Introduction
Animal microbiomes have received increased attention in recent years.
Further, the natural microbial communities that live on a host organism
are known to positively impact health, pathogen resistance, digestive
capabilities, and aid in other natural bodily functions. Environment,
dietary niche, and the health of a host all influence the diversity and
abundance of different bacterial species present in a host organism.
Avian species are globally widespread and play many important roles in
their ecosystems. Understanding the factors that contribute to health
and fitness in birds has become especially important in recent decades
due to the rapid decline in bird populations worldwide. Thus, an
understanding of the different microbiomes present within and across
avian species provides valuable insights about avian health, ecology,
evolution, and conservation. A necessary component of characterizing
such microbiomes is the accurate and effective identification of
microorganisms from specific reservoirs.
Although host species possess many reservoirs for microbial species, one
of the most important is the gut. Avian gut microbiomes and their
relationship to the behavior and fitness of the host have been of great
interest over the past two decades. Specifically, the microbiota that
exist in the gut of a bird have been linked to behavior, cognition,
metabolic function, and digestion. To study the gut microbiome, most
scientists extract bacterial DNA from fecal samples. Although fecal
samples do not perfectly represent the gut microbiome without bias, most
microbiomes have been characterized using bacterial DNA extracted from
fecal samples because it allows the characterization of the bacteria
present in the stomach and along the digestive tract as well.
In addition to gut microbiota, preen gland microbiota are important for
birds . The avian preen gland (or uropygial gland) is located at the
base of the tail and produces oils that help birds clean themselves,
protect feather health, and potentially aid in fighting against
pathogenic bacteria. Birds stimulate this gland with their beaks, and
then spread the resulting oil throughout their feathers in a behavior
called preening. Besides feather health, chemicals from preen oils vary
between species and can mediate communication between individuals in
many contexts, including species recognition and mate choice.
Additionally, preen oil chemistry has been shown to vary with seasons,
aggression levels, and reproductive success. Interestingly, recent
studies have shown that preen oil contains bacteria , which play a role
in the synthesis of the chemical compounds found in the oil. Thus, both
gut and uropygial gland microbiota play important roles in avian life.
To characterize the microbiomes present within a species, microbial DNA
must be extracted from samples collected from different reservoirs. To
facilitate comparisons of the microbiome communities present in
different reservoirs, the field would benefit from a microbial DNA
extraction method that is effective for different sample types. However,
within avian hosts, multiple sample types present particular
difficulties. Specifically, microbial DNA extractions from both avian
fecal material and preen oil present several challenges. Although feces
contains abundant bacteria, avian and reptilian organisms combine their
feces with urine, which contains minimal bacteria and therefore lowers
the overall concentrations of the bacteria present in fecal samples.
Further, urine contains PCR inhibitors, such as uric acid, which can
impede bacterial DNA detection. Additionally, preen oil displays low
bacterial abundance42 and exhibits antimicrobial
properties, leading to an expectation of even lower bacterial yields
from preen gland versus avian fecal samples61. The
preen oil microbiome has only recently begun to receive attention in the
literature and there are no standardized methodologies for preen gland
microbial DNA extraction.
Most studies on microbiomes have used commercial DNA extraction kits
that are used on other sources of microbial DNA such as human feces or
soil. For avian fecal matter however, DNA extractions from commercial
kits often do not produce visible bands on agarose gels complicating
verification of extraction success. For example, Eriksson et al. (2017)
compared the performance of six different commercial DNA extraction kits
using mallard duck (Anas platyrhynchos ) feces and few bands were
visible on gel images. Additionally, the commercially available fecal
extraction kits can be quite expensive (Table 1).
Thus, there is a need for more effective, efficient, and inexpensive
methodologies for extracting microbial DNA from multiple reservoirs.
Here, we present a new method that effectively and consistently extracts
bacterial DNA from both fecal and preen oil samples collected across a
wide range of avian species.