Discussion
Our new microbial DNA extraction method is effective for avian fecal samples (Figs. 1 and 3; Table 3; Supp. Figs. 1-4) which have been traditionally challenging materials for use in DNA extractions and preen gland oil samples (Fig. 2; Table 4; Supp. Figs. 3-4), both collected across a broad range of avian species. Further, our method successfully extracted DNA from both gram-positive and gram-negative bacteria (Table 5). Additionally, the ease and relative cost effectiveness of our method (Supplemental Table 1) makes it particularly advantageous. We hope that our method will facilitate advances in our understanding of microbiomes from multiple reservoirs across a wide range of avian species.
While the host microbiome of an organism has shown to be a crucial determinant of the overall health of an organism, the full extent of the coevolutionary relationships between a host and its symbiotic microbes is not known. Recent discoveries have shown hosts rely on microbes for health and protection against viruses and pathogens, breaking down compounds, digestion, cognitive function, growth, development, and more. Gut microbiomes are particularly important in many taxa. To sample the gut microbiome of birds, cloacal swabs have been shown to be nonrepresentative of microbial diversity, only accounting for one component of the digestive tract, whereas fecal samples travel the whole digestive tract and are thus accurate representations of the entire gut microbiome. However, existing DNA extraction methods have not been consistently successful when working with avian fecal samples . Birds and reptiles combine their uric waste with their fecal waste, thus creating added difficulty in extracting DNA from fecal samples. Our method is not only reliable with such samples but is considerably cheaper than most commercial kits used to extract avian microbiome DNA (Supplemental Table 1). The low cost and high reliability of our method will improve the feasibility and accessibility of research on avian host microbiomes.
As microbiome studies have increased popularity in the last decade, the host reservoirs in which scientists have been studying has expanded as well. However, beyond the gut, the community composition of microbiomes from additional reservoirs in host animals needs to be explored before the function of these bacterial communities can be investigated as well. Beyond the abundant and diverse gut microbiome, a potentially functionally important reservoir in avian species is the microbiome that exists within the uropygial or preen gland. The preen gland and the oils produced there are known to affect communication, species recognition, mate choice, and feather maintenance. The chemical composition of preen oil is at least partially dependent on the microbes present in this gland. The bacterial communities present in the uropygial gland have been shown to be less diverse and abundant than those within the gut microbiome; yet, they are predicted to play direct roles in the odor of avian hosts as well as potentially in feather and body health. Some preliminary studies in this field of research have observed that disease may not be directly linked to differences in preen oil microbiomes. Additionally, differences in microbial communities may correspond with population differences, and in some species, sex differences have been observed. The consistent success of our method in extracting microbial DNA from avian preen oil samples will facilitate new research on this currently understudied microbiome reservoir.
Because we have established the consistent success of our method in extracting microbial DNA from two avian sample types that are quite different, it is likely that our method will be useful across additional avian microbiome reservoirs. Using the same extraction methodology will provide more consistent and reliable results for future research efforts. Standardization of methodology is important to allow for accurate cross-study comparisons and higher confidence in the results of individual studies. Current variation in the approaches used for microbial DNA extraction and subsequent amplification means there is also sometimes variation in the bacteria that are detected. Widespread adoption of our method would increase the validity of future cross-study comparisons.
With increased interest in the relationship between hosts and their bacterial community composition and abundance across different microbiome reservoirs, the accurate identification of microbial taxa is critical. With an enhanced potential to identify and quantify the symbiotic microbes, the investigation of their impact on host health and conservation can involve deeper questions. To conclude, we have developed and tested a highly efficient microbial extraction method and verified its efficacy across multiple avian species. Our method was successful in extracting microbial DNA from the fecal samples of 15 avian species (Table 1, Figure 1, Supplemental Figures 1-4) and the preen oil samples of 11 avian species (Table 2, Figure 2, Supplemental Figures 3-4). From this, we are confident that our method will be effective and efficient across many additional avian species globally. Future work should explore the effectiveness of this method in extracting microbial DNA from reptile fecal samples as well.