Ecoimmunological Studies Research Articles

Constitutive innate immune defenses in relation to urbanization and population density in an urban bird, the feral pigeon Columba livia domestica.

Urbanization processes modulate the immunological challenges faced by animals. Urban habitat transformations reshape pathogen diversity and abundance, while high population density-common in urban exploiter species-promotes disease transmission. Responses to urbanization may include adaptive adjustments of constitutive innate immune defenses (e.g. complement system and natural antibodies [NAbs]), which serve as first-line protection against infections. Here, we investigated associations of habitat urbanization and host population density with complement and NAbs in an urban bird, the feral pigeon Columba livia domestica. To do so, we employed the hemolysis-hemagglutination assay to analyze nearly 200 plasma samples collected across urbanization and pigeon population density gradients in five major cities in Poland. We found a negative association between urbanization score and hemagglutination (i.e. NAbs activity), but not hemolysis (i.e. complement activity), indicating either immunosuppression or adaptive downregulation of this immune defense in highly transformed urban landscape. Population density was not significantly related to either immune parameter, providing no evidence for density-dependent modulation of immune defenses. At the same time, there was a negative association of hemolysis with condition (scaled mass index), suggesting resource allocation trade-offs or contrasting effects of the urban environment on immune defenses and body condition. The results demonstrate that habitat structure can be an important factor shaping the immune defenses of the feral pigeon, although these associations were not mediated by variation in population density. Our study highlights the complexity of the links between immune defenses in wildlife and urbanization and reinforces the need for comprehensive ecoimmunological studies on urban animals.

In sickness and in health: the dynamics of the fruit bat gut microbiota under a bacterial antigen challenge and its association with the immune response.

Interactions between the gut microbiome (GM) and the immune system influence host health and fitness. However, few studies have investigated this link and GM dynamics during disease in wild species. Bats (Mammalia: Chiroptera) have an exceptional ability to cope with intracellular pathogens and a unique GM adapted to powered flight. Yet, the contribution of the GM to bat health, especially immunity, or how it is affected by disease, remains unknown. Here, we examined the dynamics of the Egyptian fruit bats' (Rousettus aegyptiacus) GM during health and disease. We provoked an inflammatory response in bats using lipopolysaccharides (LPS), an endotoxin of Gram-negative bacteria. We then measured the inflammatory marker haptoglobin, a major acute phase protein in bats, and analyzed the GM (anal swabs) of control and challenged bats using high-throughput 16S rRNA sequencing, before the challenge, 24h and 48h post challenge. We revealed that the antigen challenge causes a shift in the composition of the bat GM (e.g., Weissella, Escherichia, Streptococcus). This shift was significantly correlated with haptoglobin concentration, but more strongly with sampling time. Eleven bacterial sequences were correlated with haptoglobin concentration and nine were found to be potential predictors of the strength of the immune response, and implicit of infection severity, notably Weissella and Escherichia. The bat GM showed high resilience, regaining the colony's group GM composition rapidly, as bats resumed foraging and social activities. Our results demonstrate a tight link between bat immune response and changes in their GM, and emphasize the importance of integrating microbial ecology in ecoimmunological studies of wild species. The resilience of the GM may provide this species with an adaptive advantage to cope with infections and maintain colony health.

Substrate specificity of phenoloxidase-like activity in an ecoimmunological model speciesLymnaea stagnalis

ABSTRACTEcoimmunological research on molluscs and other invertebrates frequently quantifies phenoloxidase (PO) activity to estimate the strength of the immune function. PO enzymes form different families whose relative roles in oxidative reactions are typically unknown. Understanding this could allow enzyme-specific assays with higher accuracy than in commonly used nonspecific assays. We tested the contribution of different PO enzyme families to haemolymph PO-like activity in Lymnaea stagnalis snails using substrates specific to enzymes detected in L. stagnalis transcriptome data (p-phenylenediamine, specific to laccases; L-tyrosine, specific to tyrosinases) and compared the reactions to those with a nonspecific substrate (L-dopa). We found laccase-like but no tyrosinase-like activity. However, reactions with L-dopa were the strongest, possibly due to other oxidative enzymes in snail haemolymph. Laccase-like activity is common in molluscs, and we propose the use of enzyme-specific assays in future ecoimmunological studies of this taxon. The lack of tyrosinase-like activity in L. stagnalis contradicts earlier transcriptome data, which calls for investigating the expression of PO enzymes in L. stagnalis at the proteome level.

