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 shown to directly influence host
behaviors, for example through diet choice and digestion adaptations.
Although not yet investigated in birds, gut microbiomes in non-avian
taxa are linked to cognition, resistance to disease, increased metabolic
functions, and digestion . To study the gut microbiome, most scientists
extract bacterial DNA from fecal samples. Because fecal waste begins in
the stomach and travels through the entire digestive tract, sequence
data obtained from fecal microbial DNA are effective in providing an
accurate representation of the abundance and diversity of all the
microbial communities existing along the digestive tract.
In addition to gut microbiota, preen gland microbiota are important for
birds . The avian preen gland (or uropygial gland) is located above the
tail feathers on the back and produces oils that help birds clean
themselves, protect feather health, and potentially aid in fighting
against pathogenic bacteria . Birds will 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 shown to vary with
seasonality, aggression, 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,
multiple microbial communities from different reservoirs play important
roles in avian life.
To characterize the microbiomes present within a species, DNA must be
extracted from the microbes present in samples collected from different
reservoirs. In order to compare results across studies focusing on
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
has abundant bacteria, avian and reptilian organisms combine their feces
with urine containing minimal bacteria, lowering the overall
concentrations of the bacteria present in fecal samples. Further, urine
contains PCR inhibitors such as urea, beta-human chorionic gonadotropin,
and crystals that can impede bacterial DNA detection. This combination
of digestive and uric acid waste in birds inhibits microbial DNA
extraction and subsequent PCR amplification, making it difficult to
obtain reliable and consistent results. There are many commercially
available kits for fecal extractions, but these can differ in
effectiveness and multiple extractions can quickly become cost
prohibitive. Additionally, 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 forms of eDNA such as feces or soil. For avian
fecal matter however, commercial DNA extraction kits are often
unsuccessful in producing sufficiently concentrated microbial DNA
extractions. For example, Eriksson et al. (2017) compared the
performance of six different commercial DNA extraction kits using
mallard duck (Anas platyrhynchos ) feces, and obtained low yield
of microbial DNA from all kits 64. Further, due to the
low bacterial abundance present in preen oil42, we
expect similar difficulties in microbial DNA extractions.
Thus, there is a need for more effective, efficient, and inexpensive
methodologies for extracting microbial DNA from avian feces and preen
oil samples. Here, we present an optimized method for effectively and
consistently extracting bacterial DNA from both fecal and preen oil
samples collected across a wide range of avian species.