Genetic and environmental variation in condition, cutaneous immunity, and haematocrit in house wrens.

BACKGROUND: Life-history studies of wild bird populations often focus on the relationship between an individual's condition and its capacity to mount an immune response, as measured by a commonly-employed assay of cutaneous immunity, the PHA skin test. In addition, haematocrit, the packed cell volume in relation to total blood volume, is often measured as an indicator of physiological performance. A multi-year study of a wild population of house wrens has recently revealed that those exhibiting the highest condition and strongest PHA responses as nestlings are most likely to be recruited to the breeding population and to breed through two years of age; in contrast, intermediate haematocrit values result in the highest recruitment to the population. Selection theory would predict, therefore, that most of the underlying genetic variation in these traits should be exhausted resulting in low heritability, although such traits may also exhibit low heritability because of increased residual variance. Here, we examine the genetic and environmental variation in condition, cutaneous immunity, and haematocrit using an animal model based on a pedigree of approximately 2,800 house wrens. RESULTS: Environmental effects played a paramount role in shaping the expression of the fitness-related traits measured in this wild population, but two of them, condition and haematocrit, retained significant heritable variation. Condition was also positively correlated with both the PHA response and haematocrit, but in the absence of any significant genetic correlations, it appears that this covariance arises through parallel effects of the environment acting on this suite of traits. CONCLUSIONS: The maintenance of genetic variation in different measures of condition appears to be a pervasive feature of wild bird populations, in contradiction of conventional selection theory. A major challenge in future studies will be to explain how such variation persists in the face of the directional selection acting on condition in house wrens and other species.

Neonatal body condition, immune responsiveness, and hematocrit predict longevity in a wild bird population.

Measures of body condition, immune function, and hematological health are widely used in ecological studies of vertebrate populations, predicated on the assumption that these traits are linked to fitness. However, compelling evidence that these traits actually predict long-term survival and reproductive success among individuals in the wild is lacking. Here, we show that body condition (i.e., size-adjusted body mass) and cutaneous immune responsiveness to phytohaemagglutinin (PHA) injection among neonates positively predict recruitment and subsequent longevity in a wild, migratory population of house wrens (Troglodytes aedon). However, neonates with intermediate hematocrit had the highest recruitment and longevity. Neonates with the highest PHA responsiveness and intermediate hematocrit prior to independence eventually produced the most offspring during their lifetime breeding on the study site. Importantly, the effects of PHA responsiveness and hematocrit were revealed while controlling for variation in body condition, sex, and environmental variation. Thus, our data demonstrate that body condition, cutaneous immune responsiveness, and hematocrit as a neonate are associated with individual fitness. Although hematocrit's effect is more complex than traditionally thought, our results suggest a previously underappreciated role for this trait in influencing survival in the wild.

Male song structure predicts offspring recruitment to the breeding population in a migratory bird.

Bird song is a classic example of a sexually selected trait, but much of the work relating individual song components to fitness has not accounted for song typically being composed of multiple, often-correlated components, necessitating a multivariate approach. We explored the role of sexual selection in shaping the complex male song of house wrens (Troglodytes aedon) by simultaneously relating its multiple components to fitness using multivariate selection analysis, which is widely used in insect and anuran studies but not in birds. The analysis revealed significant variation in the form and strength of selection acting on song across different selection episodes, from nest-site defense to recruitment of offspring to the breeding population. Males that sang more song typically employed in close communication sired more offspring that were subsequently recruited to the breeding population than those that sang more far-communication song. However, this relationship was not consistent across earlier selection episodes, as evidenced by non-linear selection acting on these song components in other contexts. Collectively, our results present a complex picture of multivariate selection on male song structure that would not be evident using univariate approaches and suggest possible trade-offs within and among song components at different points of the breeding season.

Sea Snacks from DNA Tracks: Using DNA Metabarcoding to Characterize the Diet of Green Turtles (Chelonia Mydas).

The green turtle (Chelonia mydas) is a circumglobal species with a wide dietary breadth that varies among regions and life history stages. Comprehensive understanding of foraging ecology over space and time is critical to inform conservation and management of this species and its habitats. Here, we used DNA metabarcoding to test candidate primer sets with 39 gut content homogenates from stranded green turtles (FL, USA) to identify primer sets that maximize detection of food items and specificity of taxonomic classifications. We tested six existing universal primer sets to detect plants, animals, and eukaryotes more broadly (CO1, 18SV1-V3, 18SV4, rbcL, UPA, ITS). The CO1 and 18SV4 primer sets produced the greatest number of dietary amplicon sequence variants (ASVs) and unique taxonomic classifications, and they were the only primer sets to amplify taxa from all three kingdoms relevant to green turtle diet (Animalia, Chromista, and Plantae). Even though the majority of CO1-derived reads were of host origin (>90%), this primer set still produced the largest number of dietary ASVs classified to species among the six primer sets. However, because the CO1 primer set failed to detect both vascular plants and green algae, we do not recommend the use of this primer set on its own to characterize green turtle diet. Instead, our findings support previous research highlighting the utility of using multiple primer sets, specifically targeting CO1 and the V4 region of the 18S gene, as doing so will provide the most comprehensive understanding of green turtle diet. More generally, our results highlight the importance of primer and loci selection and the need to validate primer sets against the study system of interest. The addition of DNA metabarcoding with optimized primer sets to the sea turtle researcher's toolbox will both increase our understanding of foraging ecology and better inform science-based conservation and ecosystem management.

