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Comparative analyses in biology rely on the quality of available data. Methodological differences among studies may introduce variation in results that obscure patterns. In the field of eco-immunology, functional immune assays such as antimicrobial capacity assays are widely used for among-species applications. Sample storage time and animal handling time can influence assay results in some species, but how sample holding time prior to freezing influences assay results is unknown. Sample holding time can vary widely in field studies on wild animals, prompting the need to understand the implications of such variation on assay results. We investigated the hypothesis that sample holding time prior to freezing influences assay results in six species (Leiocephalus carinatus, Iguana iguana, Loxodonta africana, Ceratotherium simum, Columba livia, and Buteo swainsoni) by comparing antibacterial capacity of serum with varying processing times prior to snap-freezing. Blood was collected once from each individual and aliquots were placed on ice and assigned different holding times (0, 30, 60, 180, and 240 min), after which each sample was centrifuged, then serum was separated and snap-frozen on dry ice and stored at −80ºC for 60 days prior to assaying. For each aliquot, we conducted antibacterial capacity assays with serial dilutions of serum inoculated with E. coli and extracted the dilution at 50% antibacterial capacity for analysis. We found a decrease in antibacterial capacity with increased holding time in one of the six species tested (B. swainsoni), driven in part by complete loss of antibacterial capacity in some individuals at the 240-min time point. While the majority of species’ antibacterial capacity were not affected, our results demonstrate the need to conduct pilot assays spanning the anticipated variation in sample holding times to develop appropriate field protocols.

 

ABSTRACT Powered flight has evolved several times in vertebrates and constrains morphology and physiology in ways that likely have shaped how organisms cope with infections. Some of these constraints probably have impacts on aspects of immunology, such that larger fliers might prioritize risk reduction and safety. Addressing how the evolution of flight may have driven relationships between body size and immunity could be particularly informative for understanding the propensity of some taxa to harbor many virulent and sometimes zoonotic pathogens without showing clinical disease. Here, we used a comparative framework to quantify scaling relationships between body mass and the proportions of two types of white blood cells – lymphocytes and granulocytes (neutrophils/heterophils) – across 63 bat species, 400 bird species and 251 non-volant mammal species. By using phylogenetically informed statistical models on field-collected data from wild Neotropical bats and from captive bats, non-volant mammals and birds, we show that lymphocyte and neutrophil proportions do not vary systematically with body mass among bats. In contrast, larger birds and non-volant mammals have disproportionately higher granulocyte proportions than expected for their body size. Our inability to distinguish bat lymphocyte scaling from birds and bat granulocyte scaling from all other taxa suggests there may be other ecological explanations (i.e. not flight related) for the cell proportion scaling patterns. Future comparative studies of wild bats, birds and non-volant mammals of similar body mass should aim to further differentiate evolutionary effects and other aspects of life history on immune defense and its role in the tolerance of (zoonotic) infections.