Ecoimmunology: What Unconventional Organisms Tell Us after Two Decades.

Model organisms have long been used to characterize the vertebrate immune system, which is responsible for protecting the host from mechanical and pathogen-induced injuries, pathology, and disease development (Janeway et al. 2001). As a result, today we have a deep understanding of how vertebrate defensive responses can be mediated by innate (i.e., non-specific) or adaptive (i.e., specific) mechanisms, or a mixture of both. Innate responses involve a generalized activation of immune mechanisms that can operate in response to any infectious agent (i.e., they are actionable to a diverse range of pathogens and parasites; Pham and Schneider 2008). In contrast, adaptive responses entail an activation of a subset of cells and/or systems to defend a host against a precise pathogen. While many of these mechanisms are highly conserved across vertebrate classes, intriguing distinctions have been revealed by research on non-model organisms (Demas and Nelson 2012). Ecoimmunology, or ecological immunology, emerged as a research area in the late 1990s and early 2000s (Sheldon and Verhulst 1996; Norris and Evans 2000; Zuk and Stoehr 2002; Brock et al. 2014; Downs et al. 2014). In this field, researchers seek to understand the factors leading to variation in the function of the immune system and how this variation contributes to disease susceptibility in a wide variety of taxonomic groups (Sheldon and Verhulst 1996; Norris and Evans 2000; Zuk and Stoehr 2002; Brock et al. 2014; Downs et al. 2014). Since its inception, ecoimmunological studies have predominantly focused on birds, with relatively fewer studies focused on other taxonomic groups (Martin et al. 2003; Ashley et al. 2012; Evans et al. 2015; Haase et al. 2016; Taves et al. 2017; Tieleman 2018). More recently, however, there has been an expansion in the diversity of taxa studied from an ecoimmunological perspective, with studies ranging from plants to invertebrates, ectothermic vertebrates, and non-model (i.e., lab rodents) mammalian species (Rolff and Siva-Jothy 2003; Zimmerman et al. 2010; Ardia et al. 2012; Fisher et al. 2012; Pijanowski et al. 2015; Titon et al. 2019; Sueiro et al. 2020; Zimmerman 2020). In addition, there has been a closer examination and optimization of the immune methods and techniques that are needed to conduct ecoimmunology studies in diverse organisms (e.g., Claunch et al. 2022).

Oxidative damage increases with degree of simulated bacterial infection, but not ectoparasitism, in tree swallow nestlings.

The purpose of mounting an immune response is to destroy pathogens, but this response comes at a physiological cost, including the generation of oxidative damage. However, many studies on the effects of immune challenges employ a single high dose of a simulated infection, meaning that the consequences of more mild immune challenges are poorly understood. We tested whether the degree of immunological challenge in tree swallows (Tachycineta bicolor) affects oxidative physiology and body mass, and whether these metrics correlate with parasitic nest mite load. We injected 14day old nestlings with 0, 0.01, 0.1 or 1 mg lipopolysaccharide (LPS) per kg body mass, then collected a blood sample 24h later to quantify multiple physiological metrics, including oxidative damage (i.e. d-ROMs), circulating amounts of triglyceride and glycerol, and levels of the acute phase protein haptoglobin. After birds had fledged, we identified and counted parasitic nest mites (Dermanyssus spp. and Ornithonyssus spp.). We found that only nestlings injected with 1 mg LPSkg-1 body mass, which is a common dosage in ecoimmunological studies, lost more body mass than individuals from other treatment groups. However, every dose of LPS resulted in a commensurate increase in oxidative damage. Parasitic mite abundance had no effect on oxidative damage across treatments. The amount of oxidative damage correlated with haptoglobin levels, suggesting compensatory mechanisms to limit self-damage during an immune response. We conclude that while only the highest-intensity immune challenges resulted in costs related to body mass, even low-intensity immune challenges result in detectable increases in oxidative damage.

Examining adaptive evolution of immune activity: opportunities provided by gastropods in the age of 'omics'.

