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.