Host avian species and environmental conditions influence the microbial ecology of brood parasitic brown-headed cowbird nestlings: What rules the roost?

The role of species interactions, as well as genetic and environmental factors, all likely contribute to the composition and structure of the gut microbiome; however, disentangling these independent factors under field conditions represents a challenge for a functional understanding of gut microbial ecology. Avian brood parasites provide unique opportunities to investigate these questions, as brood parasitism results in parasite and host nestlings being raised in the same nest, by the same parents. Here we utilized obligate brood parasite brown-headed cowbird nestlings (BHCO; Molothrus ater) raised by several different host passerine species to better understand, via 16S rRNA sequencing, the microbial ecology of brood parasitism. First, we compared faecal microbial communities of prothonotary warbler nestlings (PROW; Protonotaria citrea) that were either parasitized or non-parasitized by BHCO and communities among BHCO nestlings from PROW nests. We found that parasitism by BHCO significantly altered both the community membership and community structure of the PROW nestling microbiota, perhaps due to the stressful nest environment generated by brood parasitism. In a second dataset, we compared faecal microbiotas from BHCO nestlings raised by six different host passerine species. Here, we found that the microbiota of BHCO nestlings was significantly influenced by the parental host species and the presence of an inter-specific nestmate. Thus, early rearing environment is important in determining the microbiota of brood parasite nestlings and their companion nestlings. Future work may aim to understand the functional effects of this microbiota variability on nestling performance and fitness.

Convergent remodelling of the gut microbiome is associated with host energetic condition over long-distance migration.

The gut microbiome can be thought of as a virtual organ given its immense metabolic capacity and profound effects on host physiology. Migratory birds are capable of adaptively modulating many aspects of their physiology to facilitate long-distance movements, raising the hypothesis that their microbiome may undergo a parallel remodeling process that helps to meet the energetic demands of migration.To test this hypothesis, we investigated changes in gut microbiome composition and function over the fall migration of the Blackpoll Warbler (Setophaga striata), which exhibits one of the longest known autumnal migratory routes of any songbird and rapidly undergoes extensive physiological remodeling during migration.Overall, our results showed that the Blackpoll Warbler microbiome differed significantly across phases of fall migration. This pattern was driven by a dramatic increase in the relative abundance of Proteobacteria, and more specifically a single 16S rRNA gene amplicon sequence variant belonging to the family Enterobacteriaceae. Further, Blackpoll Warblers exhibited a progressive reduction in microbiome diversity and within-group variance over migration, indicating convergence of microbiome composition among individuals during long-distance migration. Metagenomic analysis revealed that the gut microbiome of staging individuals was enriched in bacterial pathways involved in vitamin, amino acid, and fatty acid biosynthesis, as well as carbohydrate metabolism, and that these pathways were in turn positively associated with host body mass and subcutaneous fat deposits.Together, these results provide evidence that the gut microbiome of migratory birds may undergo adaptive remodeling to meet the physiological and energetic demands of long-distance migration.

Two is better than one: Coupling DNA metabarcoding and stable isotope analysis improves dietary characterizations for a riparian-obligate, migratory songbird.

While an increasing number of studies are adopting molecular and chemical methods for dietary characterization, these studies often employ only one of these laboratory-based techniques; this approach may yield an incomplete, or even biased, understanding of diet due to each method's inherent limitations. To explore the utility of coupling molecular and chemical techniques for dietary characterizations, we applied DNA metabarcoding alongside stable isotope analysis to characterize the dietary niche of breeding Louisiana waterthrush (Parkesia motacilla), a migratory songbird hypothesized to preferentially provision its offspring with pollution-intolerant, aquatic arthropod prey. While DNA metabarcoding was unable to determine if waterthrush provision aquatic and terrestrial prey in different abundances, we found that specific aquatic taxa were more likely to be detected in successive seasons than their terrestrial counterparts, thus supporting the aquatic specialization hypothesis. Our isotopic analysis added greater context to this hypothesis by concluding that breeding waterthrush provisioned Ephemeroptera and Plecoptera, two pollution-intolerant, aquatic orders, in higher quantities than other prey groups, and expanded their functional trophic niche when such prey were not abundantly provisioned. Finally, we found that the dietary characterizations from each approach were often uncorrelated, indicating that the results gleaned from a diet study can be particularly sensitive to the applied methodologies. Our findings contribute to a growing body of work indicating the importance of high-quality, aquatic habitats for both consumers and their pollution-intolerant prey, while also demonstrating how the application of multiple, laboratory-based techniques can provide insights not offered by either technique alone.

