Bat cellular immunity varies by year and dietary habit amidst land conversion.

Monitoring the health of wildlife populations is essential in the face of increased agricultural expansion and forest fragmentation. Loss of habitat and habitat degradation can negatively affect an animal's physiological state, possibly resulting in immunosuppression and increased morbidity or mortality. We sought to determine how land conversion may differentially impact cellular immunity and infection risk in Neotropical bats species regularly infected with bloodborne pathogens, and to evaluate how effects may vary over time and by dietary habit. We studied common vampire bats (Desmodus rotundus), northern yellow-shouldered bats (Sturnira parvidens) and Mesoamerican mustached bats (Pteronotus mesoamericanus), representing the dietary habits of sanguivory, frugivory and insectivory respectively, in northern Belize. We compared estimated total white blood cell count, leukocyte differentials, neutrophil to lymphocyte ratio and infection status with two bloodborne bacterial pathogens (Bartonella spp. and hemoplasmas) of 118 bats captured in a broadleaf, secondary forest over three years (2017-2019). During this period, tree cover decreased by 14.5% while rangeland expanded by 14.3%, indicating increasing habitat loss and fragmentation. We found evidence for bat species-specific responses of cellular immunity between years, with neutrophil counts significantly decreasing in S. parvidens from 2017 to 2018, but marginally increasing in D. rotundus. However, the odds of infection with Bartonella spp. and hemoplasmas between 2017 and 2019 did not differ between bat species, contrary to our prediction that pathogen prevalence may increase with land conversion. We conclude that each bat species invested differently in cellular immunity in ways that changed over years of increasing habitat loss and fragmentation. We recommend further research on the interactions between land conversion, immunity and infection across dietary habits of Neotropical bats for informed management and conservation.

Expanded diversity of novel hemoplasmas in rare and undersampled Neotropical bats.

Hemotropic mycoplasmas are emerging as a model system for studying bacterial pathogens in bats, but taxonomic coverage of sampled host species remains biased. We leveraged a long-term field study in Belize to uncover novel hemoplasma diversity in bats by analyzing 80 samples from 19 species, most of which are infrequently encountered. PCR targeting the partial 16S rRNA gene found 41% of bats positive for hemoplasmas. Phylogenetic analyses found two novel host shifts of hemoplasmas, four entirely new hemoplasma genotypes, and the first hemoplasma detections in four bat species. One of these novel hemoplasmas (from Neoeptesicus furinalis) shared 97.6% identity in the partial 16S rRNA gene to a human hemoplasma (Candidatus Mycoplasma haemohominis). Additional analysis of the partial 23S rRNA gene allowed us to also designate two novel hemoplasma species, in Myotis elegans and Phyllostomus discolor, with the proposed names Candidatus Mycoplasma haematomyotis sp. nov. and Candidatus Mycoplasma haematophyllostomi sp. nov., respectively. Our analyses show that additional hemoplasma diversity in bats can be uncovered by targeting rare or undersampled host species.

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.

Leukocyte Concentrations Are Isometric in Reptiles Unlike in Endotherms.

AbstractHow do large and small reptiles defend against infections, given the consequences of body mass for physiology and disease transmission? Functionally equivalent mammalian and avian granulocytes increased disproportionately with body mass (i.e., scaled hypermetrically), such that large organisms had higher concentrations than expected by a prediction of proportional protection across sizes. However, as these scaling relationships were derived from endothermic animals, they do not necessarily inform the scaling of leukocyte concentration for ectothermic reptiles that have a different physiology and evolutionary history. Here, we asked whether and how lymphocyte and heterophil concentrations relate to body mass among more than 120 reptile species. We compared these relationships to those found in birds and mammals and to existing scaling frameworks (i.e., protecton, complexity, rate of metabolism, or safety factor hypotheses). Both lymphocyte and heterophil concentrations scaled almost isometrically among reptiles. In contrast, functionally equivalent granulocytes scaled hypermetrically and lymphocytes scaled isometrically in birds and mammals. Life history traits were also poor predictors of variation in reptilian heterophil and lymphocyte concentrations. Our results provide insight into differences in immune protection in birds and mammals relative to that in reptiles through a comparative lens. The shape of scaling relationships differs, which should be considered when modeling disease dynamics among these groups.

Hemolytic parasites affect survival in migrating red-tailed hawks.

