UC Berkeley

Microbes occur in extraordinary abundance and diversity in vertebrates, and a growing body of work, particularly on humans and mice, has highlighted the pervasive role that microbiota may play in host physiology and health. Yet factors underlying the assembly of microbiota and the potential for microbiota to mediate host fitness in free-living vertebrate populations remain understudied. Investigating host-microbial interactions in natural systems is necessary to understand the extent to which ecologically relevant levels of environmental heterogeneity shape host-microbial relationships and to gain insight into the role that microbiota may play in the ecology and evolution of non-model host taxa. The neonatal vertebrate life stage is of particular interest during which substantial physiological development occurs and through which the initial microbial colonization can have longlasting effects on metabolic programming. To better understand mechanisms underlying microbial variation and associations with host condition during early periods of vertebrate development, this study characterized the gut microbiota of nestlings and their mothers in a natural population of Western Bluebirds Sialia mexicana. A novel method based on solid phase reversible immobilization beads was developed to extract metagenomic DNA from minimally invasive avian fecal and swab specimens. Illumina sequence analysis of bacterial 16S rRNA amplicons was performed on oral, cloacal, and fecal samples collected longitudinally from nestlings throughout the nestling period across multiple nests and over three consecutive breeding seasons (2011-2013). Environmental selection appeared to drive variation in microbiota across gut sites within individuals as well as between nests among individuals. Ambient temperature and sampling year correlated substantially with variation in the gut microbiota. In contrast, surprisingly no association was recovered between host age and alpha diversity, and increased similarity of microbiota across gut sites with host age was observed. Also unexpectedly, shared nest environment and geographical distance were weak predictors of microbial similarity among individuals. Variation in the gut microbiota significantly associated with morphological metrics of nestling growth, even after accounting for the influence of several temporal and environmental variables known to mediate nestling development. However, gut microbiota comprised a less robust predictor of nestling morphology than host age, sampling year, and ambient temperature. Furthermore, perturbation in the form of a viral immune challenge caused measurable shifts in the bacterial gut microbiota, which has previously been undocumented, but no measured effect of the immune challenge was observed on morphological metrics. These data inform intriguing hypotheses for future research on the drivers of avian host-microbial associations and the extent to which microbiota may impact host growth in natural systems. Discerning how microbiota may generate phenotypic or fitness differences among vertebrate host organisms is essential to understanding the evolution and maintenance of biodiversity.