Understanding spatiotemporal effects of food supplementation on host-parasite interactions using community-based science.

Supplemental feeding can increase the overall health of animals but also can have variable effects on how animals defend themselves against parasites. However, the spatiotemporal effects of food supplementation on host-parasite interactions remain poorly understood, likely because large-scale, coordinated efforts to investigate them are difficult. Here, we introduce the Nest Parasite Community Science Project, which is a community-based science project that coordinates studies with bird nest box 'stewards' from the public and scientific community. This project was established to understand broad ecological patterns between hosts and their parasites. The goal of this study was to determine the effect of food supplementation on eastern bluebirds (Sialia sialis) and their nest parasite community across the geographic range of the bluebirds from 2018 to 2021. We received 674 nests from 69 stewards in 26 states in the eastern United States. Nest box stewards reported whether or not they provided mealworms or suet near nesting bluebirds, then they followed the nesting success of the birds (number of eggs laid and hatched, proportion that hatched, number and proportion of nestlings that successfully fledged). We then identified and quantified parasites in the nests. Overall, we found that food supplementation increased fledging success. The most common nest parasite taxon was the parasitic blow fly (Protocalliphora sialia), but a few nests contained fleas (Ceratophyllus idius, C. gallinae and Orchopeas leucopus) and mites (Dermanyssus spp. and Ornithonyssus spp.). Blow flies were primarily found at northern latitudes, where food supplementation affected blow fly prevalence. However, the direction of this effect varied substantially in direction and magnitude across years. More stewards fed bluebirds at southern latitudes than at northern latitudes, which contradicts the findings of other community-based science projects. Overall, food supplementation of birds was associated with increased host fitness but did not appear to play a consistent role in defence against these parasites across all years. Our study demonstrates the importance of coordinated studies across years and locations to understand the effects of environmental heterogeneity, including human-based food supplementation, on host-parasite dynamics.

Direct and indirect effects of sexual signal loss on female reproduction in the Pacific field cricket (Teleogryllus oceanicus).

Sexual signal evolution may present fitness consequences for the non-signaling sex due to shared genes and altered social conditions, but this is rarely studied in natural populations. On the Hawaiian Island of Kauai, most male Teleogryllus oceanicus (Pacific field crickets) lack the ability to sing because of a novel wing mutation (flatwing) that arose and spread in <20 generations. Obligately silent flatwing males have been highly successful because they avoid detection by a deadly, acoustically-orienting parasitoid fly. Little is known about how the flatwing mutation and resulting song-less acoustic environment affects female fitness. We found that Kauai females carrying the flatwing allele invested less in reproductive tissues and experienced more instances of mating failure than normal-wing-carrying females, though total offspring production did not differ between female genotypes. Females from Oahu (HI, where the parasitoid and flatwing also occur) and Mangaia (an island in the Cook Islands which harbors neither the parasitoid nor flatwing) invested less in reproductive tissues when reared in a song-less acoustic environment. Kauai females did not exhibit this plasticity, perhaps because they have experienced nearly song-less conditions for the past ~15 years following the establishment of flatwing. We show that female T. oceanicus experience a mix of costly and beneficial effects of sexual signal loss, which should help maintain the wing polymorphism in the wild. Our results demonstrate that the non-signaling sex can experience a nuanced set of phenotypic consequences resulting from signal evolution, which can further shape dynamics of sexual signal evolution.

Rapid evolution of disease resistance is accompanied by functional changes in gene expression in a wild bird.

Wild organisms are under increasing pressure to adapt rapidly to environmental changes. Predicting the impact of these changes on natural populations requires an understanding of the speed with which adaptive phenotypes can arise and spread, as well as of the underlying mechanisms. However, our understanding of these parameters is poor in natural populations. Here we use experimental and molecular approaches to investigate the recent emergence of resistance in eastern populations of North American house finches (Carpodacus mexicanus) to Mycoplasma galliseptum (MG), a severe conjunctivitis-causing bacterium. Two weeks following an experimental infection that took place in 2007, finches from eastern US populations with a 12-y history of exposure to MG harbored 33% lower MG loads in their conjunctivae than finches from western US populations with no prior exposure to MG. Using a cDNA microarray, we show that this phenotypic difference in resistance was associated with differences in splenic gene expression, with finches from the exposed populations up-regulating immune genes postinfection and those from the unexposed populations generally down-regulating them. The expression response of western US birds to experimental infection in 2007 was more similar to that of the eastern US birds studied in 2000, 7 y earlier in the epizootic, than to that of eastern birds in 2007. These results support the hypothesis that resistance has evolved by natural selection in the exposed populations over the 12 y of the epizootic. We hypothesize that host resistance arose and spread from standing genetic variation in the eastern US and highlight that natural selection can lead to rapid phenotypic evolution in populations when acting on such variation.

Temporal genomics in Hawaiian crickets reveals compensatory intragenomic coadaptation during adaptive evolution.

