Challenges associate with microbiome diversity, glucocorticoids, and condition in a wild songbird.

Wild animals experience daily fitness challenges, and the resulting stress responses can disrupt an animal’s gut microbiome. Given the links between health and microbiome composition, it is essential to understand how challenges affect microbial communities. We showed that a fitness challenge significantly alters the cloacal microbiome of free-living Northern cardinals (Cardinalis cardinalis), and these shifts covary with changes to the glucocorticoid stress response, condition, and beak ornamentation. Treatment significantly related to beta diversity, while it only influenced alpha diversity indirectly through interactions with health-related traits. Birds held for an extended time before release showed greater cloacal microbiome community changes 11 days later vs. birds administered a simulated territorial intrusion challenge, or control birds administered no challenge. We also detected relationships between beta diversity and change in body condition and beak ornamentation. Birds showing the greatest alpha diversity decrease and largest beta diversity between timepoints experienced the greatest corticosterone response to handling. Finally, several Amplicon Sequence Variants were differentially abundant in challenged birds compared to control birds. To our knowledge, this study is the first to demonstrate proximate effects of fitness challenges on the microbiome of an adult, free-living songbird, while simultaneously tracking changes in glucocorticoid levels, body condition, and ornamentation—providing a uniquely integrative perspective on how stress shapes host-microbe interactions in the wild.