The Promise of a Pointillist Perspective for Comparative Immunology.

Most studies in comparative immunology involve investigations into the detailed mechanisms of the immune system of a non-model organism. Although this approach has been insightful, it has promoted a deep understanding of only a handful of species, thus inhibiting the recognition of broad taxonomic patterns. Here, we call for investigating the immune defenses of numerous species within a pointillist framework, that is, the meticulous, targeted collection of data from dozens species and investigation of broad patterns of organismal, ecological, and evolutionary forces shaping those patterns. Without understanding basic immunological patterns across species, we are limited in our ability to extrapolate and/or translate our findings to other organisms, including humans. We illustrate this point by focusing predominantly on the biological scaling literature with some integrations of the pace of life literature, as these perspectives have been the most developed within this framework. We also highlight how the more traditional approach in comparative immunology works synergistically with a pointillist approach, with each approach feeding back into the other. We conclude that the pointillist approach promises to illuminate comprehensive theories about the immune system and enhance predictions in a wide variety of domains, including host-parasite dynamics and disease ecology.

Bacterial sepsis triggers stronger transcriptomic immune responses in larger primates.

Empirical data relating body mass to immune defence against infections remain limited. Although the metabolic theory of ecology predicts that larger organisms would have weaker immune responses, recent studies have suggested that the opposite may be true. These discoveries have led to the safety factor hypothesis, which proposes that larger organisms have evolved stronger immune defences because they carry greater risks of exposure to pathogens and parasites. In this study, we simulated sepsis by exposing blood from nine primate species to a bacterial lipopolysaccharide (LPS), measured the relative expression of immune and other genes using RNAseq, and fitted phylogenetic models to determine how gene expression was related to body mass. In contrast to non-immune-annotated genes, we discovered hypermetric scaling in the LPS-induced expression of innate immune genes, such that large primates had a disproportionately greater increase in gene expression of immune genes compared to small primates. Hypermetric immune gene expression appears to support the safety factor hypothesis, though this pattern may represent a balanced evolutionary mechanism to compensate for lower per-transcript immunological effectiveness. This study contributes to the growing body of immune allometry research, highlighting its importance in understanding the complex interplay between body size and immunity over evolutionary timescales.

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.

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.

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.

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.

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.

Macroevolutionary Patterning in Glucocorticoids Suggests Different Selective Pressures Shape Baseline and Stress-Induced Levels.

Glucocorticoid (GC) hormones are important phenotypic mediators across vertebrates, but their circulating concentrations can vary markedly. Here we investigate macroevolutionary patterning in GC levels across tetrapods by testing seven specific hypotheses about GC variation and evaluating whether the supported hypotheses reveal consistent patterns in GC evolution. If selection generally favors the "supportive" role of GCs in responding effectively to challenges, then baseline and/or stress-induced GCs may be higher in challenging contexts. Alternatively, if selection generally favors "protection" from GC-induced costs, GCs may be lower in environments where challenges are more common or severe. The predictors of baseline GCs were all consistent with supportive effects: levels were higher in smaller organisms and in those inhabiting more energetically demanding environments. During breeding, baseline GCs were also higher in populations and species with fewer lifetime opportunities to reproduce. The predictors of stress-induced GCs were instead more consistent with the protection hypothesis: during breeding, levels were lower in organisms with fewer lifetime reproductive opportunities. Overall, these patterns indicate a surprising degree of consistency in how some selective pressures shape GCs across broad taxonomic scales; at the same time, in challenging environments selection appears to operate on baseline and stress-induced GCs in distinct ways.

Speeding up Growth: Selection for Mass-Independent Maximal Metabolic Rate Alters Growth Rates.

Investigations into relationships between life-history traits, such as growth rate and energy metabolism, typically focus on basal metabolic rate (BMR). In contrast, investigators rarely examine maximal metabolic rate (MMR) as a relevant metric of energy metabolism, even though it indicates the maximal capacity to metabolize energy aerobically, and hence it might also be important in trade-offs. We studied the relationship between energy metabolism and growth in mice (Mus musculus domesticus Linnaeus) selected for high mass-independent metabolic rates. Selection for high mass-independent MMR increased maximal growth rate, increased body mass at 20 weeks of age, and generally altered growth patterns in both male and female mice. In contrast, there was little evidence that the correlated response in mass-adjusted BMR altered growth patterns. The relationship between mass-adjusted MMR and growth rate indicates that MMR is an important mediator of life histories. Studies investigating associations between energy metabolism and life histories should consider MMR because it is potentially as important in understanding life history as BMR.

Flea fitness is reduced by high fractional concentrations of CO2 that simulate levels found in their hosts' burrows.

Nidicolous ectoparasites such as fleas and gamasid mites that feed on small and medium-sized mammals spend much of their time in their hosts' burrows, which provide an environment for living, and often feeding, to their pre-imaginal and/or adult stages. Thus, these ectoparasites should be adapted to environmental conditions in burrows, including high fractional concentrations of CO2 (F(CO2)). We examined how a high F(CO2) (0.04) affected survival and reproductive success of a hematophagous ectoparasite of burrowing rodents using fleas Xenopsylla ramesis and Sundevall's jirds Meriones crassus. In the first experiment, fleas fed on hosts housed in high-CO2 (F(CO2) =0.04) or atmospheric-CO2 (F(CO2) ≈0.0004) air, and were allowed to breed. In a second experiment, fleas were maintained in high CO2 or CO2-free air with no hosts to determine how CO2 levels affect survival and activity levels. We found that at high F(CO2) fleas laid fewer eggs, reducing reproductive success. In addition, at high F(CO2), activity levels and survival of fleas were reduced. Our results indicate that fleas do not perform well in the F(CO2) used in this experiment. Previous research indicated that the type and intensity of the effects of CO2 concentration on the fitness of an insect depend on the F(CO2) used, so we advise caution when generalizing inferences drawn to insects exposed to other F(CO2). If, however, F(CO2) found in natural mammal burrows brings about reduced fitness in fleas in general, then burrowing hosts may benefit from reduced parasite infestation if burrow air F(CO2) is high.