Theory predicts that compensatory genetic changes reduce negative indirect effects of selected variants during adaptive evolution, but evidence is scarce. Here, we test this in a wild population of Hawaiian crickets using temporal genomics and a high-quality chromosome-level cricket genome. In this population, a mutation, flatwing, silences males and rapidly spread due to an acoustically-orienting parasitoid. Our sampling spanned a social transition during which flatwing fixed and the population went silent. We find long-range linkage disequilibrium around the putative flatwing locus was maintained over time, and hitchhiking genes had functions related to negative flatwing-associated effects. We develop a combinatorial enrichment approach using transcriptome data to test for compensatory, intragenomic coevolution. Temporal changes in genomic selection were distributed genome-wide and functionally associated with the population's transition to silence, particularly behavioural responses to silent environments. Our results demonstrate how 'adaptation begets adaptation'; changes to the sociogenetic environment accompanying rapid trait evolution can generate selection provoking further, compensatory adaptation.

Experimental evidence for distinct costs of pathogenesis and immunity against a natural pathogen in a wild bird.

Protective immunity is expected to evolve when the costs of mounting an immune response are less than those of harbouring pathogens. Estimating the costs of immunity vs. pathogenesis in natural systems is challenging, however, because they are typically closely linked. Here we attempt to disentangle the relative cost of each using experimental infections in a natural host-parasite system in which hosts (house finches, Carpodacus mexicanus) differ in resistance to a bacterium (Mycoplasma gallisepticum, MG), depending on whether they originate from co-evolved or unexposed populations. Experimental infection with a 2007-strain of MG caused finches from co-evolved populations to lose significantly more mass relative to controls, than those from unexposed populations. In addition, infected co-evolved finches that lost the most mass harboured the least amounts of MG, whereas the reverse was true in finches from unexposed populations. Finally, within co-evolved populations, individuals that displayed transcriptional evidence of higher protective immune activity, as indicated by changes in the expression of candidate immune and immune-related genes in a direction consistent with increased resistance to MG, showed greater mass loss and lower MG load. Thus, mass loss appeared to reflect the costs of immunity vs. pathogenesis in co-evolved and unexposed populations, respectively. Our results suggest that resistance can evolve even when the short-term energetic costs of protective immunity exceed those of pathogenesis, providing the longer-term fitness costs of infection are sufficiently high.

Innate immunity and the evolution of resistance to an emerging infectious disease in a wild bird.

Innate immunity is expected to play a primary role in conferring resistance to novel infectious diseases, but few studies have attempted to examine its role in the evolution of resistance to emerging pathogens in wild vertebrate populations. Here, we used experimental infections and cDNA microarrays to examine whether changes in the innate and/or acquired immune responses likely accompanied the emergence of resistance in house finches (Carpodacus mexicanus) in the eastern United States subject to a recent outbreak of conjunctivitis-causing bacterium (Mycoplasma gallisepticum-MG). Three days following experimental infection with MG, we observed differences in the splenic transcriptional responses between house finches from eastern U.S. populations, with a 12-year history of MG exposure, versus western U.S. populations, with no history of exposure to MG. In particular, western birds down-regulated gene expression, while eastern finches showed no expression change relative to controls. Studies involving poultry have shown that MG can manipulate host immunity, and our observations suggest that pathogen manipulation occurred only in finches from the western populations, outside the range of MG. Fourteen days after infection, eastern finches, but not western finches, up-regulated genes associated with acquired immunity (cell-mediated immunity) relative to controls. These observations suggest population differences in the temporal course of the response to infection with MG and imply that innate immune processes were targets of selection in response to MG in the eastern U.S. population.

Immunogenetic and tolerance strategies against a novel parasitoid of wild field crickets.

Among the parasites of insects, endoparasitoids impose a costly challenge to host defenses because they use their host's body for the development and maturation of their eggs or larvae, and ultimately kill the host. Tachinid flies are highly specialized acoustically orienting parasitoids, with first instar mobile larvae that burrow into the host's body to feed. We investigated the possibility that Teleogryllus oceanicus field crickets employ postinfestation strategies to maximize survival when infested with the larvae of the parasitoid fly Ormia ochracea. Using crickets from the Hawaiian Islands of Kauai, where the parasitoid is present, and crickets from the Cook Islands (Mangaia), where the parasitoid is absent, we evaluated fitness consequences of infestation by comparing feeding behavior, reproductive capacity, and survival of males experimentally infested with O. ochracea larvae. We also evaluated mechanisms underlying host responses by comparing gene expression in crickets infested with fly larvae for different lengths of time with that of uninfested control crickets. We observed weak population differences in fitness (spermatophore production) and survival (total survival time postinfestation). These responses generally did not show an interaction between population and the number of larva hosts carried or by host body condition. Gene expression patterns also revealed population differences in response to infestation, but we did not find evidence for consistent differences in genes associated with immunity or stress response. One possibility is that any postinfestation evolved resistance does not involve genes associated with these particular functional categories. More likely, these results suggest that coevolution with the fly does not strongly select for either postinfestation resistance or tolerance of parasitoid larvae in male crickets.

