The gastrointestinal (GI) microbiome is an important mediator and indicator of host physiology, health, and fitness in many vertebrate systems, but sources of variation in microbiome composition within species are poorly understood, particularly in free-ranging birds. Hummingbirds have GI anatomy and physiology similar to other avian species, but they have a relatively rapid gastrointestinal transit rate, which could affect microbiome structure. To date, it has not been elucidated if microbiomes isolated from feces and samples from sections of the GI tract differ in an avian organism that has a rapid gastrointestinal time. Here, we test the hypothesis that the microbiome in free-ranging and wildlife rehabilitation-sourced Anna’s Hummingbirds (Calypte anna) differ between fecal and samples from three sections of the GI tract and between birds that differ in sex, age and pox virus infection status. We characterized bacterial composition in fecal and GI tract samples from Anna’s Hummingbirds by amplicon sequencing of the 16S rRNA gene. We found strong evidence of differentiation in bacterial composition among GI tract regions and compared to fecal samples. Actinobacteria, primarily genus Corynebacterium, Firmicutes and Fusobacteria were abundant in bird samples from regions of the GI tract. In contrast, fecal bacterial communities were more diverse and variable compared to GI tract samples. Bacterial community composition differed between male and female hummingbirds, and with bird age (hatch year vs. after-hatch year). Finally, birds with symptoms of avian poxvirus infection had higher relative abundance of Staphylococcus spp. than birds with no symptoms of pox. Our results suggest that Anna’s Hummingbirds host differentiated microbiome among GI tract regions that is consistent among individual birds. The GI bacterial community also contained taxa not represented in fecal samples. This provides evidence for the possibility of a residential gut microbiome in Anna’s Hummingbirds, although functional significance of the bacterial microbiome remains unknown.

Sample Collection

To examine if the bacterial microbiome composition differs with bird age or sex, fecal samples from 48 Anna’s Hummingbirds (Calypte anna) were obtained from three locations in northern California: two sites at private residences in Winters, CA and one site at a commercial business in Acampo, CA. Although 48 birds were sampled, only a subset was successfully sequenced (see below). All three sites were chosen based on observed pre-existing populations of Anna’s Hummingbirds and collected between 7am and 12pm during mild weather conditions of partly cloudy and 21-26°C throughout June 2018. Hall feeder traps were used to capture the hummingbirds, and fecal samples were collected directly from the cloaca into a haematocrit tube. All sampled birds were aged, sexed, weighed, banded, and checked for presence of suspected pox viral lesions before release. Birds were evaluated for the presence of suspected pox lesions given that poxviridae infections have been described in hummingbirds (Baek et al. 2020; Godoy et al. 2013) and it is unknown if this disease process has impacts on the gastrointestinal microbiome. All birds with pox lesions used in the study were confirmed positive by a method utilizing a real-time quantitative PCR assay developed for detecting the hummingbird-specific Avipox 4b core protein gene, as described by Baek et al. (2020). No recaptured birds were used for this study. All live birds sampled for fecal collection (n=48) were observed using hummingbird feeders. In addition, sugar-water solution samples from the feeders were collected on the same day as fecal samples were collected for additional comparison and potential food items (pooled insect samples) were also collected. The feeders were filled with a 1:4 sugar-water solution made with granulated pure cane white sugar (C&H®) and residential tap water. Permission to obtain the hummingbird carcasses for scientific study was approved by the United States Fish and Wildlife Service (Permit: MB55944B-2) and the California Department of Fish and Wildlife (Permit: SC-013066).

