DOI: 10.25338/b81d0x
title: Anna's hummingbird microbiome analysis

description: 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.
description: <strong><i>Sample
 Collection</i></strong> To examine if the
 bacterial microbiome composition differs with bird age or sex, fecal
 samples from 48 Anna’s Hummingbirds (<i>Calypte anna)</i> 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
 <i>Avipox</i> 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<sup>o</sup>C 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<sup>o</sup>C 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. <strong><i>Microbiome characterization</i></strong> 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).<b> </b>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.<b> </b>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<b>. </b>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).
description: See details on metadata included in the readme file.