Migrating birds face a myriad of hazards, including higher exposure to parasites and numerous competing energy demands. It follows that migration may act as a selective filter and limit population growth. Understanding how individual-level physiological condition and disease status scale up to population dynamics through differential survival of individuals is necessary to identify threats and management interventions for migratory populations, many of which face increasing conservation challenges. However, linking individual physiological condition, parasite infection status and survival can be difficult. We examined the relationship among two measures of physiological condition [scaled-mass index and heterophil/leukocyte (H/L) ratio], hematozoa (i.e. hemoparasites) presence and abundance, and constitutive immunity in 353 autumn migrating red-tailed hawks (Buteo jamaicensis calurus) from 2004 to 2018. Hematazoa (i.e. Haemoproteus and Leucocytozoon) were in the blood smears from 139 red-tailed hawks (39.4%). H/L ratio decreased with scaled-mass index. Adults had a significantly higher H/L ratio than juveniles. Our two measures of immune defences, hemolytic-complement activity and bacteria-killing ability, were highly positively correlated. Our most notable finding was a negative relationship between Haemoproteus parasitemia and survival (i.e. documented individual mortality), indicating that haemosporidian parasites influence survival during a challenging life stage. The effect of haemosporidian parasites on individuals is often debated, and we provide evidence that parasitemia can affect individual survival. In contrast, we did not find evidence of trade-offs between survival and immune defences.

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.

Leukocyte allometries in birds are not affected by captivity.

Body size affects many traits, but often in allometric, or disproportionate ways. For example, large avian and mammalian species circulate far more of some immune cells than expected for their size based on simple geometric principles. To date, such hypermetric immune scaling has mostly been described in zoo-dwelling individuals, so it remains obscure whether immune hyper-allometries have any natural relevance. Here, we asked whether granulocyte and lymphocyte allometries in wild birds differ from those described in captive species. Our previous allometric studies of avian immune cell concentrations were performed on animals kept for their lifetimes in captivity where conditions are benign and fairly consistent. In natural conditions, infection, stress, nutrition, climate, and myriad other forces could alter immune traits and hence mask any interspecific scaling relationships between immune cells and body size. Counter to this expectation, we found no evidence that immune cell allometries differed between captive and wild species, although we had to rely on cell proportion data, as insufficient concentration data were available for wild species. Our results indicate that even in variable and challenging natural contexts, immune allometries endure and might affect disease ecology and evolution.

Trade-Offs (and Constraints) in Organismal Biology.

AbstractTrade-offs and constraints are inherent to life, and studies of these phenomena play a central role in both organismal and evolutionary biology. Trade-offs can be defined, categorized, and studied in at least six, not mutually exclusive, ways. (1) Allocation constraints are caused by a limited resource (e.g., energy, time, space, essential nutrients), such that increasing allocation to one component necessarily requires a decrease in another (if only two components are involved, this is referred to as the Y-model, e.g., energy devoted to size versus number of offspring). (2) Functional conflicts occur when features that enhance performance of one task decrease performance of another (e.g., relative lengths of in-levers and out-levers, force-velocity trade-offs related to muscle fiber type composition). (3) Shared biochemical pathways, often involving integrator molecules (e.g., hormones, neurotransmitters, transcription factors), can simultaneously affect multiple traits, with some effects being beneficial for one or more components of Darwinian fitness (e.g., survival, age at first reproduction, fecundity) and others detrimental. (4) Antagonistic pleiotropy describes genetic variants that increase one component of fitness (or a lower-level trait) while simultaneously decreasing another. (5) Ecological circumstances (or selective regime) may impose trade-offs, such as when foraging behavior increases energy availability yet also decreases survival. (6) Sexual selection may lead to the elaboration of (usually male) secondary sexual characters that improve mating success but handicap survival and/or impose energetic costs that reduce other fitness components. Empirical studies of trade-offs often search for negative correlations between two traits that are the expected outcomes of the trade-offs, but this will generally be inadequate if more than two traits are involved and especially for complex physiological networks of interacting traits. Moreover, trade-offs often occur only in populations that are experiencing harsh environmental conditions or energetic challenges at the extremes of phenotypic distributions, such as among individuals or species that have exceptional athletic abilities. Trade-offs may be (partially) circumvented through various compensatory mechanisms, depending on the timescale involved, ranging from acute to evolutionary. Going forward, a pluralistic view of trade-offs and constraints, combined with integrative analyses that cross levels of biological organization and traditional boundaries among disciplines, will enhance the study of evolutionary organismal biology.