Parasites threaten all free-living organisms, including molluscs. Understanding the evolution of immune defence traits in natural host populations is crucial for predicting their long-term performance under continuous infection risk. Adaptive trait evolution requires that traits are subject to selection (i.e. contribute to organismal fitness) and that they are heritable. Despite broad interest in the evolutionary ecology of immune activity in animals, the understanding of selection on and evolutionary potential of immune defence traits is far from comprehensive. For instance, empirical observations are only rarely in line with theoretical predictions of immune activity being subject to stabilizing selection. This discrepancy may be because ecoimmunological studies can typically cover only a fraction of the complexity of an animal immune system. Similarly, molecular immunology/immunogenetics studies provide a mechanistic understanding of immunity, but neglect variation that arises from natural genetic differences among individuals and from environmental conditions. Here, we review the current literature on natural selection on and evolutionary potential of immune traits in animals, signal how merging ecological immunology and genomics will strengthen evolutionary ecological research on immunity, and indicate research opportunities for molluscan gastropods for which well-established ecological understanding and/or 'immune-omics' resources are already available. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Transcriptome profiling of Lymnaea stagnalis (Gastropoda) for ecoimmunological research

BackgroundHost immune function can contribute to numerous ecological/evolutionary processes. Ecoimmunological studies, however, typically use one/few phenotypic immune assays and thus do not consider the complexity of the immune system. Therefore, “omics” resources that allow quantifying immune activity across multiple pathways are needed for ecoimmunological models. We applied short-read based RNAseq (Illumina NextSeq 500, PE-81) to characterise transcriptome profiles of Lymnaea stagnalis (Gastropoda), a multipurpose model snail species. We used a genetically diverse snail stock and exposed individuals to immune elicitors (injury, bacterial/trematode pathogens) and changes in environmental conditions that can alter immune activity (temperature, food availability).ResultsImmune defence factors identified in the de novo assembly covered elements broadly described in other gastropods. For instance, pathogen-recognition receptors (PRR) and lectins activate Toll-like receptor (TLR) pathway and cytokines that regulate cellular and humoral defences. Surprisingly, only modest diversity of antimicrobial peptides and fibrinogen related proteins were detected when compared with other taxa. Additionally, multiple defence factors that may contribute to the phenotypic immune assays used to quantify antibacterial activity and phenoloxidase (PO)/melanisation-type reaction in this species were found. Experimental treatments revealed factors from non-self recognition (lectins) and signalling (TLR pathway, cytokines) to effectors (e.g., antibacterial proteins, PO enzymes) whose transcription depended on immune stimuli and environmental conditions, as well as components of snail physiology/metabolism that may drive these effects. Interestingly, the transcription of many factors (e.g., PRR, lectins, cytokines, PO enzymes, antibacterial proteins) showed high among-individual variation.ConclusionsOur results indicate several uniform aspects of gastropod immunity, but also apparent differences between L. stagnalis and some previously examined taxa. Interestingly, in addition to immune defence factors that responded to immune elicitors and changes in environmental conditions, many factors showed high among-individual variation across experimental snails. We propose that such factors are highly important to be included in future ecoimmunological studies because they may be the key determinants of differences in parasite resistance among individuals both within and between natural snail populations.

Quantifying phagocytic lymphocytes in ectothermic vertebrates: A simplified technique for assessing immune function

Abstract Many measures of immune function used in ecoimmunological studies were optimized for endotherms and may not adequately characterize immunocompetence in ectothermic species. Lymphocytes are interpreted as functioning in adaptive immunity despite evidence that large proportions of these cells have innate immune functions, including phagocytosis, in ectotherms. We modified previously published protocols to remove many barriers that inhibited the wide application of assays to quantify phagocytic activity in lymphocytes. These barriers included the use of species‐specific reagents, high cost and incompatibility with field work. We statistically compared published methods to our simplified technique using blood samples collected from tortoises. We show the efficacy of the new technique and offer an outline for optimizing these methods for different species and to address a wide variety of ecoimmunological questions. A full understanding of immune function in ectothermic vertebrates is needed to comprehend complex epidemiological patterns. As our assay disentangles the relationship between effector cells (lymphocytes) and their function (phagocytosis), these methods provide a foundation for more thorough analyses of ecoimmunological patterns.

Genetic background and thermal environment differentially influence the ontogeny of immune components during early life in an ectothermic vertebrate.