To sample bacteria directly from the GI tract, 20 deceased hummingbird carcasses, with no evidence of disease, were obtained from a wildlife rehabilitation center (Lindsay Wildlife Experience, Walnut Creek, CA). Birds were brought in from local good Samaritans but data regarding the exact locations was limited. As with fecal samples, some samples from regions of the GI tract failed to yield sufficient sequence for analysis (described below). These carcasses had no external signs of illness or injury, did not receive medication while in care, and all died within 24 hours of arrival to the center. In addition, body conditions were aligned with healthy birds with high metabolic rates and living on an energetic edge and there were no abnormalities on gross dissection. However, it could not be definitively confirmed that birds were completely devoid of any health, nutrition, or toxicological conditions without running extensive diagnostic tests, which are limited for hummingbirds. As a comparison to birds that did not have any evidence of disease, seven hummingbird carcasses with obvious abnormalities on physical examination were evaluated. The seven hummingbirds had visual evidence of disease in the form of external pox-like lesions on either the bill, wing joints, legs, or a combination of all. All seven birds were confirmed to be positive for pox virus using a previously published PCR method (Baek et al. 2020). Immediately after euthanasia or death, carcasses were placed in a -20^oC freezer. The species, age and sex of the hummingbirds were determined as previously described (Pyle 1997). Three hummingbirds with no external signs of disease received minimal amounts of a commercial food (Nekton Nektar Plus, NEKTON, Kelden Germany), therefore dry samples of the food powder were additionally obtained for DNA extraction. Once transferred to the research laboratory, carcasses were stored at -70^oC until dissection of digestive organs. Carcass freezing has been a technique employed by other microbiome studies (Capunitan et al. 2020; Song et al. 2020) and, given their small body size, hummingbird carcasses freeze rapidly, minimizing the time for change in bacterial community composition.

For each bird, organs were removed for DNA extraction using aseptic technique. The acidic proventriculus and ventriculus (“proventriculus/ventriculus” hereafter; King & McLelland 1984) were sampled by cutting the esophageal-proventricular and ventricular-duodenal junctions. The small intestine sample was obtained by cutting the ventricular-duodenal junction and extending distally into the small intestine by approximately 0.5cm. Similarly, the lower intestine sample was obtained by starting with the colonic-cloacal junction and cutting approximately 0.5cm proximally into the large intestine.

Dissected organs were removed and collected in sterile microcentrifuge tubes and flushed using a syringe of sterile PCR-grade water, ensuring it passed through the entirety of the interior of the intestinal tract, in order to remove any potential dietary products. Following the flush, the intestinal tract was divided into upper and lower segments, which were sampled separately.

Microbiome characterization

To characterize the bacterial communities in the fecal and GI tract samples and potential food items, we used metabarcoding of the 16S rRNA. Briefly, DNA was extracted from samples from regions of the GI tract using the DNeasy PowerSoil Kit (Qiagen, Hilden Germany) and following manufacturer’s instructions, modified by the addition of initial bead-beating and overnight lysis (Rubin et al. 2014). We also sequenced a sample of the commercial food (Nekton Nektar Plus, NEKTON, Kelden Germany), as some birds from Lindsay Wildlife Experience used received minimal amounts of powder prior to being euthanized, and two blank control samples to ensure the quality of the sequencing data. From each extraction, DNA was sent to the Dalhousie IMR facility, and sequenced using the Illumina MiSeq platform. Bacteria were characterized using the V6-V8 region of the 16S rRNA gene using 926F-1392R primers to avoid contamination by bird host tissues (Tremblay et al. 2015). The raw sequence data was submitted to the Sequence Read Archive (SRA) at the NCBI and can be located under access code PRJNA646258.

Reads were error-corrected and assembled into amplicon sequence variants (ASVs) using the R package DADA2 version 1.10.1 (Callahan et al. 2016) to represent the microbial taxa in and across all samples using recommended parameters. We chose to use ASVs rather than OTUs because of their greater precision and ability to compare across studies (Callahan et al. 2016). Briefly, primers were removed and reads filtered and trimmed using settings maxEE=2,2 and length (forward=280, reverse=190). Reads were error-corrected and merged and chimeras detected and removed using the ‘consensus’ method. Bacteria were assigned taxonomy using SILVA training set v.132 (Quast et al. 2013).

See details on metadata included in the readme file.