Applied ecoimmunology: using immunological tools to improve conservation efforts in a changing world.

Ecoimmunology is a rapidly developing field that explores how the environment shapes immune function, which in turn influences host-parasite relationships and disease outcomes. Host immune defence is a key fitness determinant because it underlies the capacity of animals to resist or tolerate potential infections. Importantly, immune function can be suppressed, depressed, reconfigured or stimulated by exposure to rapidly changing environmental drivers like temperature, pollutants and food availability. Thus, hosts may experience trade-offs resulting from altered investment in immune function under environmental stressors. As such, approaches in ecoimmunology can provide powerful tools to assist in the conservation of wildlife. Here, we provide case studies that explore the diverse ways that ecoimmunology can inform and advance conservation efforts, from understanding how Galapagos finches will fare with introduced parasites, to using methods from human oncology to design vaccines against a transmissible cancer in Tasmanian devils. In addition, we discuss the future of ecoimmunology and present 10 questions that can help guide this emerging field to better inform conservation decisions and biodiversity protection. From better linking changes in immune function to disease outcomes under different environmental conditions, to understanding how individual variation contributes to disease dynamics in wild populations, there is immense potential for ecoimmunology to inform the conservation of imperilled hosts in the face of new and re-emerging pathogens, in addition to improving the detection and management of emerging potential zoonoses.

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.

High Body Temperature is an Unlikely Cause of High Viral Tolerance in Bats.

The global SARS-CoV-2 pandemic and the role of bats in zoonotic spillover have renewed interest in the flight-as-fever hypothesis, which posits that high body temperatures experienced by bats during flight contribute to their high viral tolerance. We argue that flight-as-fever is unlikely to explain why bats harbor more viruses than other mammals on the basis of two lines of reasoning. First, flight temperatures reported in the literature overestimate true flight temperatures because of methodologic limitations. Second, body temperatures in bats are only high relative to humans, and not relative to many other mammals. We provide examples of mammals from diverse habitats to show that temperatures in excess of 40 C during activity are quite common in species with lower viral diversity than bats. We caution scientists against stating the flight-as-fever hypothesis as unquestioned truth, as has repeatedly occurred in the popular media in the wake of the SARS-CoV-2 pandemic.

Cellular metabolism and IL-6 concentrations during stimulated inflammation in primary fibroblasts from small and large dog breeds as they age.

The immune system undergoes marked changes during aging characterized by a state of chronic, low-grade inflammation termed 'inflammaging'. We explore this phenomenon in domestic dogs, which are the most morphologically and physiologically diverse group of mammals, with the widest range in body sizes for a single species. Additionally, smaller dogs tend to live significantly longer than larger dogs across all breeds. Body size is intricately linked to mass-specific metabolism and aging rates, which suggests that dogs are exemplary for studies in inflammaging. Dermal fibroblast cells play an important role in skin inflammation, making them a good model for inflammatory patterns across dog breed, body sizes and ages. Here, we examined aerobic and glycolytic cellular metabolism, and IL-6 concentrations in primary fibroblast cells isolated from small and large dog breeds, that were either recently born puppies or old dogs after death. We found no differences in cellular metabolism when isolated fibroblasts were treated with lipopolysaccharide (LPS) from Escherichia coli to stimulate an inflammatory phenotype. Unlike responses observed in mice and humans, there was a less drastic amplification of IL-6 concentration after LPS treatment in the geriatric population of dogs compared with recently born dogs. In young dogs, we also found evidence that untreated fibroblasts from large breeds had significantly lower IL-6 concentrations than observed for smaller breeds. This implies that the patterns of inflammaging in dogs may be distinct and different from other mammals commonly studied.

The impacts of body mass on immune cell concentrations in birds.