Integrating DNA-Based Prey Occurrence Probability into Stable Isotope Mixing Models.

The introduction of laboratory methods to animal dietary studies has allowed researchers to obtain results with accuracy and precision, not possible with observational techniques. For example, DNA barcoding, or the identification of prey with taxon-specific DNA sequences, allows researchers to classify digested prey tissues to the species-level, while stable isotope analysis paired with Bayesian mixing models can quantify dietary contributions by comparing a consumer's isotopic values to those derived from their prey. However, DNA-based methods are currently only able to classify, but not quantify, the taxa present in a diet sample, while stable isotope analysis can only quantify dietary taxa that are identified a priori as prey isotopic values are a result of life history traits, not phylogenetic relatedness. Recently, researchers have begun to couple these techniques in dietary studies to capitalize on the reciprocal benefits and drawbacks offered by each approach, with some even integrating DNA-based results directly into Bayesian mixing models as informative priors. As the informative priors used in these models must represent known dietary compositions (e.g., percentages of prey biomasses), researchers have scaled the DNA-based frequency of occurrence of major prey groups so that their normalized frequency of occurrence sums to 100%. Unfortunately, such an approach is problematic as priors stemming from binomial, DNA-based data do not truly reflect quantitative information about the consumer's diet and may skew the posterior distribution of prey quantities as a result. Therefore, we present a novel approach to incorporate DNA-based dietary information into Bayesian stable isotope mixing models that preserves the binomial nature of DNA-based results. This approach uses community-wide frequency of occurrence or logistic regression-based estimates of prey occurrence to dictate the probability that each prey group is included in each mixing model iteration, and, in turn, the probability that each iteration's results are included in the posterior distribution of prey composition possibilities. Here, we demonstrate the utility of this method by using it to quantify the prey composition of nestling Louisiana waterthrush (Parkesia motacilla).

The gut microbiome influences host diet selection behavior.

Diet selection is a fundamental aspect of animal behavior with numerous ecological and evolutionary implications. While the underlying mechanisms are complex, the availability of essential dietary nutrients can strongly influence diet selection behavior. The gut microbiome has been shown to metabolize many of these same nutrients, leading to the untested hypothesis that intestinal microbiota may influence diet selection. Here, we show that germ-free mice colonized by gut microbiota from three rodent species with distinct foraging strategies differentially selected diets that varied in macronutrient composition. Specifically, we found that herbivore-conventionalized mice voluntarily selected a higher protein:carbohydrate (P:C) ratio diet, while omnivore- and carnivore-conventionalized mice selected a lower P:C ratio diet. In support of the long-standing hypothesis that tryptophan­the essential amino acid precursor of serotonin­serves as a peripheral signal regulating diet selection, bacterial genes involved in tryptophan metabolism and plasma tryptophan availability prior to the selection trial were significantly correlated with subsequent voluntary carbohydrate intake. Finally, herbivore-conventionalized mice exhibited larger intestinal compartments associated with microbial fermentation, broadly reflecting the intestinal morphology of their donor species. Together, these results demonstrate that gut microbiome can influence host diet selection behavior, perhaps by mediating the availability of essential amino acids, thereby revealing a mechanism by which the gut microbiota can influence host foraging behavior.

Robustness of Mammalian Gut Microbiota to Humanization in Captivity.

In mammals, the composition of the gut microbiota is associated with host phylogenetic history, and host-lineage specific microbiota have been shown, in some cases, to contribute to fitness-related traits of their hosts. However, in primates, captivity can disrupt the native microbiota through a process of humanization in which captive hosts acquire gut microbiota constituents found in humans. Despite the potential importance of this process for the health of captive hosts, the degree to which captivity humanizes the gut microbiota of other mammalian taxa has not been explored. Here, we analyzed hundreds of published gut microbiota profiles generated from wild and captive hosts spanning seven mammalian families to investigate the extent of humanization of the gut microbiota in captivity across the mammalian phylogeny. Comparisons of these hosts revealed compositional convergence between captive mammal and human gut microbiota in the majority of mammalian families examined. This convergence was driven by a diversity of microbial lineages, including members of the Archaea, Clostridium, and Bacteroides. However, the gut microbiota of two families-Giraffidae and Bovidae-were remarkably robust to humanization in captivity, showing no evidence of gut microbiota acquisition from humans relative to their wild confamiliars. These results demonstrate that humanization of the gut microbiota is widespread in captive mammals, but that certain mammalian lineages are resistant to colonization by human-associated gut bacteria.