DNA Metabarcoding Across Disciplines: Sequencing Our Way to Greater Understanding Across Scales of Biological Organization.

DNA metabarcoding describes the use of targeted DNA (i.e., amplicon) sequencing to identify community constituents from a complex sample containing genetic material from multiple organisms, such as water, soil, gut contents, microbiomes, or biofilms. This molecular approach for characterizing mixed DNA samples relies on the development of "universal primers" that allow for effective amplification of target sequences across a broad range of taxa. Armed with optimized lab protocols and rigorous bioinformatics tools, DNA metabarcoding can produce a wealth of information about the hidden biodiversity of various sample types by probing for organisms' molecular footprints. DNA metabarcoding has received considerable popular press over the last few years because of gut microbiome studies in humans and beyond. However, there are many other applications that are continually integrating molecular biology with other fields of study to address questions that have previously been unanswerable, for both prokaryotic and eukaryotic targets. For example, we can now sample mostly digested gut contents from virtually any organism to learn about ontogeny and foraging ecology. Water samples collected from different locations can be filtered to extract eDNA (i.e., environmental DNA), revealing the biodiversity of fish and other taxa targeted by carefully selected primer sets. This universal primer metabarcoding approach has even been extended to looking at diverse gene families within single species, which is particularly useful for complex immune system genetics. The purpose of this SICB symposium was to bring together researchers using DNA metabarcoding approaches to (a) showcase the diversity of applications of this technique for addressing questions spanning ecology, evolution, and physiology, and (b) to spark connections among investigators from different fields that are utilizing similar approaches to facilitate optimization and standardization of metabarcoding methods and analyses. The resulting manuscripts from this symposium represent a great diversity of metabarcoding applications and taxonomic groups of interest.

Developmental assays using invasive cane toads, Rhinella marina, reveal safety concerns of a common formulation of the rice herbicide, butachlor.

Identifying the adverse impacts of pesticide exposure is essential to guide regulations that are protective of wildlife and human health. Within rice ecosystems, amphibians are valuable indicators because pesticide applications coincide with sensitive reproductive and developmental life stages. We conducted two experiments using wild cane toads (Rhinella marina) to test 1) whether environmentally relevant exposure to a commercial formulation of butachlor, an acetanilide herbicide used extensively in rice, affects amphibian development and 2) whether cane toad tadpoles are capable of acclimatizing to sub-lethal exposure. First, we exposed wild cane toads to 0.002, 0.02, or 0.2 mg/L of butachlor (Machete EC), during distinct development stages (as eggs and hatchlings, as tadpoles, or continuously) for 12 days. Next, we exposed a subset of animals from the first experiment to a second, lethal concentration and examined survivorship. We found that cane toads exposed to butachlor developed slower and weighed less than controls, and that development of the thyroid gland was affected: exposed individuals had smaller thyroid glands and thyrocyte cells, and more individual follicles. Analyses of the transcriptome revealed that butachlor exposure resulted in downregulation of transcripts related to metabolic processes, anatomic structure development, immune system function, and response to stress. Last, we observed evidence of acclimatization, where animals exposed to butachlor early in life performed better than naïve animals during a second exposure. Our findings indicate that the commercial formulation of butachlor, Machete EC, causes thyroid endocrine disruption in vertebrates, and suggest that exposure in lowland irrigated rice fields presents a concern for wildlife and human health. Furthermore, we establish that developmental assays with cane toads can be used to screen for adverse effects of pesticides in rice fields.

Full lifetime perspectives on the costs and benefits of lay-date variation in tree swallows.

Animals must balance various costs and benefits when deciding when to breed. The costs and benefits of breeding at different times have received much attention, but most studies have been limited to investigating short-term season-to-season fitness effects. However, breeding early, versus late, in a season may influence lifetime fitness over many years, trading off in complex ways across the breeder's lifespan. In this study, we examined the complete life histories of 867 female tree swallows (Tachycineta bicolor) breeding in Ithaca, New York, between 2002 and 2016. Earlier breeders outperformed later breeders in short-term measures of reproductive output and offspring quality. Though there were weak indications that females paid long-term future survival costs for breeding early, lifetime fledgling output was markedly higher overall in early-breeding birds. Importantly, older females breeding later in the season did not experience compensating life history advantages that suggested an alternative equal-fitness breeding strategy. Rather, most or all of the swallows appear to be breeding as early as they can, and differences in lay dates appear to be determined primarily by differences in individual quality or condition. Lay date had a significant repeatability across breeding attempts by the same female, and the first lay date of females fledged in our population was strongly influenced by the first lay date of their mothers, indicating the potential for ongoing selection on lay date. By examining performance over the entire lifespan of a large number of individuals, we were able to clarify the relationship between timing of breeding and fitness and gain new insight into the sources of variability in this important life history trait.