An understudied aspect of vertebrate ecoimmunology has been the relative contributions of environmental factors (E), genetic background (G) and their interaction (G×E) in shaping immune development and function. Environmental temperature is known to affect many aspects of immune function and alterations in temperature regimes have been implicated in emergent disease outbreaks, making it a critical environmental factor to study in the context of immune phenotype determinants of wild animals. We assessed the relative influences of environmental temperature, genetic background and their interaction on first-year development of innate and adaptive immune defences of captive-born garter snakes Thamnophis elegans using a reciprocal transplant laboratory experiment. We used a full-factorial design with snakes from two divergent life-history ecotypes, which are known to differ in immune function in their native habitats, raised under conditions mimicking the natural thermal regime-that is, warmer and cooler-of each habitat. Genetic background (ecotype) and thermal regime influenced innate and adaptive immune parameters of snakes, but in an immune-component specific manner. We found some evidence of G×E interactions but no indication of adaptive plasticity with respect to thermal environment. At the individual level, the effects of thermal environment on resource allocation decisions varied between the fast- and the slow-paced life-history ecotypes. Under warmer conditions, which increased food consumption of individuals in both ecotypes, the former invested mostly in growth, whereas the latter invested more evenly between growth and immune development. Overall, immune parameters were highly flexible, but results suggest that other environmental factors are likely more important than temperature per se in driving the ecotype differences in immunity previously documented in the snakes under field conditions. Our results also add to the understanding of investment in immune development and growth during early postnatal life under different thermal environments. Our finding of immune-component specific patterns strongly cautions against oversimplification of the highly complex immune system in ecoimmunological studies. In conjunction, these results deepen our understanding of the degree of immunological flexibility wild animals present, information that is ever more vital in the context of rapid global environmental change.

Low-dose immune challenges result in detectable levels of oxidative damage.

Infection can result in substantial costs to animals, so they frequently respond by removing infectious agents with an immune response. However, immune responses entail their own costs, including upregulation of processes that destroy pathogens (e.g. the production of reactive oxygen species) and processes that limit the extent of self-damage during the immune response (e.g. production of anti-inflammatory proteins such as haptoglobin). Here, we simulated bacterial infection across a 1000-fold range using lipopolysaccharide (LPS) administered to northern bobwhite quail (Colinus virginianus), and quantified metrics related to pro-inflammatory conditions [i.e. generation of oxidative damage (d-ROMs), depletion of antioxidant capacity], anti-inflammatory mechanisms (i.e. production of haptoglobin, expression of the enzyme heme oxygenase, production of the organic molecule biliverdin) and nutritional physiology (e.g. circulating triglyceride levels, maintenance of body mass). We detected increases in levels of haptoglobin and d-ROMs even at LPS doses that are 1/1000th the concentration of doses frequently used in ecoimmunological studies, while loss of body mass and decreases in circulating triglycerides manifested only in individuals receiving the highest dose of LPS (1 mg LPSkg-1 body mass), highlighting variation among dose-dependent responses. Additionally, individuals that lost body mass during the course of the experiment had lower levels of circulating triglycerides, and those with more oxidative damage had greater levels of heme oxygenase expression, which highlights the complex interplay between pro- and anti-inflammatory processes. Because low doses of LPS may simulate natural infection levels, variation in dose-dependent physiological responses may be particularly important in modeling how free-living animals navigate immune challenges.

Do Life History Traits Influence Patterns of Maternal Immune Elements in New World Blackbirds (Icteridae)?

AbstractAvian immunology developed originally by investigating domesticated poultry species (Galliformes), but in recent decades eco-immunological studies of wild bird species have revealed that avian immune systems are more diverse than initially assumed. This study compares six immunological elements in eggs of six species within the same family, the New World blackbirds (Icteridae),whose members differ most notably in two life history parameters, brood parasitism and body size. We measured the maternal immune investment of passive immune components in both yolk and albumen: lysozyme, ovotransferrin, and immunoglobulins (Igs), and LPS-specific Igs. We predicted that brood parasites would have higher levels of immune activity for both innate and adaptive immunity compared with non-brood parasites, and that increased body size could increase microbial exposure of larger animals, resulting in an increase in some adaptive immune responses, such as LPS-specific Igs. We found that brood parasites had significantly higher levels of Igs and lysozyme levels in albumen, but significantly lower levels of Igs in yolk compared with non-brood parasites. Igs in yolk scaled according to body size, with the smallest organisms (the brood parasites) having the lowest levels, and the largest organism (common grackle) having the highest. Our results confirm the findings of other studies of comparative immunity among species in a single taxon that (1) similarities in immune investment cannot be assumed among closely related species and (2) single measures of immune defense cannot be assumed to be indicators of a species’ overall immune strategy, as life history traits can differentially affect immune responses.