Context-dependent effects of glucocorticoids on the lizard gut microbiome.

The vertebrate gut microbiota (bacterial, archaeal and fungal communities of the gastrointestinal tract) can have profound effects on the physiological processes of their hosts. Although relatively stable, changes in microbiome structure and composition occur due to changes in the environment, including exposure to stressors and associated increases in glucocorticoid hormones. Although a growing number of studies have linked stressor exposure to microbiome changes, few studies have experimentally explored the specific influence of glucocorticoids on the microbiome in wild animals, or across ecologically important processes (e.g., reproductive stages). Here we tested the response of the gut microbiota of adult female Sceloporus undulatus across gestation to ecologically relevant elevations of a stress-relevant glucocorticoid hormone (CORT) in order to determine (i) how experimentally elevated CORT influenced microbiome characteristics, and (ii) whether this relationship was dependent on reproductive context (i.e., whether females were gravid or not, and, in those that were gravid, gestational stage). We show that the effects of CORT on gut microbiota are complex and depend on both gestational state and stage. CORT treatment altered microbial community membership and resulted in an increase in microbiome diversity in late-gestation females, and microbial community membership varied according to treatment. In nongravid females, CORT treatment decreased interindividual variation in microbial communities, but this effect was not observed in late-gestation females. Our results highlight the need for a more holistic understanding of the downstream physiological effects of glucocorticoids, as well as the importance of context (here, gestational state and stage) in interpreting stress effects in ecology.

Two's company, three's a crowd: Exploring how host-parasite-microbiota interactions may influence disease susceptibility and conservation of wildlife.

A large body of research has demonstrated that host-associated microbiota-the archaeal, bacterial, fungal and viral communities residing on and inside organisms-are critical to host health (Cho & Blaser, 2012). Although the vast majority of these studies focus on humans or model organisms in laboratory settings (Pascoe, Hauffe, Marchesi, & Perkins, 2017), they nevertheless provide important conceptual evidence that the disruption of host-associated microbial communities (termed "dysbiosis") among wild animals may reduce host fitness and survival under natural environmental conditions. Among the myriad of environmental factors capable of inducing dysbiosis among wild animals (Trevelline, Fontaine, Hartup, & Kohl, 2019), parasitic infections represent a potentially potent, yet poorly understood, factor influencing microbial community dynamics and animal health. The study by DeCandia et al. in this issue of Molecular Ecology is a rare example of a host-parasite-microbiota interaction that impacts the health, survival and conservation of a threatened wild animal in its natural habitat. Using culture-independent techniques, DeCandia et al. found that the presence of an ectoparasitic mite (Otodectes cynotis) in the ear canal of the Santa Catalina Island fox (Urocyon littoralis catalinae) was associated with significantly reduced ear canal microbial diversity, with the opportunistic pathogen Staphylococcus pseudintermedius dominating the community. These findings suggest that parasite-induced inflammation may contribute to the formation of ceruminous gland tumours in this subspecies of Channel Island fox. As a rare example of a host-parasite-microbiota interaction that may mediate a lethal disease in a population of threatened animals, their study provides an excellent example of how aspects of disease ecology can be integrated into studies of host-associated microbiota to advance conservation science and practice.

A bird's-eye view of phylosymbiosis: weak signatures of phylosymbiosis among all 15 species of cranes.

