16S and 18S rRNA gene sequencing of fecal microbial extracts for: Variation in gut microbial contribution of essential amino acids to host protein metabolism in a wild small mammal community

Herbivory is a dominant feeding strategy among animals, yet herbivores are often protein limited. The gut microbiome is hypothesized to help maintain host protein balance by provisioning essential macromolecules, but this has never been tested in wild consumers. Using amino acid carbon and nitrogen stable isotope analysis, we estimated the proportional contributions of essential amino acids synthesized by gut microbes to five co-occurring desert rodents representing herbivorous, omnivorous, and insectivorous functional groups. We found that herbivorous rodents occupying lower trophic positions (Dipodomys spp.) routed nearly half of their essential amino acids from gut microbes, while higher trophic level omnivores (Peromyscus spp.) and insectivores (Onychomys arenicola) obtained most of their essential amino acids from plant-based energy channels but still received approximately one-fifth of their essential amino acids from gut microbes. These findings empirically demonstrate that gut microbes play a key functional role in host protein metabolism in wild animals.To characterize the amino acid carbon and nitrogen stable isotope values of gut microbes, we isolated microbial cells from fecal samples collected from D. merriami, D. ordii, D. spectabilis, and Peromyscus spp. using a Nycodenz density gradient and centrifugation. A subset of these samples were also aliquoted for 16S and 18S rRNA gene sequencing. DNA was extracted using a modified CTAB method with phenol:chloroform:IAA purification. PCR was was used to amplify the V4 region of the 16S rRNA gene using Illumina 515f and 806r primers to identify Bacteria and Archaea and the V9 region of the 18S rRNA gene using Illumina Euk1391f and EukBr primers to identify Eukaryota. Dual-indexed, paired-end amplicons were sequenced on an Illumina MiSeq at the University of New Mexico.