Body mass affects many biological traits, but its impacts on immune defences are fairly unknown. Recent research on mammals found that neutrophil concentrations disproportionately increased (scaled hypermetrically) with body mass, a result not predicted by any existing theory. Although the scaling relationship for mammals might predict how leucocyte concentrations scale with body mass in other vertebrates, vertebrate classes are distinct in many ways that might affect their current and historic interactions with parasites and hence the evolution of their immune systems. Subsequently, here, we asked which existing scaling hypothesis best-predicts relationships between body mass and lymphocyte, eosinophil and heterophil concentrations-the avian functional equivalent of neutrophils-among more than 100 species of birds. We then examined the predictive power of body mass relative to life-history variation, as extensive literature indicates that the timing of key life events has influenced immune system variation among species. Finally, we ask whether avian scaling patterns differ from the patterns we observed in mammals. We found that an intercept-only model best explained lymphocyte and eosinophil concentrations among birds, indicating that the concentrations of these cell types were both independent of body mass. For heterophils, however, body mass explained 31% of the variation in concentrations among species, much more than life-history variation (4%). As with mammalian neutrophils, avian heterophils scaled hypermetrically (b= 0.19 ± 0.05), but more steeply than mammals (approx. 1.5 ×; 0.11 ± 0.03). As such, we discuss why birds might require more broadly protective cells compared to mammals of the same body size. Overall, body mass appears to have strong influences on the architecture of immune systems.

Untangling life span and body mass discrepancies in canids: phylogenetic comparison of oxidative stress in blood from domestic dogs and wild canids.

Canids are a morphological and physiological diverse group of animals, with the most diversity found within one species, the domestic dog. Underlying observed morphological differences, there must also be differences at other levels of organization that could lead to elucidating aging rates and life span disparities between wild and domestic canids. Furthermore, small-breed dogs live significantly longer lives than large-breed dogs, while having higher mass-specific metabolic rates and faster growth rates. At the cellular level, a clear mechanism underlying whole animal traits has not been fully elucidated, although oxidative stress has been implicated as a potential culprit of the disparate life spans of domestic dogs. We used plasma and red blood cells from known aged domestic dogs and wild canids, and measured several oxidative stress variables: total antioxidant capacity (TAC), lipid damage, and enzymatic activities of catalase, superoxide dismutase, and glutathione peroxidase (GPx). We used phylogenetically informed general linear mixed models and nonphylogenetically corrected linear regression analysis. We found that lipid damage increases with age in domestic dogs, whereas TAC increases with age and TAC and GPx activity increases as a function of age/maximum life span in wild canids, which may partly explain longer potential life spans in wolves. As body mass increases, TAC and GPx activity increase in wild canids, but not domestic dogs, highlighting that artificial selection may have decreased antioxidant capacity in domestic dogs. We found that small-breed dogs have significantly higher circulating lipid damage compared with large-breed dogs, concomitant to their high mass-specific metabolism and higher growth rates, but in opposition to their long life spans.

Intercontinental test of constraint-breaking adaptations: Testing behavioural plasticity in the face of a predator with novel hunting strategies.

Constraint-breaking adaptations are evolutionary tools that provide a mechanism for incumbent-replacement between species filling similar ecological roles. In common-garden experiments, we exposed populations of two desert rodents to two different viper species, testing their ability to adjust to novel predators that use different hunting strategies. We aimed to understand whether both predators and prey with constraint-breaking adaptations actually manifest comparative advantage over their counterparts. We used convergent species from desert dunes in the Mojave Desert in North America, Merriam's kangaroo rat Dipodomys merriami and the sidewinder rattlesnake Crotalus cerastes, and from the Negev Desert in the Middle East, the greater Egyptian gerbil Gerbillus pyramidum and the Saharan horned viper Cerastes cerastes. Both Mojave species hold constraint-breaking adaptations in relation to their counterparts from the Negev. The rattlesnakes have heat sensing organs (pits) and the kangaroo rats have fur-lined cheek pouches that allow for greater foraging efficiency and food preservation. Using patch-use theory, we evaluated the rodents' risk-assessment from each snake-separately, together and in combination with barn owls. Initially each rodent species foraged less in the presence of its familiar snake, but within a month both foraged less in the presence of the pit-viper (sidewinder). Our findings indicate a level of learning, and behavioural plasticity, in both rodents and ability to assess the risk from novel predators. The kangaroo rats were capable of harvesting far greater amounts of resources under the same conditions of elevated risk. However, the reason for their advantage may lie in bi-pedal agility and not only their ability collect food more efficiently.

Effects of Selection for Mass-Independent Maximal Metabolic Rate on Food Consumption.

Metabolic rates potentially regulate the pace of important physiological and life-history traits. Natural selection has shaped the evolution of metabolic rates and the physiology that supports them, including digestibility and the rate of food consumption. Understanding the relationship between metabolic rates and energy internalization is central to understanding how resources are allocated among competing physiological functions. We investigated how artificial selection on mass-independent basal metabolic rate (BMR) and mass-independent aerobic maximal metabolic rate (MMR) affected food consumption and apparent digestibility in mice. Evolved changes in mass-corrected BMR-but not mass-corrected MMR-corresponded with changes in food consumption. This result is consistent with previous work showing that BMR constitutes a large portion of an animal's daily energy budget and thus that BMR might provide a better indicator of daily food requirements than MMR. In contrast, digestive efficiencies did not differ among selection treatments and did not evolve in these mice. This study provides insights into how evolution of metabolic rates may affect food consumption and overall energy use.