In numerous animal clades, the evolutionary history of host species drives patterns of gut microbial community structure, resulting in more divergent microbiota with increasing phylogenetic distance between hosts. This phenomenon, termed phylosymbiosis, has been observed in diverse evolutionary lineages, but has been difficult to detect in birds. Previous tests of phylosymbiosis among birds have been conducted using wild individuals, and thus interspecific differences in diet and environment may have masked a phylogenetic signal. Therefore, we tested for phylosymbiosis among all 15 species of cranes (family Gruidae) housed in the same captive environment and maintained on identical diets. 16S rRNA sequencing revealed that crane species harbour distinct gut microbiota. Overall, we detected marginally significant patterns of phylosymbiosis, the strength of which was increased when including the estimates of absolute microbial abundance (rather than relative abundance) derived from microbial densities determined by flow cytometry. Using this approach, we detected the statistically significant signatures of phylosymbiosis only after removing male cranes from our analysis, suggesting that using mixed-sex animal cohorts may prevent the detection of phylosymbiosis. Though weak compared with mammals (and especially insects), these results provide evidence of phylosymbiosis in birds. We discuss the potential differences between birds and mammals, such as transmission routes and host filtering, that may underlie the differences in the strength of phylosymbiosis.

Gestation alters the gut microbiota of an oviparous lizard.

Mammalian pregnancy can alter the diversity, membership and structure of the maternal gut microbiota, but it is unclear whether this phenomenon occurs in vertebrates with different reproductive strategies. We conducted 16S rRNA bacterial inventories to investigate whether oviparous lizards exhibit shifts in gut microbiota similar to those observed in mammals. Using wild-caught eastern fence lizards from Alabama, USA, we collected and extracted fecal DNA from gravid and non-gravid individuals over 54 days in captivity. We predicted that, like mammals, the alpha diversity of lizard gut microbiota would decrease over gestation, and that inter-individual variation in community composition would increase. Indeed, we found that individuals in late-gestation harbored lower gut bacterial richness compared to non-gravid females. Lizard gut microbial communities of late-gestational females exhibited higher pairwise distances for both community membership and community structure compared to earlier gestation stages, indicating a higher degree of inter-individual variation as gestation progressed. Additionally, we found that the relative abundance and prevalence of the candidate phylum Melainabacteria tended to decrease over the course of gestation. While the consequences of these specific alterations are unknown, our results suggest that a general restructuring of gut microbial communities over gestation may be widespread across vertebrate reproductive strategies.

Conservation biology needs a microbial renaissance: a call for the consideration of host-associated microbiota in wildlife management practices.

The central aim of conservation biology is to understand and mitigate the effects of human activities on biodiversity. To successfully achieve this objective, researchers must take an interdisciplinary approach that fully considers the effects, both direct and indirect, of anthropogenic disturbances on wildlife physiology and health. A recent surge in research has revealed that host-associated microbiota-the archaeal, bacterial, fungal and viral communities residing on and inside organisms-profoundly influence animal health, and that these microbial communities can be drastically altered by anthropogenic activities. Therefore, conservation practitioners should consider the disruption of host-associated microbial diversity as a serious threat to wildlife populations. Despite the tremendous potential for microbiome research to improve conservation outcomes, few efforts have been made to truly integrate these fields. In this review, we call for the microbial renaissance of conservation biology, where biodiversity of host-associated microbiota is recognized as an essential component of wildlife management practices. Using evidence from the existing literature, we will examine the known effects of anthropogenic activities on the diversity of host-associated microbial communities and integrate approaches for maintaining microbial diversity to successfully achieve conservation objectives.

Stream acidification and reduced aquatic prey availability are associated with dietary shifts in an obligate riparian Neotropical migratory songbird.

Streams and their surrounding riparian habitats are linked by reciprocal exchanges of insect prey essential to both aquatic and terrestrial consumers. Aquatic insects comprise a large proportion of total prey in riparian habitats and are opportunistically exploited by terrestrial insectivores; however, several species of songbirds are known to preferentially target aquatic prey via specialized foraging strategies. For these songbirds, reduced availability of aquatic insects via stream acidification may result in compensatory changes in provisioning during the nesting period, thereby influencing both adult and nestling diet composition. In this study, we used DNA metabarcoding to test the hypothesis that an obligate riparian Neotropical migratory songbird, the Louisiana Waterthrush (Parkesia motacilla), expands its diet to compensate for the loss of preferred aquatic prey taxa (primarily pollution-sensitive Ephemeroptera, Plecoptera, and Trichoptera) as a result of stream acidification. Our results revealed that both adult and nestling waterthrush exhibited an increase in dietary richness and niche breadth resulting from the consumption of terrestrial prey taxa in acidified riparian habitats. In contrast, compensatory dietary shifts were not observed in syntopic Neotropical migrant species known to primarily provision terrestrial prey taxa. In addition to providing support for our hypothesis that waterthrush compensate for stream acidification and aquatic prey limitations by expanding their diet, our findings highlight the vulnerability of Louisiana Waterthrush to anthropogenic disturbances that compromise stream quality or reduce the availability of pollution-sensitive aquatic insects.

