Adapting flow cytometry for studying immune tradeoffs in wild bats

Abstract

Understanding the differences in the immune systems of wildlife is critical for assessing the impacts of ecological stressors and zoonotic disease risk as many animals differ in the ways they mitigate infection while managing ecological challenges. However, our understanding of immune dynamics in wild species remains limited due to the challenges involved in measuring cellular immune function such as cold chain maintenance and a lack of species-specific reagents. This study adapts flow cytometry methods, which have been traditionally confined to laboratory settings, by optimizing protocols to enable immune profiling with small blood volumes and extended sample holding times to facilitate the study of cellular immunity in wildlife. We then applied these methods to a field research setting in rural Oklahoma to analyze trends in immune function in a migratory Mexican free-tailed bat (Tadarida brasiliensis) colony using a single-cell RNA sequencing–confirmed antibody panel (CD3 T-cells, CD79a B-cells, MCHII macrophages, and CD11b neutrophils) developed by a study in Egyptian fruit bats (Rousettus aegyptiacus). We first sought to compare flow cytometry with traditional white blood cell counts from blood smears, to demonstrate how flow cytometry could offer greater precision in immune cell identification. Then, through seasonal sampling, we identified trade-offs not only between innate and adaptive immune responses, but also within the innate and adaptive arms, revealing how energetic stressors like migration and reproduction influence specific immune cell strategies. Specifically, we found that, similarly to other animals, innate immunity dominates immediately following migration, while adaptive immunity becomes more prominent during non-energetically demanding life stages. More interestingly however, we found that within the innate arm of the immune system macrophage activity peaks just after migrating bats arrive in Oklahoma, as does T-cell abundance within the adaptive arm, while neutrophil activity peaks during the pup-rearing season, and B-cell activity peaks as pups become more self reliant. This suggests that within the innate and adaptive systems, cellular activity shows seasonal fluctuations that could better inform the strategy of reliance on innate immunity during energetically demanding life stages in bats, as well as strategies of adaptive cell use during less energetically stressful periods. Understanding these inter-system immune dynamics is crucial for understanding and predicting disease risk, particularly in light of the zoonotic potential of many bat-borne pathogens. These findings provide valuable insights into the immune ecology of bats and lay the groundwork for expanding the application of flow cytometry in wildlife immunology.