Ectoparasite performance when feeding on reproducing mammalian females: an unexpected decrease when on pregnant hosts.

Reproduction is an energy-demanding activity in mammalian females, with increased energy requirements during pregnancy and, especially, during lactation. To better understand the interactions between parasitism and host reproduction, we investigated feeding and reproductive performance of fleas (Xenopsylla ramesis) parasitizing non-reproducing, pregnant or lactating gerbilline rodents (Meriones crassus). Based on energetic considerations, we predicted that feeding and reproductive performance of fleas would be lowest on non-breeding females, moderate on pregnant females and highest on lactating females. We estimated feeding performance of the fleas via absolute and mass-specific bloodmeal size and reproductive performance via egg production and latency to peak oviposition. Host reproductive status had no effect on either absolute or mass-specific bloodmeal size or the day of peak oviposition, but significantly affected the daily number of eggs produced by a female flea. Surprisingly, and contrary to our predictions, egg production of fleas fed on pregnant rodents was significantly lower than that of fleas on non-reproducing and lactating rodents, while no difference in egg production between fleas feeding on non-reproducing and lactating hosts was found. Our results suggest that differences in parasite reproduction when feeding on hosts of different reproductive status are not associated with the different energy requirements of the hosts at non-breeding, pregnancy and lactation but rather with variation in hormonal and/or immune status during these periods.

Host reproductive status and reproductive performance of a parasite: offspring quality and trade-offs in a flea parasitic on a rodent.

We investigated offspring quality in fleas (Xenopsylla ramesis) feeding on non-reproducing, pregnant or lactating rodents (Meriones crassus) and asked whether (a) quality of flea offspring differs dependent on host reproductive status; (b) fleas trade off offspring quantity for quality; and (c) quality variables are inter-correlated. Emergence success was highest when parents exploited pregnant hosts, while development time was longest when parents exploited lactating hosts. Male offspring from fleas fed on non-reproductive and pregnant hosts were larger than those from lactating hosts whereas female offspring from fleas fed on pregnant hosts were larger than those from both lactating and non-reproductive hosts. Male offspring survived under starvation the longest when their parents exploited lactating hosts and the shortest when their parents exploited pregnant hosts. Female offspring of parents that exploited lactating hosts survived under starvation longer than those that exploited non-reproductive and pregnant hosts. Emergence success and development time decreased as mean number of eggs laid by mothers increased. Fleas that were larger and took longer to develop lived significantly longer under starvation. These results indicate the presence of a trade-off between offspring quantity and quality in fleas exploiting female Sundevall's jird in varying reproductive condition but this trade-off depended on the quality trait considered.

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.

Selection for increased mass-independent maximal metabolic rate suppresses innate but not adaptive immune function.

Both appropriate metabolic rates and sufficient immune function are essential for survival. Consequently, eco-immunologists have hypothesized that animals may experience trade-offs between metabolic rates and immune function. Previous work has focused on how basal metabolic rate (BMR) may trade-off with immune function, but maximal metabolic rate (MMR), the upper limit to aerobic activity, might also trade-off with immune function. We used mice artificially selected for high mass-independent MMR to test for trade-offs with immune function. We assessed (i) innate immune function by quantifying cytokine production in response to injection with lipopolysaccharide and (ii) adaptive immune function by measuring antibody production in response to injection with keyhole limpet haemocyanin. Selection for high mass-independent MMR suppressed innate immune function, but not adaptive immune function. However, analyses at the individual level also indicate a negative correlation between MMR and adaptive immune function. By contrast BMR did not affect immune function. Evolutionarily, natural selection may favour increasing MMR to enhance aerobic performance and endurance, but the benefits of high MMR may be offset by impaired immune function. This result could be important in understanding the selective factors acting on the evolution of metabolic rates.

Immune response to a Trichinella spiralis infection in house mice from lines selectively bred for high voluntary wheel running.

Four lines of mice bred for high voluntary wheel running (HR lines) have high baseline circulating corticosterone levels and increased daily energy expenditure as compared with four non-selected control (C) lines. High corticosterone may suppress immune function and competing energy demands may limit ability to mount an immune response. We hypothesized that HR mice have a reduced immune response and therefore a decreased ability to fight an infection by Trichinella spiralis, an ecologically relevant nematode common in mammals. Infections have an acute, intestinal phase while the nematode is migrating, reproducing and traveling throughout the bloodstream, followed by a chronic phase with larvae encysted in muscles. Adult males (generation 55 of the selection experiment) were sham-infected or infected by oral gavage with ~300 J1 T. spiralis larvae. During the chronic phase of infection, mice were given wheel access for 6 days, followed by 2 days of maximum aerobic performance trials. Two weeks post-infection, infected HR had significantly lower circulating immunoglobulin E levels compared with infected C mice. However, we found no statistical difference between infected HR and C mice in numbers of encysted larvae within the diaphragm. As expected, both voluntary running and maximum aerobic performance were significantly higher in HR mice and lower in infected mice, with no line type-by-infection interactions. Results complement those of previous studies suggesting decreased locomotor abilities during the chronic phase of T. spiralis infection. However, despite reduced antibody production, breeding for high voluntary wheel exercise does not appear to have a substantial negative impact on general humoral function.

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.

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.