Female house wrens (Troglodytes aedon) increase the size, but not immunocompetence, of their offspring through extra-pair mating.

House wrens are typically socially monogamous, but frequently engage in extra-pair matings leading to multisired broods. Because females do not appear to acquire direct material benefits from their extra-pair mates, we tested the hypothesis that female house wrens derive indirect genetic benefits, such as enhanced immunocompetence (cutaneous immune activity, humoral immunity, and plasma bactericidal activity) and condition (size and haematoserological traits) for their offspring, by mating polyandrously. We predicted that extra-pair young (EPY) should show greater immune responsiveness and better body condition than their within-pair maternal half-siblings (WPY). Contrary to our prediction, WPY had higher cutaneous immune activity than their EPY brood-mates in two of three years, and EPY and WPY did not differ in measures of innate and humoral immunity. WPY also had higher albumin to gamma-globulin ratios than EPY; however, they were not in better condition based on other measures. EPY had consistently longer tarsi (a measure of long-bone size) than their WPY half-siblings, suggesting that females engage in extra-pair copulations with larger males. The benefits of large structural size in the study population is unknown, but based on evidence from other passerines, we suggest that structural size may be an important fitness-related trait in house wrens. We conclude that our results are not consistent with the hypothesis that females gain immune-related benefits for their offspring by engaging in extra-pair matings. Further study of the fitness consequences of differences in tarsus length is needed to determine whether females acquire size-related benefits for their offspring from extra-pair mates.

Draft Genome Sequence of Xenophilus sp., a Novel Bacterium Isolated from the Skin of a Southern Leopard Frog (Rana sphenocephala) in Florida, USA.

We report here the draft genome sequence of a novel Xenophilus species cultured from the skin of a southern leopard frog (Rana sphenocephala). Compared to previously sequenced bacterial genomes, our novel isolate showed the most significant homology with Xenophilus azovorans The assembled genome is 3,978,285 bp, with 3,704 predicted genes and one predicted plasmid.

Increased extra-pair paternity in broods of aging males and enhanced recruitment of extra-pair young in a migratory bird.

Despite keen interest in extra-pair mating in birds, its adaptive significance remains unresolved. Here, we use a multi-year dataset to test whether traits of a female's social mate influence her propensity to produce extra-pair offspring in a population of house wrens, and whether producing extra-pair young has consequences for a female's fitness through effects on offspring survival. Females were most likely to produce extra-pair offspring when paired with old males and when paired with males on poor-quality territories, although this latter effect was marginally nonsignificant. Among offspring, the cutaneous immunity of within-pair young decreased as the age of their sires increased, but cutaneous immunity of extra-pair young was not affected by the age of their extra-pair sires or by the age of the males rearing them. Extra-pair offspring were more likely than within-pair offspring to return as breeding adults to the local population, with extra-pair sons being more likely to return as a breeder for multiple years. Our findings support the hypothesis that females produce extra-pair offspring to enhance their inclusive fitness beyond what they are capable of given the male with which they are socially paired.

Cutaneous immune activity, but not innate immune responsiveness, covaries with mass and environment in nestling house wrens (Troglodytes aedon).

Immunological measures are increasingly being applied to ecological and evolutionary studies of wild vertebrates, yet frequently it is not clear how condition and environmental factors correlate with various immune parameters. We used mixed-model ANOVA to examine the effects of several measures of condition (both morphological and physiological) and environmental factors on two measures of immune responsiveness in nestling house wrens (Troglodytes aedon L.) to test the hypothesis that nestlings in good condition mount stronger immune responses than those in poor condition. Based on previous studies, we predicted that the innate bactericidal response would be less likely to be affected by condition-related factors than the cutaneous response, which includes both innate and the more costly adaptive components. Both cutaneous immune activity (i.e., phytohaemagglutinin [PHA] response) and innate immune responsiveness (i.e., plasma bactericidal activity) varied significantly among broods. Nestling PHA response was significantly influenced by year, mass, and the time of day that the challenge was administered. However, besides nest of origin, no other variable examined had a significant effect on bactericidal activity. Morphological condition, assessed as body mass adjusted for structural size, differed significantly among nests and years and was positively correlated with hematocrit but not plasma albumin/gamma-globulin proteins, indicating that these are measures of different aspects of health state.