DOI: 10.5061/dryad.3n5tb2rkf
title: Disentangling relationships between physiology, morphology, diet, and gut microbial diversity in American Kestrel nestlings

description: Gut microbiota are increasingly recognized as important drivers of host
 health and fitness across vertebrate taxa. Given that gut microbial
 composition is directly influenced by the environment, gut microbiota may
 also serve as an eco-physiological mechanism connecting host ecology, such
 as diet, and physiology. Although gut microbiota have been well-studied in
 mammalian systems, little is known about how gut microbial diversity and
 composition impact morphological and physiological development in wild
 birds. Here, we characterized both diet and gut microbial diversity of
 free-living American kestrel (Falco sparverius) nestlings throughout
 development to test whether gut microbial diversity predicts host
 morphological and physiological traits in either contemporary or
 time-lagged manners. Gut microbial alpha diversity on day 21 of nestling
 development was positively correlated with diet alpha diversity
 representative of the majority of nestling development (days 5–20). Gut
 microbial alpha diversity early in development was negatively correlated
 with body mass in both contemporary and time-lagged manners. Gut microbial
 alpha diversity early in development was positively correlated with blood
 glucose later in development. As nestlings experience rapid growth demands
 in preparation to fledge, these time-lagged associations may indicate that
 gut microbial diversity at early critical developmental windows may
 determine the future trajectory of morphological and physiological traits
 underlying metabolism that ultimately impact fitness.
description: <strong>Sample
 Collection</strong> We monitored American kestrel (<em>Falco
 sparverius</em>) nest-boxes in Berks, Lehigh, and Schuylkill
 Counties, Pennsylvania, USA beginning in early May 2018 and 2019 to
 document occupancy, egg-laying date, and hatching as described in Cornell
 et al. (2021). In 2018, we sampled 12 nestlings from 4 nests, and in 2019
 we sampled 32 nestlings from 10 nests (n = 44 nestlings total). On days 7,
 14, and 21 after hatching (hatch day = day 0), we removed nestlings from
 the nest and collected morphometrics, blood samples, and fecal samples
 between 8:00 and 12:00 hours (following university IACUC and US Fish and
 Wildlife Service banding permits). Birds were sexed on day 21 from
 coloration of primary feathers: blue for males and brown for females
 (Smallwood and Bird 2020). We measured mass to the nearest 0.01 g using a
 digital scale. We banded nestlings with uniquely colored temporary leg
 bands (Darvic Wraparound 1FB, 5.5 mm, Avinet) on day 7 for identification
 within nests and banded with USGS aluminum bands on day 21. To
 characterize gut microbial diversity, we collected fecal samples (n = 131
 total) from nestlings by abdominal palpation in sterile collection
 containers. We swabbed fecal matter with sterile flocked swabs (Puritan
 Medical Products Company LLC, Guilford, ME, USA) placed in autoclaved
 microcentrifuge tubes. We stored fecal samples on ice in the field and
 then at -80°C until DNA extractions. 
 <strong>DNA Extractions,
 PCR, and Sequencing</strong>
 We extracted DNA from whole swabs
 using DNeasy PowerSoil Pro DNA Isolation Kits (Qiagen Inc., Valencia, CA,
 USA) following the manufacturer’s protocol. We amplified the V4 region of
 the 16S rRNA gene (i.e., a universal marker gene for bacteria and archaea)
 using the primers 515F and 806R with Illumina adapters added. We followed
 the Earth Microbiome Project 16S Illumina Amplicon protocol (Caporaso et
 al. 2011, Caporaso et al. 2012) except for using 10 μl total reaction
 volumes instead of 25 μl. Each PCR reaction was run in triplicate and
 included 5 μl of 2x Platinum Hot Start Master Mix (Invitrogen, Waltham,
 MA, USA), 0.5 μl of 10 μM primers, 3 μl of nuclease free water, and 1 μl
 of template DNA. Cycling conditions were 3 minutes at 94°C followed by 35
 cycles of 94°C for 45 seconds, 50°C for 60 seconds, and 72°C for 90
 seconds before a final extension at 72°C for 10
 minutes. We pooled the three replicate reactions for each
 sample and ran a 1% agarose gel to confirm that amplification was
 successful for the V4 region of the 16S rRNA gene (~350 bp). Each PCR run
 included negative controls (nuclease free water in place of template DNA
 but were not sequenced). We also extracted, amplified, and sequenced 4
 negative kit reagent controls. We submitted our final pooled PCR products
 to the Cornell Biotechnology Resource Center for quantification,
 normalization, library preparation, and sequencing in one Illumina MiSeq
 paired-end 2 x 250 bp run (n = 131 fecal samples, n = 4 negative
 controls). The sequences included in this dataset are the raw
 forward and reverse FASTQ files associated with each
 sample.
description: We have included scripts for QIIME2 (version 2021.11) and R
 (version 4.2.1). See READMe for more information.