Effect of sex and reproductive status on the immunity of the temperate bat Myotis daubentonii

Studies of immunity in bat species are rare. However, it is important to determine immunological variations to identify factors influencing the health status of these endangered mammals from an evolutionary, ecological, conservation, and public health point of view. Immunity is highly variable and can be influenced by both internal (e.g. hormone levels, energy demand) and external factors (e.g. pathogens, climate). As bats have some peculiar ecological, energetic, and putative immunological characteristics, they are outstanding study organisms for ecoimmunological studies. We tested if (i) female bats have a higher immunity than males similar to most other mammalian species and (ii) individuals differ according to their energy demand (e.g. reproductive status). To study these questions, we sampled female and male Myotis daubentonii with different reproductive states and estimated their bacterial killing activity, hemolysis/hemagglutination titer, immunoglobulin G (IgG) concentration, and total and differential white blood cell counts. These methods characterize the cellular and humoral branches of both the adaptive and the innate immune responses of these individuals. Reproductively active males had lower cellular immunity compared to non-reproductive individuals. Pregnant females had increased IgG concentrations while hemolysis was enhanced during lactation. No clear trade-off between immunity and reproduction was found; instead immunity of males and female bats seems to be modulated differently due to varying hormonal and energetic states. Our data suggest that both adaptive and innate immunity as well as individual differences (i.e. sex and reproductive state) need to be considered to get a comprehensive overall picture of immunity in wild mammals.

Parasite-altered feeding behavior in insects: integrating functional and mechanistic research frontiers.

Research on parasite-altered feeding behavior in insects is contributing to an emerging literature that considers possible adaptive consequences of altered feeding behavior for the host or the parasite. Several recent ecoimmunological studies show that insects can adaptively alter their foraging behavior in response to parasitism. Another body of recent work shows that infection by parasites can change the behavior of insect hosts to benefit the parasite; manipulations of host feeding behavior may be part of this phenomenon. Here, we address both the functional and the underlying physiological frontiers of parasite-altered feeding behavior in order to spur research that better integrates the two. Functional categories of parasite-altered behavior that are adaptive for the host include prophylaxis, therapy and compensation, while host manipulation is adaptive for the parasite. To better understand and distinguish prophylaxis, therapy and compensation, further study of physiological feedbacks affecting host sensory systems is especially needed. For host manipulation in particular, research on mechanisms by which parasites control host feedbacks will be important to integrate with functional approaches. We see this integration as critical to advancing the field of parasite-altered feeding behavior, which may be common in insects and consequential for human and environmental health.

Immune responsiveness to phytohemagglutinin displays species but not sex differences in three anuran species.

Phytohemagglutinin (PHA)-induced skin swelling response is widely used as a rough surrogate of integrative cell-mediated and innate immunity across multiple vertebrate taxa due to its simplification and feasibility. However, little is known whether there are sex and interspecific differences of immune responsiveness to PHA in ectotherms, especially for anurans. Therefore, we studied sex and species differences of PHA response in three anurans, Asiatic toads (Bufo gargarizans), Dark-spotted frogs (Pelophylax nigromaculatus) and Mongolian toads (Pseudepidalea raddei), captured in northern regions of Anhui Province (China). Footpad thickness was measured prior to (0 h) and after (6, 12, 24, 48 and 72 h) a PHA injection and normalized against saline injection in the opposite footpad. Body mass was recorded at the beginning (0 h) and end of each assay (72 h). Results showed effects of PHA assay, sex and taxa on body mass. Relative maximum swelling response (PHAmax) ranged from 18.58–29.75%, 9.77 to 20.56% and 21.97 to 31.78% and its occurrence over time was apparent 10.6–19.72 h , 7.74–14.01 h and 17.39–23.94 h postinjection for Asiatic toads, Dark-spotted frogs and Mongolian toads, respectively. Finally, the magnitude or timing of PHAmax in Dark-spotted frogs was significantly thinner and faster than in Mongolian toads, and Asiatic toads had an in-between value, not different from the other two species. The magnitude of PHAmax was significantly positively correlated with the timing of PHAmax considering individuals altogether, but not when analyzed within species. Our results indicate that male and female anuran species respond similarly to PHA antigen stimulation, but the magnitude and timing of PHAmax is species-specific. Briefly, we provide new evidence for the suitability of PHA assay in non-model anuran species with different body sizes, and exhort the need to further investigate the nature of PHA assay at the hematological and histological levels in order to extend its application in ecoimmunological studies of amphibians.

Parasite infection negatively affects PHA-triggered inflammation in the subterranean rodent Ctenomys talarum.

