Evaluation of DNA Barcoding as a Technique for Elucidating the Diet of Louisiana Waterthrush Nestlings

The Louisiana Waterthrush (Parkesia motacilla) (LOWA) is a Neotropical-Nearctic migrant songbird that annually breeds in the Eastern United States. As an obligate riparian species, it preys upon a diverse community of benthic macroinvertebrates along headwater streams. Fecal material potentially contains residual DNA that can be used to molecularly identify prey species. The objective of this study was to develop a non-invasive technique capable of elucidating the diet of Waterthrush nestlings from residual DNA present in fecal sacs via DNA barcoding. Our limited analysis revealed that the majority of the analyzed fecal sacs were comprised of an acid-sensitive family of Ephemerotperan (Heptageniidae) in addition to Megalopterans and Dipterans. These results suggest that the technique of DNA barcoding can be utilized to accurately identify prey species from residual DNA found in avian fecal samples, which may improve our understanding of landscape-level factors affecting riparian bird communities and guide future conservation efforts

DNA metabarcoding reveals provisioning of pollution-sensitive aquatic insects, resource partitioning, and dietary shifts among breeding Neotropical migratory songbirds in a riparian habitat

Elucidating the diet of Neotropical migratory birds is essential to our understanding of their ecology and to their long-term conservation. Beyond broad taxonomic or morphological categories, however, the diet of Neotropical migrants is poorly documented. Using the molecular techniques of DNA barcoding and next-generation sequencing, we elucidated the diet of Neotropical migratory songbirds breeding in the riparian zones of headwater Appalachian streams. This approach resulted in a genus- or species-level description of diets that improved the current understanding of how songbirds utilize aquatic prey resources in riparian habitats. Furthermore, our approach revealed that breeding songbirds partition prey resources within a shared riparian habitat. Despite substantial differences in foraging strategy, we provide evidence that syntopic riparian species opportunistically prey upon pollution-sensitive emergent aquatic insects, thus emphasizing the importance of aquatic resource subsidies for songbirds breeding in riparian habitats. For the stream-dependent Louisiana Waterthrush, the provisioning of aquatic insects was significantly higher than other riparian songbirds. As a result, waterthrush breeding in riparian habitats with reduced availability of aquatic arthropods expanded their diet by targeting a more diverse array of insects that included significantly more terrestrial taxa. In addition to providing support for our hypothesis that Louisiana Waterthrush compensate for food shortages by targeting terrestrial arthropods in degraded riparian habitats, our findings emphasize the vulnerability of Louisiana Waterthrush to anthropogenic disturbances that compromise stream quality and the availability of pollution-sensitive aquatic insects

How should we store avian faecal samples for microbiota analyses? Comparing efficacy and cost-effectiveness

Analyses of bacterial DNA in faecal samples are becoming ever more common, yet we still do not know much about bird microbiomes. These challenges partly lie in the unique chemical nature of their faeces, and in the choice of sample storage method, which affects DNA preservation and the resulting microbiome composition. However, there is little information available on how best to preserve avian faeces for microbial analyses. This study evaluates five widely used methods for preserving nucleic acids and inferring microbiota profiles, for their relative efficacy, cost, and practicality. We tested the five methods (in-situ bead-beating with a TerraLyzer instrument, silica-bead desiccation, ethanol, refrigeration and RNAlater buffer) on 50 fresh faecal samples collected from captive House sparrows (Passer domesticus). In line with other studies, we find that different storage methods lead to distinct bacterial profiles. Storage method had a large effect on community composition and the relative abundance of dominant phyla such as Firmicutes and Proteobacteria, with the most significant changes observed for refrigerated samples. Furthermore, differences in the abundance of aerobic or facultatively aerobic taxa, particularly in refrigerated samples and those stored in ethanol, puts limits on comparisons of bacterial communities across different storage methods. Finally, the methods that did not include in-situ bead-beating did not recover comparable levels of microbiota to the samples that were immediately processed and preserved using a TerraLyzer device. However, this method is also less practical and more expensive under field work circumstances. Our study is the most comprehensive analysis to date on how storage conditions affect subsequent molecular assays applied to avian faeces and provides guidance on cost and practicality of methods under field conditions

How can the MHC mediate social odor via the microbiota community? A deep dive into mechanisms

Genes of the major histocompatibility complex (MHC) have long been linked to odor signaling and recently researchers’ attention has focused on MHC structuring of microbial communities and how this may in turn impact odor. However, understanding of the mechanisms through which the MHC could affect the microbiota to produce a chemical signal that is both reliable and strong enough to ensure unambiguous transmission of behaviorally important information remains poor. This is largely because empirical studies are rare, predictions are unclear, and the underlying immunological mechanisms governing MHC-microbiota interactions are often neglected. Here we review the immunological processes involving MHC class II (MHC-II) that could affect the commensal community. Focusing on immunological and medical research, we provide background knowledge for non-immunologists by describing key players within the vertebrate immune system relating to MHC-II molecules (which present extracellular-derived peptides, and thus interact with extracellular commensal microbes). We then systematically review the literature investigating MHC-odor-microbiota interactions in animals and identify areas for future research. These insights will help to design studies that are able to explore the role of MHC-II and the microbiota in the behavior of wild populations in their natural environment and consequently propel this research area forward

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

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

Supplemental Molecular Protocols from <i>In ovo</i> microbial communities: a potential mechanism for the initial acquisition of gut microbiota among oviparous birds and lizards

Detailed protocols for DNA extraction, PCR amplification, library preparation, and Illumina sequencing

Supplemental Tables and Figures from <i>In ovo</i> microbial communities: a potential mechanism for the initial acquisition of gut microbiota among oviparous birds and lizards

Supplemental Tables S1-S3 and Supplemental Figures S1-S2