key: cord-0324011-x8y26ny2 authors: Ruhs, Emily Cornelius; Becker, Daniel J.; Oakey, Samantha J.; Ogunsina, Ololade; Fenton, M. Brock; Simmons, Nancy B.; Martin, Lynn B.; Downs, Cynthia J. title: Body size affects immune cell proportions in birds and non-volant mammals, but not bats date: 2020-12-21 journal: bioRxiv DOI: 10.1101/2020.12.18.423538 sha: 326912f3805a52607c96b72d7028f1b1c3345529 doc_id: 324011 cord_uid: x8y26ny2 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 likely 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 scaling framework to quantify scaling relationships between body mass and the proportions of two types of white blood cells--lymphocytes, and granulocytes (neutr-/heterophils)--across 60 bat species, 414 bird species, and 256 non-volant mammal species. By using phylogenetically-informed statistical models on field-collected data from wild Neotropical bats, data gleaned from other wild bats available in the literature, and data from captive 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. 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. Summary statement Powered flight might constrain morphology such that certain immunological features are prioritized. We show that bats largely have similar cell proportions across body mass compared to strong allometric scaling relationships in birds and non-flying mammals. Powered flight has evolved at least three times in the evolutionary history of vertebrates and yet 21 is one of the most energetically costly modes of transportation (Rayner, 1988) . Birds and bats 22 experience a 6-14 fold and >25 fold increase over resting metabolic rate, respectively, in 23 metabolic expenditure during flight, whereas a similarly-sized mammal only experiences a 6-8 24 fold increase during sustained running (Schmidt-Nielsen, 1972; Thomas, 1975) . Although there 25 is some debate over whether bats or birds are more efficient fliers (Muijres, Johansson, Bowlin, 26 Winter, and Hedenström, 2012; Swartz et al., 2007; Tian et al., 2006) , there are clear functional 27 and physiological constraints associated with this costly activity (Maurer et al., 2004; Muijres et 28 al., 2012) . One of the most evident constraints is body size. Exceptionally large and small body 29 sizes have apparently been selected against in the evolution of flying vertebrates due to demands 30 imposed by the physics of flight (Stanley, 1973) ; however, the constraining factors for bats and 31 birds likely differ, as the largest bats are much smaller than the largest flying birds. The 32 evolution of flight and body size constraints may have had numerous direct and indirect effects 33 on evolution of the immune system in flying vertebrates. For example, evolution of a lightened 34 skeleton (Feduccia and Feduccia, 1999; Dumont, 2010) (Ruhs et al., 2020) . It should be noted that the high energetic costs of flight have varying 38 impacts on the immune system (Hasselquist et al. 2007; Voigt et al. 2020; Nebel et al. 2012) . 39 While birds and bats have much in common in terms of constraints that accommodate the ability 40 to fly, the evolution of flight likely impacted the dynamics between body size, physiological 41 traits, and the exposure risk to pathogens relative to non-flying birds and mammals. viruses appear to not kill and rarely cause clinical disease in bats (Williamson et al., 2000) . 83 Whereas the high diversity of zoonotic viruses in Chiroptera might be partly driven by 84 the speciose nature of this order (Mollentze and Streicker, 2020) , bat tolerance of particular 85 viruses may be shaped by specialized immune mechanisms in these flying mammals (Brook et 86 al., 2020; Zhang et al., 2013) . Bat immunoglobulins and leukocytes are structurally similar to 87 those of humans and mice (Baker et al. 2013) , but bats also have unique immune system traits 88 such as complement proteins robust to temperature change, lack of fever with bacterial 89 (lipopolysaccharide) challenge, high constitutive expression of type I interferons, and dampened 90 inflammation (Ahn et al., 2019; Hatten et al., 1973; Pavlovich et al., 2018; Stockmaier et al., 91 2015; Zhou et al., 2016) . Flight may explain these distinctions, including increased metabolic 92 rates that enable stronger immune responses and elevated body temperature that could mirror 93 febrile responses to control infection (O'Shea et al. 2014 ; but see Levesque et al. 