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.