Disentangling relationships between physiology, morphology, diet, and gut microbial diversity in American Kestrel nestlings

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.We have included scripts for QIIME2 (version 2021.11) and R (version 4.2.1). See READMe for more information. Funding provided by: Blake-Nuttall Avian Research Grant*Crossref Funder Registry ID: Award Number: Funding provided by: Beta Beta Beta Student Research Grant*Crossref Funder Registry ID: Award Number: Funding provided by: Penn State Altoona Research and Development Grant*Crossref Funder Registry ID: Award Number:Sample Collection We monitored American kestrel (Falco sparverius) 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. DNA Extractions, PCR, and Sequencing 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