In ovo microbial communities: a potential mechanism for the initial acquisition of gut microbiota among oviparous birds and lizards.

Vertebrate gut microbiota mediate critical physiological processes known to affect host fitness, but the mechanisms that expose wildlife to pioneer members of this important microbial community are not well understood. For example, oviparous vertebrates are thought to acquire gut microbiota through post-natal exposure to the external environment, but recent evidence from placental mammals suggests that the vertebrate reproductive tract harbours microbiota that may inoculate offspring in utero These findings suggest that oviparous vertebrates may be capable of acquiring pioneer microbiota in ovo, but this phenomenon remains unexplored. To fill this knowledge gap, we used culture-independent inventories to determine if the eggs of wild birds and lizards harboured in ovo microbial communities. Our approach revealed distinct in ovo bacterial communities, but fungal communities were indistinguishable from controls. Further, lizard eggs from the same clutch had bacterial community structures that were more similar to each other than to unrelated individuals. These results suggest that oviparous vertebrates may acquire maternal microbiota in ovo, possibly through the inoculation of egg yolk prior to shelling. Therefore, this study may provide a first glimpse of a phenomenon with substantial implications for our understanding of the ecological and evolutionary factors shaping gut microbial communities.

DNA metabarcoding of nestling feces reveals provisioning of aquatic prey and resource partitioning among Neotropical migratory songbirds in a riparian habitat.

Riparian habitats are characterized by substantial flows of emergent aquatic insects that cross the stream-forest interface and provide an important source of prey for insectivorous birds. The increased availability of prey arising from aquatic subsidies attracts high densities of Neotropical migratory songbirds that are thought to exploit emergent aquatic insects as a nestling food resource; however, the prey preferences and diets of birds in these communities are only broadly understood. In this study, we utilized DNA metabarcoding to investigate the extent to which three syntopic species of migratory songbirds-Acadian Flycatcher, Louisiana Waterthrush, and Wood Thrush-breeding in Appalachian riparian habitats (Pennsylvania, USA) exploit and partition aquatic prey subsidies as a nestling food resource. Despite substantial differences in adult foraging strategies, nearly every nestling in this study consumed aquatic taxa, suggesting that aquatic subsidies are an important prey resource for Neotropical migrants nesting in riparian habitats. While our results revealed significant interspecific dietary niche divergence, the diets of Acadian Flycatcher and Wood Thrush nestlings were strikingly similar and exhibited significantly more overlap than expected. These results suggest that the dietary niches of Neotropical migrants with divergent foraging strategies may converge due to the opportunistic provisioning of non-limiting prey resources in riparian habitats. In addition to providing the first application of DNA metabarcoding to investigate diet in a community of Neotropical migrants, this study emphasizes the importance of aquatic subsidies in supporting breeding songbirds and improves our understanding of how anthropogenic disturbances to riparian habitats may negatively impact long-term avian conservation.

Elevated gastrin-releasing peptide receptor mRNA expression in buccal mucosa: association with head and neck squamous cell carcinoma.

BACKGROUND: Expression of gastrin-releasing peptide receptor (GRPR) is elevated in mucosa adjacent to head and neck squamous cell carcinoma (HNSCC) compared with mucosa from cancer-free controls, suggesting elevated GRPR expression may indicate presence of HNSCC. METHODS: We measured GRPR mRNA levels in histologically normal buccal mucosa from 65 surgical patients with HNSCC and 75 cancer-free control subjects using quantitative polymerase chain reaction (PCR). We tested for association between GRPR expression and HNSCC and evaluated differences in patient progression-free survival (PFS). RESULTS: Buccal GRPR expression was higher in cases but not controls who were active smokers (p = .04). High GRPR expression was associated with HNSCC (odds ratio [OR] = 3.55; 95% confidence interval [CI] = 1.15-10.93), even after adjustment for age, sex, tobacco use, and sample storage time. PFS did not differ between patients with HNSCC with high versus low GRPR expression (p = .22). CONCLUSION: Elevated buccal GRPR expression was significantly associated with HNSCC independent of known risk factors but was not an indicator of disease prognosis.