2020) . However, the primary hypothesis for how bats can tolerate viruses is that they evolved 95 mechanisms to minimize or repair the negative effects of oxidative stress generated as a 96 consequence of flight (Zhang et al., 2013) . For example, some bat species show resistance to This propensity to resist acute oxidative stress and repair oxidative damage could have also 100 helped bats cope with viral replication that would have otherwise caused cell damage (Kacprzyk 101 et al., 2017; Xie et al., 2018; Zhang et al., 2013) . Here, we first asked whether leukocyte proportion scaling in bats is distinct compared to 103 other taxa already described. Then, we asked whether the ability to fly (i.e. bats and birds) 104 explains immune cell proportion allometries across extant vertebrate endotherms. Most studies 105 assessing immunity in bats have been limited to few species (but see Schneeberger et al., 2013) . 106 We combined field-collected data from Neotropical bats with data from the primary literature to 107 maximize sample sizes as well as phylogenetic and body size diversity. We then quantified 108 scaling relationships for proportions of two primary leukocytes for which abundant data were 109 available, lymphocytes and granulocytes. Lymphocytes include B and T cells, which provide 110 specific, but time-lagged, protection through antibody production and coordination of cascading 111 immune responses. Granulocytes (neutrophils in mammals and heterophils in birds) are Diagnostics Quick III). All bats were released after processing. Sampling followed guidelines for Bat leukocyte data 151 We used light microscopy (1000X) to quantify the proportion of neutrophils and lymphocytes 152 from 100 leukocytes from each field sample (Schneeberger et al., 2013) . As Neotropical bats are (Dunning Jr., 2007) and/or publicly available databases such as AnAge (Tacutu et al., 2013) , the mammals 178 Our modeling progressed in two stages. First, to test hypotheses about allometric scaling of 179 leukocytes in bats only, we used phylogenetic generalized mixed-effects models (GLMMs) with 180 the ape and MCMCglmm packages in R (Hadfield & Others, 2010; Paradis et al., 2004) . All 181 models included phylogenetic effects from a phylogeny produced in PhyloT using data from the 182 National Center for Biotechnology Information (Letunic, 2015) and with resolved polytomies. We used that tree to create two phylogenetic covariance matrices, one for bat-only analyses and 184 one that we used later for direct comparisons of scaling slopes across taxa. We set the inverse-185 gamma priors to 0.01 for the random effect of phylogenetic variance and default priors for the 186 fixed effects in all models. All models were run for 260k iterations with 60k burn-in and a 200-187 iteration thinning interval Ruhs et al., 2020) . For all models, we used 188 Deviance Information Criterion (ΔDIC) to identify the best-fit GLMM. We defined the top 189 model as that with the lowest DIC, and we considered models within Δ DIC<5 as having 190 equivalent support (Richards, 2005) . For all models, we also calculated Pagel's unadjusted λ and 191 conditional and marginal R 2 (Housworth et al., 2004; Nakagawa and Schielzeth, 2013) . We then 192 used this approach to determine the scaling relationship for lymphocyte and neutrophil 193 proportions, separately, across 60 bat species. Also, because previously published slopes for 194 mammal and bird leukocyte scaling used cell concentrations Ruhs et al., 195 2020), we determined the scaling relationships of cell proportion data for both birds (n=414) and For birds, the mass model (model 2) was best-supported (Table S2) Table S3 ). For non-volant mammals, the 243 lymphocyte and neutrophil mass models were also the best-supported (Table S4) Although bats and birds represent two independent evolutionary origins of flight (Rayner, 1988) , Are bats immunologically different? 