Large Mammals Have More Powerful Antibacterial Defenses Than Expected from Their Metabolic Rates.

AbstractTerrestrial mammals span seven orders of magnitude in body size, ranging from the <2-g Etruscan pygmy shrew (Suncus etruscus) to the >3,900-kg African elephant (Loxodonta africana). Although body size profoundly affects the behavior, physiology, ecology, and evolution of species, how investment in functional immune defenses changes with body size across species is unknown. Here, we (1) developed a novel 12-point dilution curve approach to describe and compare antibacterial capacity against three bacterial species among >160 terrestrial species of mammals and (2) tested published predictions about the scaling of immune defenses. Our study focused on the safety factor hypothesis, which predicts that broad, early-acting immune defenses should scale hypermetrically with body mass. However, our three statistical approaches demonstrated that antibacterial activity in sera across mammals exhibits isometry; killing capacity did not change with body size across species. Intriguingly, this result indicates that the serum of a large mammal is less hospitable to bacteria than would be predicted by its metabolic rates. In other words, if metabolic rates underlie the rates of physiological reactions as postulated by the metabolic theory of ecology, large species should have disproportionately lower antibacterial capacity than small species, but they do not. These results have direct implications for effectively modeling the evolution of immune defenses and identifying potential reservoir hosts of pathogens.

Snap-freezing in the Field: Effect of Sample Holding Time on Performance of Bactericidal Assays.

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.

Body size affects immune cell proportions in birds and non-volant mammals, but not bats.

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.

Understanding metrics of stress in the context of invasion history: the case of the brown treesnake (Boiga irregularis).

Invasive species can exert rapid depletion of resources after introduction and, in turn, affect their own population density. Additionally, management actions can have direct and indirect effects on demography. Physiological variables can predict demographic change but are often restricted to snapshots-in-time and delayed confirmation of changes in population density reduces their utility. To evaluate the relationships between physiology and demography, we assessed metrics of individual and demographic stress (baseline and 1-h corticosterone (CORT), body condition and bacterial killing ability) in the invasive snake Boiga irregularis on Guam collected in intervals of 10-15 years. We also assessed potential discrepancies between different methods of measuring hormones [radioimmunoassay (RIA) versus enzyme immunoassay (EIA)]. The magnitude of difference between RIA and EIA was negligible and did not change gross interpretation of our results. We found that body condition was higher in recent samples (2003 and 2018) versus older (1992-93) samples. We found corresponding differences in baseline CORT, with higher baseline CORT in older, poorer body condition samples. Hormonal response to acute stress was higher in 2018 relative to 2003. We also found a weak relationship between circulating CORT and bacterial killing ability among 2018 samples, but the biological significance of the relationship is not clear. In an effort to develop hypotheses for future investigation of the links between physiology and demography in this and other systems, we discuss how the changes in CORT and body condition may reflect changes in population dynamics, resource availability or management pressure. Ultimately, we advocate for the synchronization of physiology and management studies to advance the field of applied conservation physiology.