The Effects of Body Mass on Immune Cell Concentrations of Mammals.

Theory predicts that body mass should affect the way organisms evolve and use immune defenses. We investigated the relationship between body mass and blood neutrophil and lymphocyte concentrations among more than 250 terrestrial mammalian species. We tested whether existing theories (e.g., protecton theory, immune system complexity, and rate of metabolism) accurately predicted the scaling of immune cell concentrations. We also evaluated the predictive power of body mass for these leukocyte concentrations compared to sociality, diet, life history, and phylogenetic relatedness. Phylogeny explained >70% of variation in both lymphocytes and neutrophils, and body mass appeared more informative than other interspecific trait variation. In the best-fit mass-only model, neutrophils scaled hypermetrically (b=0.11) with body mass, whereas lymphocytes scaled just shallow of isometrically. Extrapolating to total cell numbers, this exponent means that an African elephant circulates 13.3 million times the neutrophils of a house mouse, whereas their masses differ by only 250,000-fold. We hypothesize that such high neutrophil numbers might offset the (i) higher overall parasite exposure that large animals face and/or (ii) the higher relative replication capacities of pathogens to host cells.

Multi-Scale Drivers of Immunological Variation and Consequences for Infectious Disease Dynamics.

The immune system is the primary barrier to parasite infection, replication, and transmission following exposure, and variation in immunity can accordingly manifest in heterogeneity in traits that govern population-level infectious disease dynamics. While much work in ecoimmunology has focused on individual-level determinants of host immune defense (e.g., reproductive status and body condition), an ongoing challenge remains to understand the broader evolutionary and ecological contexts of this variation (e.g., phylogenetic relatedness and landscape heterogeneity) and to connect these differences into epidemiological frameworks. Ultimately, such efforts could illuminate general principles about the drivers of host defense and improve predictions and control of infectious disease. Here, we highlight recent work that synthesizes the complex drivers of immunological variation across biological scales of organization and scales these within-host differences to population-level infection outcomes. Such studies note the limitations involved in making species-level comparisons of immune phenotypes, stress the importance of spatial scale for immunology research, showcase several statistical tools for translating within-host data into epidemiological parameters, and provide theoretical frameworks for linking within- and between-host scales of infection processes. Building from these studies, we highlight several promising avenues for continued work, including the application of machine learning tools and phylogenetically controlled meta-analyses to immunology data and quantifying the joint spatial and temporal dependencies in immune defense using range expansions as model systems. We also emphasize the use of organismal traits (e.g., host tolerance, competence, and resistance) as a way to interlink various scales of analysis. Such continued collaboration and disciplinary cross-talk among ecoimmunology, disease ecology, and mathematical modeling will facilitate an improved understanding of the multi-scale drivers and consequences of variation in host defense.

Early Life Conditions and Immune Defense in Nestling Swainson's Hawks.

The quality of perinatal conditions directly influences the physical and immunological development of nestlings, yet it is inherently variable across space and time. Long-term breeding data for a population of Swainson's hawks (Buteo swainsoni) in northern California show a continuum of territory occupancy and productivity values of individual territories and nests. Here we explore effects of variation among territories on immune system development. We hypothesize that nestlings benefitting from favorable conditions will invest in stronger immune systems, a trait with long-term benefits. We used two immunological assays, a bactericidal assay and a hemolytic-complement activity assay, with leukocyte differentials (heterophil∶lymphocyte ratio) to evaluate the constitutive innate immune system. We examined whether early brood-rearing conditions (i.e., number of siblings, hatch date, endoparasite prevalence) were associated with immunological development. Linear mixed-effects models indicated a positive relationship between extended territory occupancy history-an index of habitat quality-and nestling immune function during years with poorer reproduction. There was no association during an exceptionally good reproductive year. Hence, at least under some circumstances, nestling environments or territory characteristics may affect immune function of nestlings. Our study contributes to the growing body of evidence highlighting the importance of facultative allocation to immune traits using long-term demographic data of a top avian predator.