Discussion
Our new microbial DNA extraction method is effective for avian fecal samples (Figure 1), which have been traditionally challenging materials for use in DNA extractions. Our method is also effective for preen gland oil samples (Figure 1), which likely harbor lower bacterial abundance due to antimicrobial characteristics of preen oil. Both sample types were collected across a broad range of avian species. Further, our method successfully extracted DNA from both gram-positive and gram-negative bacteria (Table 4). Other studies have found both gram positive and negative bacteria, supporting the efficacy of this method. The ease and relative cost effectiveness of our method (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 an organism’s host microbiome is a crucial determinant of its overall health, 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, providing enzymes that break down molecules. Additionally hosts, rely on microbes for 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 contain inputs from the whole gastrointestinal tract and are thus a more 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 (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 in popularity over the last decade, the range of host reservoirs scientists are studying has expanded 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 oil are thought to be less 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 chemically different, it is likely that our method will be useful across additional avian microbiome reservoirs. 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. Using the same extraction methodology across samples from different reservoirs will allow more consistent and confident comparisons of microbiomes from different reservoirs for future research efforts. 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. Widespread adoption of our method would therefore increase the validity of future cross-study and cross-reservoir comparisons.
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 on the first attempt from 25 of 25 fecal samples collected from 15 avian species, and from 11 of 12 preen oil samples collected from 12 avian species (Figure 1, Tables 2-3). It is also relatively inexpensive compared with commercial fecal DNA extraction kits (Table 1). 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 reservoirs in other taxa, including reptile fecal samples as well.