesis, we experimentally manipulated the abundance of P. downsi in nests, then measured baseline and acute stress-induced corticosterone levels, body condition, and hematocrit (red blood cell content). Acute stress-induced corticosterone levels increased over baseline levels, but this response did not differ significantly with parasite treatment. There was also no significant difference in the body condition or hematocrit of females from parasitized versus non-parasitized nests. Our results suggest that the lower reproductive success of females from parasitized nests is not mediated by a physiological stress response.

Experimental demonstration of a parasite-induced immune response in wild birds: Darwin's finches and introduced nest flies.

Ecological immunology aims to explain variation among hosts in the strength and efficacy of immunological defenses. However, a shortcoming has been the failure to link host immune responses to actual parasites under natural conditions. Here, we present one of the first experimental demonstrations of a parasite-induced immune response in a wild bird population. The recently introduced ectoparasitic nest fly Philornis downsi severely impacts the fitness of Darwin's finches and other land birds in the Galápagos Islands. An earlier study showed that female medium ground finches (Geospiza fortis) had P. downsi-binding antibodies correlating with presumed variation in fly exposure over time. In the current study, we experimentally manipulated fly abundance to test whether the fly does, in fact, cause changes in antibody levels. We manipulated P. downsi abundance in nests and quantified P. downsi-binding antibody levels of medium ground finch mothers, fathers, and nestlings. We also quantified host behaviors, such as preening, which can integrate with antibody-mediated defenses against ectoparasites. Philornis downsi-binding antibody levels were significantly higher among mothers at parasitized nests, compared to mothers at (fumigated) nonparasitized nests. Mothers with higher antibody levels tended to have fewer parasites in their nests, suggesting that antibodies play a role in defense against parasites. Mothers showed no behavioral changes that would enhance the effectiveness of the immune response. Neither adult males, nor nestlings, had P. downsi-induced immunological or behavioral responses that would enhance defense against flies. None of the parasitized nests fledged any offspring, despite the immune response by mothers. Thus, this study shows that, while the immune response of mothers appeared to be defensive, it was not sufficient to rescue current reproductive fitness. This study further shows the importance of testing the fitness consequences of immune defenses, rather than assuming that such responses increase host fitness. Host immune responses can protect against the negative fitness consequences of parasitism; however, the strength and effectiveness of these responses vary among hosts. Strong host immune responses are often assumed to correlate with greater host fitness. This study investigates the relationship between host immune response, parasite load, and host fitness using Darwin's finches and an invasive nest parasite. We found that while the immune response of mothers appeared defensive, it did not rescue current reproductive fitness.