305 We predicted that bats, like birds, might need a disproportionately large amount of broadly 306 protective cells as they are more likely to be exposed to more and diverse parasites than non- differences between scaling patterns of total leukocytes or neutrophil counts, despite captive animals having higher mean lymphocyte counts than wild animals (Tian et al., 2015) . Taken in 329 sum, the lack of scaling patterns found here are unlikely driven by variation in wild bat cell Increased spillover of zoonotic viruses, such as henipaviruses and coronaviruses, has renewed 356 public and scientific interest in whether bats are immunologically unique reservoir hosts (Brook 357 and Dobson, 2015; Halpin et al., 2011; Li et al., 2005; Luis et al., 2013) . Investigating allometric physiological traits that impact host ability to tolerate virulent pathogens. We here demonstrate 360 some differences in the scaling patterns of innate immune cell proportions between taxa of 361 endotherms; however, we did not observe substantial effects of body size on cell proportions in 362 bats. It is important to note that it is likely more difficult to find allometric patterns of cell 363 proportions using the methods employed here compared to the previous discovery of hypermetric 364 scaling of cell concentrations Ruhs et al., 2020) ; therefore, future studies Lastly, we focused on cell proportion allometry and the potential for body mass alone to 372 explain immunological differences among species . Flying endotherms can 373 vary in other ecological traits besides body mass that also shape pathogen exposure and immune 374 investment, such as diet, coloniality, and roost type (Minias, Whittingham, & Dunn, 2017; 375 Schneeberger et al., 2013) . Dampened NLRP3-mediated inflammation in bats and implications for a special viral reservoir host Reproduction has different costs for immunity and parasitism in a wild mammal Animal migration and infectious disease risk Oral shedding of Marburg virus in experimentally infected Egyptian fruit bats (Rousettus aegyptiacus) Antiviral immune responses of bats: a review Bats and lyssaviruses Handling Stress and Sample Storage Are Associated with Weaker Complement-Mediated Bactericidal Ability in Birds but Not Bats Livestock abundance predicts vampire bat demography, immune profiles and bacterial infection risk. Of the Royal … Costs of immune responses are related to host body size and lifespan Prey Detection, Dietary Niche Breadth, and Body Size in Bats: Why are Aerial Insectivorous Bats so Small? Accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence. eLife Bats as "special"reservoirs for emerging zoonotic pathogens Toward a metabolic theory of ecology Size, Function, and Life History Social Grooming in Bats: Are Vampire Bats Exceptional? How Birds Combat Ectoparasites The Protection: The Unit of Humoral Immunity Selected by Evolution Reproductive Biology of Bats The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists Preening and associated comfort behavior in birds Allometric scaling of the active hematopoietic stem cell pool across mammals The Effects of Body Mass on Immune Cell Concentrations of Mammals Scaling of Host Competence Bone density and the lightweight skeletons of birds CRC Handbook of Avian Body Masses The Origin and Evolution of Birds Disease alters macroecological patterns of North American bats Evolutionary History of Bats: Fossils, Molecules and Morphology Host phylogenetic distance drives trends in virus virulence and transmissibility across the animal-human interface MCMC methods for multi-response generalized linear mixed models: the MCMCglmm R package Pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission Western Bats as a Reservoir of Novel Streptomyces Species with Antifungal Activity Infectious disease transmission and behavioural allometry in wild mammals Do Performance-Safety Tradeoffs Cause Hypometric Metabolic Scaling in Animals? A serologic comparison of bat complements Ecology and mode-of-life explain lifespan variation in birds and mammals The changing ecology of primate parasites: Insights from wild captive comparisons Disassembly of Fragmented Bat Communities in Orange Walk District The phylogenetic mixed model Bats Are an Untapped System for Understanding Microbiome Evolution in Mammals Does Cellular Metabolism from Primary Fibroblasts and Oxidative Stress in Blood Differ between Mammals and Birds? The (Lack-thereof) Scaling of Oxidative Stress The evolution of echolocation in bats A potent anti-inflammatory response in bat macrophages may be linked to extended longevity and viral tolerance Causes and Consequences of Sociality in Bats Changing resource landscapes and spillover of henipaviruses Body size and metabolism Roosting Ecology of Bats Ecosystem services provided by bats Size of bats at birth and maternal investment during pregnancy. Symposia of the Zoological Society