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.