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 phytohemagglutinin (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.

Single components of the immune system are widely used to assess immune function in free‐living vertebrates. However, as different immunological components are triggered by different types of threats and may be regulated independently, there is little reason to assume that they should respond similarly if challenged. We investigated whether three commonly assayed immune responses, cutaneous immune activity (phytohaemagglutinin assay), antibody response (tetanus toxoid immunization), and plasma bactericidal activity (Escherichia colikilling) are positively related in nestling house wrens (Troglodytes aedon). Multivariate analysis revealed significant differences in overall immune responsiveness among broods (i.e. nests), primarily attributable to differences in plasma bactericidal activity. Among broods, humoral immune response was negatively related to cutaneous immune activity and positively related to plasma bactericidal activity. We found no significant relationships among these measures of immunity among individual nestlings within broods. Our results suggest that different broods (i.e. families) invest differentially in the various branches of the immune system. Further study is needed to characterize the roles of maternal, genetic and environmental effects on the expression of this physiological bias.

DNA metabarcoding is a molecular technique frequently used to characterize diet composition of insectivorous birds. However, results are sensitive to methodological decisions made during sample processing, with primer selection being one of the most critical. The most frequently used DNA metabarcoding primer set for avian insectivores is ZBJ. However, recent studies have found that ZBJ produces significant biases in prey classification that likely influence our understanding of foraging ecology. A new primer set, ANML, has shown promise for characterizing insectivorous bat diets with fewer taxonomic biases than ZBJ, but ANML has not yet been used to study insectivorous birds. Here, we evaluate the ANML primer set for use in metabarcoding of avian insectivore diets through comparison with the more commonly used ZBJ primer set. Fecal samples were collected from both adult and nestling Purple Martins (Progne subis subis) at 2 sites in the USA and 1 site in Canada to maximize variation in diet composition and to determine if primer selection impacts our understanding of diet variation among sites. In total, we detected 71 arthropod prey species, 39 families, and 10 orders. Of these, 40 species were uniquely detected by ANML, whereas only 11 were uniquely detected by ZBJ. We were able to classify 54.8% of exact sequence variants from ANML libraries to species compared to 33.3% from ZBJ libraries. We found that ANML outperformed ZBJ for PCR efficacy, taxonomic coverage, and specificity of classification, but that using both primer sets together produced the most comprehensive characterizations of diet composition. Significant variation in both alpha- and beta-diversity between sites was found using each primer set separately and in combination. To our knowledge, this is the first published metabarcoding study using ANML primers to describe avian diet, and also the first to directly compare results returned by ANML and ZBJ primer sets. • DNA metabarcoding is a popular technique for characterizing diet composition by sequencing DNA of prey items found in fecal samples. • Molecular primer sets used to detect and identify prey DNA differ in efficacy, resulting in different conclusions about diet composition depending on the primer set selected. • We tested whether the recently developed ANML primer set may improve our ability to comprehensively describe diet composition for insectivorous Purple Martins as compared to the commonly used ZBJ primer set. • ANML has been shown to outperform ZBJ for characterizing diet in insectivorous bats, but ANML has not yet been tested against ZBJ for characterizing diets of insectivorous birds. • We found that the new ANML primer set detected and identified more insect prey than the ZBJ primer set, but that using both together provided the most comprehensive approach. • This study is the first to test ANML primers with bird diet samples and also to compare results between the use of ANML and ZBJ.

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.