Molecular biogeography and host relations of a parasitoid fly.

Successful geographic range expansion by parasites and parasitoids may also require host range expansion. Thus, the evolutionary advantages of host specialization may trade off against the ability to exploit new host species encountered in new geographic regions. Here, we use molecular techniques and confirmed host records to examine biogeography, population divergence, and host flexibility of the parasitoid fly, Ormia ochracea (Bigot). Gravid females of this fly find their cricket hosts acoustically by eavesdropping on male cricket calling songs; these songs vary greatly among the known host species of crickets. Using both nuclear and mitochondrial genetic markers, we (a) describe the geographical distribution and subdivision of genetic variation in O. ochracea from across the continental United States, the Mexican states of Sonora and Oaxaca, and populations introduced to Hawaii; (b) demonstrate that the distribution of genetic variation among fly populations is consistent with a single widespread species with regional host specialization, rather than locally differentiated cryptic species; (c) identify the more-probable source populations for the flies introduced to the Hawaiian islands; (d) examine genetic variation and substructure within Hawaii; (e) show that among-population geographic, genetic, and host song distances are all correlated; and (f) discuss specialization and lability in host-finding behavior in light of the diversity of cricket songs serving as host cues in different geographically separate populations.

Mechanisms of evolutionary change in structural plumage coloration among bluebirds (Sialia spp.).

Combinations of microstructural and pigmentary components of barbs create the colour displays of feathers. It follows that evolutionary changes in colour displays must reflect changes in the underlying production mechanisms, but rarely have the mechanisms of feather colour evolution been studied. Among bluebirds in the genus Sialia, male rump colour varies among species from dark blue to light blue while breast colour varies from blue to rusty. We use spectrometry, transmission electron microscopy and Fourier analysis to identify the morphology responsible for these divergent colour displays. The morphology of blue rump barbs is similar among the three species, with an outer keratin cortex layer surrounding a medullary 'spongy layer' and a basal row of melanin granules. A spongy layer is also present in blue breast barbs of mountain bluebirds Sialia currucoides and in rusty breast barbs of western Sialia mexicana and eastern bluebirds Sialia sialis. In blue barbs melanin is basal to the spongy layer, but is not present in the outer cortex or spongy layer, while in rusty barbs, melanin is present only in the cortex. The placement of melanin in the cortex masks expression of structural blue, creating a rusty display. Such shifts in microstructures and pigments may be widespread mechanisms for the evolutionary changes in the colours of feathers and other reflective structures across colourful organisms.

Testing the Hamilton-Zuk hypothesis: past, present, and future.

Hamilton and Zuk proposed a good-genes model of sexual selection in which genetic variation can be maintained when females prefer ornaments that indicate resistance to parasites. When trait expression depends on a male's resistance, the co-adaptive cycles between host resistance and parasite virulence provide a mechanism in which genetic variation for fitness is continually renewed. The model made predictions at both the intraspecific and interspecific levels. In the three decades since its publication, these predictions have been theoretically examined in models of varying complexity, and empirically tested across many vertebrate and invertebrate taxa. Despite such prolonged interest, however, it has turned out to be extremely difficult to empirically demonstrate the process described, in part because we have not been able to test the underlying mechanisms that would unequivocally identify how parasites act as mediators of sexual selection. Here, we discuss how the use of high-throughput sequencing datasets available from modern genomic approaches might improve our ability to test this model. We expect that important contributions will come through the ability to identify and quantify the suite of parasites likely to influence the evolution of hosts' resistance, to confidently reconstruct phylogenies of both host and parasite taxa, and, perhaps most exciting, to detect generational cycles of heritable variants in populations of hosts and parasites. Integrative approaches, building on systems undergoing parasite-mediated selection with genomic resources already available, will be particularly useful in moving toward robust tests of this hypothesis. We finish by presenting case studies of well-studied host-parasite relationships that represent promising avenues for future research.

Primer design and transcript quantification of a highly multiplexed RT-PCR for a nonmodel avian species.

Multiplexed qRT-PCR assays are currently lacking for nearly all species without genome or transcriptome resources. Here, we present a strategy for primer design of highly multiplexed qRT-PCR assays, evaluate Beckman Coulter's Quant Tool gene expression quantification software and provide details of our assay for the North American songbird Carpodacus mexicanus (house finch), for which only small sections of genome sequence are available. We combined Beckman Coulter's eXpress Designer module for creating custom multiplex primers with the free, online program Amplify 3 to design and evaluate primers computationally before testing them empirically. We also generated a standard curve for each gene included in the final multiplex. We compared models using cubic and quadratic polynomial estimators that did and did not force the intercept through zero. Ultimately, we used the sequences available for 316 clones differentially expressed in cDNA macroarray and microarray comparisons, and from these sequences, we were able to generate a set of transcript-specific primers for use with the GeXP analyser for 20 house finch genes.