of London Shades of grey -the blurring view of innate and adaptive immunity Linking immune defenses and life history at the levels of the individual and the species Phylot : Phylogenetic Tree Generator High body temperature is an unlikely cause of high tolerance in bats Bats are natural reservoirs of SARS-like coronaviruses A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? Going to Bat(s) for Studies of Disease Tolerance Seasonal changes in vertebrate immune activity: mediation by physiological trade-offs Similarities in body size distributions of small-bodied flyingvertebrates What Can Birds Tell Us about the Migration Physiology of Bats Coloniality and migration are related to selection on MHC genes in birds. Evolution Viral zoonotic risk is homogenous among taxonomic orders of mammalian and avian reservoir hosts Comparing aerodynamic efficiency in birds and bats suggests better flight performance in birds Bats and birds: Exceptional longevity despite high metabolic rates A general and simple method for obtaining R2 from generalized linear mixed-effects models Cultured renal epithelial cells from birds and mice: enhanced resistance of avian cells to oxidative stress and DNA damage Exceptional cellular resistance to oxidative damage in long-lived birds requires active gene expression Bat flight and zoonotic viruses APE: Analyses of Phylogenetics and Evolution in R language The Encyclopedia of Life v2: Providing Global Access to Knowledge About Life on Earth The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity Synchronous shedding of multiple bat paramyxoviruses coincides with peak periods of Hendra virus spillover Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus) A new field record for bat longevity The evolution of vertebrate flight A field guide to the mammals of Central America and Southeast México Testing ecological theory using the information-theoretic approach: examples and cautionary results Constraints on Reproduction by Flying Vertebrates: Energy and Calcium The impacts of body mass on immune cell concentrations in birds Locomotion: energy cost of swimming, flying, and running Scaling: Why is Animal Size So Important? Measures of the constitutive immune system are linked to diet and roosting habits of neotropical bats Seasonal Fluctuations of Astrovirus, But Not Coronavirus Shedding in Bats Inhabiting Human-Modified Tropical Forests Habitat disturbance results in chronic stress and impaired health status in forest-dwelling paleotropical bats Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses Chemical warfare? Effects of uropygial oil on feather-degrading bacteria Guidelines of the American Society of Mammalogists for the use of wild mammals in research Bat Species of the World: A taxonomic and geographic database The physiological costs of reproduction in small mammals AN EXPLANATION FOR COPE'S RULE. Evolution No fever and leucocytosis in response to a lipopolysaccharide challenge in an insectivorous bat Wing Structure and the Aerodynamic Basis of Flight in Bats Human Ageing Genomic Resources: integrated databases and tools for the biology and genetics of ageing Bat Biology, Genomes, and the Bat1K Project: To Generate Chromosome-Level Genomes for All Living Bat Species Metabolism during flight in two species of bats, Phyllostomus hastatus and Pteropus gouldii Circulating white blood cell counts in captive and wild rodents are influenced by body mass rather than testes mass, a correlate of mating promiscuity Direct measurements of the kinematics and dynamics of bat flight From Immunology to Eco-Immunology: More than a New Name Viral Richness is Positively Related to Group Size, but Not Mating System Scaling in biology: patterns and processes, causes and consequences Some scaling principles for the immune system Recurrent evolution of extreme longevity in bats Life history, ecology and longevity in bats Experimental hendra virus infectionin pregnant guinea-pigs and fruit Bats (Pteropus poliocephalus) EltonTraits 1.0: Species-level foraging attributes of the world's birds and mammals: Ecological Archives E095-178 Dampened STING-Dependent Interferon Activation in Bats Comparative analysis of bat genomes provides insight into the evolution of flight and immunity Contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats ZIMS: Zoo aquarium animal management software -Species360 The authors declare no competing interests.