The human gut microbiome has been found to play major roles in organismal metabolism, nutrition, and immunity (Shulzhenko, Morgun et al. 2011, Cho and Blaser 2012, Consortium 2012). Consequently, biomedical research has prioritized understanding the development, composition, function of the gut microbial community (Palmer, Bik et al. 2007, Dominguez-Bello, Blaser et al. 2011, Kau, Ahern et al. 2011, Koenig, Spor et al. 2011) under the premise that an enhanced understanding of the gut microbiome may provide insight into chronic pathological conditions such as colitis, Crohn’s disease, and inflammatory bowel disease (Peterson, Frank et al. 2008). These same mechanisms have the potential to lend insight into the dynamics, health and natural history of a wide range of wildlife species. Conversely, understanding the composition of microbial communities associated with different wildlife species has the potential to contribute insights into the effects of phylogeny, ecology and evolution on both composition and function of the human gut microbiome (Barmann and Moran 1977, Ley, Hamady et al. 2008, Koch and Schmid-Hempel 2011, Mattila, Rios et al. 2012, Franzenburg, Fraune et al. 2013). Avian brood parasites offer the potential to investigate how exposure to a range of different host microbiomes shapes an animal's micro biome and in turn how those micro biomes influence aspects of the birds physiology and immunity (Johnsgard 1997, Ortega 1998, Davies 2000).
The brown-headed cowbird (Molothrus after, hereafter cowbird), a brood parasite, provides a unique case study for observing principles governing development and composition of the gut microbiome. The cowbird’s life history consists of a parasitic phase during early development when it associates with non-conspecifics, followed by a non-parasitic phase as juvenile and adult, when cowbirds associate primarily with conspecifics. Female cowbirds exploit approximately 250 other songbird species, laying eggs in their nests and appropriating parental care for their young during the first 4-6 weeks (Ortega 1999). Consequently, a young cowbird may be exposed during development to any one of 250 species and the diverse habitats where they breed (Hahn and O'Connor 2002). The cowbird is an extreme host-generalist, at one end of the brood parasite continuum, in contrast to host-specialists at the other end of the brood parasite continuum (e.g. the common cuckoo Cuculus canorus), which lays its eggs in the nests of only a single species (Davies 2000). The cowbird, therefore, offers wider exposure to other species and habitat types than any other brood parasite.
Interestingly, cowbirds have been shown to have an enhanced immune resistance compared to other phylogenetically related bird species from the same region (REF). Previous work has shown that the brown-headed cowbird showed significantly greater resistance to infection with the West Nile Virus (WNV) than did three closely related, nonparasitic blackbird species (Reisen and Hahn 2007, Hahn and Reisen 2011). Great resistance to infection with a virulent pathogen like WNV, an invasive species that emerged in the US in 1999 and to which no domestic songbirds had previously been exposed is a strong indicator of unusually effective immunity. In addition, in experimental infections of western equine encephalitis virus (WEEV) and the saint louis encephalitis (SLEV), the cowbird also displayed more effective immuno response compared other closely related bird species (REF). A separate survey of several parasitic cowbird species evaluated immune response showed that a level of immunity that corresponded to the level of host diversity among species (REF). Brown-headed cowbirds with the broadest host selection (>100 host species) demonstrated higher resistance to infection than other cowbirds whose life history consists of a more selective (12-20 host species) host range. Thus, while not mechanisist, these results suggest that the diversity of the cowbird micro biome may provide a higher level of resistance to disease.
To test how life history of brood-parasitism affects gut microbiome diversity and membership we evaluated whether the cowbird microbial diversity is more diverse than a related non-parasitic species the red-winged blackbird (Agelaius phoeniceus). The two species have similar range size, diet, migration, flocking and roosting patterns, all traits that affect microbial exposure patterns (Lowther 1993, Yasukawa and Searcy 1995). Many comparative studies of brown-headed cowbird have been carried out with red-winged blackbird, perhaps more than with any other species (Yasukawa and Searcy 1995). If the parasitic nature of cowbirds is the predominant factor influencing microbiome composition and diversity, then the adult cowbird microbiome may be composed instead of microorganisms characteristic of an individual’s foster parents and have a higher diversity than other birds with similar but non-parastitic life histories. Conversely, if genetic predisposition plays the dominant determinative role (Zoetendal 2001, Tims et al. 2011), then adult cowbirds should not be any more diverse than the gut microbiome of other avian species. In order characterize the gut microbiome of a bird, the intestinal track of the individual must be removed and sampled and cannot be achieved without killing the individual bird. As such, non-destructive methods of characterizing microbial gut communities are preferred. It has been proposed that the cloacal microbiome is an index of the gut microbial community, (Lombardo et al. 1999) and could be highly useful as proxy for the microbiome and one that can be sampled without killing the study animal. A second objective of our study was to compare the gut microbiome composition and diversity with that of the cloacal community within the same individual. Finally, other factors such as diet and geography influenced gut microbiome composition and diversity as has been documented in a several wildlife species (Godoy-Vitorino, Ley et al. 2008, Ley, Hamady et al. 2008, Hird SM 2014). To test for the effects of geography and gender on microbiome communities we collected birds of both genders from Maryland and in Michigan. We examined the diversity and composition of the cowbird microbiome by (1) sequencing small subunit (SSU) rRNA genes from the gut microbiome of the cowbird and (2) by sequencing and comparing SSU rRNA genes from the cowbird cloacal microbiome with the red-winged blackbird (Agelaius phoeniceus), and (3) testing for differences in microbiome communities from birds collected in Michigan and Maryland.