Magnitude and effectiveness of immune responses vary greatly between and within species. Among factors reported to determine this variation, parasitism is a critical one, although controversial effects of parasites over immunological indices have been reported. Information regarding immune strategies in species with different life histories is crucial to better understand the role of immune defenses in an ecological and evolutionary context. Here, we examine the influence of the parasite community on immune responsiveness of a solitary subterranean rodent, Ctenomys talarum. To do this, we assessed the impact of the natural parasite community and the experimental infection with Eimeria sp. on the phytohemagglutinin (PHA)-response, as well as other immune, condition, nutrition, and stress parameters. PHA-triggered inflammation was similarly impaired by Eimeria sp. infection alone or co-occurring with a number of gastrointestinal nematodes. None of the other physiological parameters studied were affected by parasitism. This indicates that parasitism is a general key factor modulating immune responsiveness of the host, and in particular for C. talarum, it could explain the great inter-individual variation previously observed in the PHA-response. Thus, our results highlight the importance of taking the parasite community into account in ecoimmunological studies, particularly when using immunological indices.

Fecal antibody levels as a noninvasive method for measuring immunity to gastrointestinal nematodes in ecological studies.

Among-individual variation in antibody-associated immunity to gastrointestinal nematode parasites (GIN) is known be associated with life-history traits and vital rates in wild vertebrate systems. To date, measurement of levels of antibodies against GIN antigens in natural populations has exclusively been based on invasive blood sampling techniques. Previous work in laboratory rodents and ruminant livestock suggests that antibody measures from feces may provide a viable noninvasive approach. We measured total and anti-GIN antibodies of different isotypes (immunoglobulin (Ig) G, IgA and IgE) from paired samples of plasma and feces from free-living Soay sheep of different ages and sexes. We tested the correlations among these measures as well as their associations with body mass and Strongyle nematode fecal egg counts (FEC). Significant positive correlations were present among plasma and fecal anti-GIN antibody levels for IgG and IgA. Generally, correlations between total antibody levels in plasma and feces were weaker and not significant. No significant relationships were found between any antibody measures and body mass; however, fecal anti-GIN antibody levels were significantly negatively correlated with FEC. Our data clearly demonstrate the feasibility of measuring anti-GIN antibodies from fecal samples collected in natural populations. Although associations of fecal antibody levels with their plasma counterparts and FEC were relatively weak, the presence of significant correlations in the predicted direction in a relatively small and heterogeneous sample suggests fecal antibody measures could be a useful, noninvasive addition to current eco-immunological studies.

Beyond mice and men: environmental change, immunity and infections in wild ungulates.

In the face of rapid environmental change, anticipating shifts in microparasite and macroparasite dynamics, including emergence events, is an enormous challenge. We argue that immunological studies in natural populations are pivotal to meeting this challenge: many components of environmental change--shifts in biotic assemblages, altered climate patterns and reduced environmental predictability--may affect host immunity. We suggest that wild ungulates can serve as model systems aiding the discovery of immunological mechanisms that link environmental change with parasite transmission dynamics. Our review of eco-immunological studies in wild ungulates reveals progress in understanding how co-infections affect immunity and parasite transmission and how environmental and genetic factors interact to shape immunity. Changes in bioavailability of micronutrients have been linked to immunity and health in wild ungulates. Although physiological stress in response to environmental change has been assessed, downstream effects on immunity have not been studied. Moreover, the taxonomic range of ungulates studied is limited to bovids (bighorn sheep, Soay sheep, chamois, musk oxen, bison, African buffalo) and a few cervids (red deer, black-tailed deer). We discuss areas where future studies in ungulates could lead to significant contributions in understanding the patterns of immunity and infection in natural populations and across species.

Activation of an inflammatory response is context-dependent during early development of the California sea lion.

Variations in immune function can arise owing to trade-offs, that is, the allocation of limited resources among costly competing physiological functions. Nevertheless, there is little information regarding the ontogeny of the immune system within an ecological context, and it is still unknown whether development affects the way in which resources are allocated to different immune effectors. We investigated changes in the inflammatory response during early development of the California sea lion (Zalophus californianus) and examined its association with body condition, as a proxy for the availability of energetic resources. We found that the relationship between inflammation and body condition varied according to developmental stage and circulating levels of leucocyte populations, a proxy for current infection. Body condition was related to the magnitude of the inflammatory response during two of the three developmental periods assessed, allowing for the possibility that the availability of pup energetic reserves can limit immune function. For older pups, the ability to mount an inflammatory response was related to their circulating levels of neutrophils and the neutrophil to lymphocyte ratio, implying that the infection status of an individual will influence its ability to respond to a new challenge. Our results suggest that trade-offs may occur within the immune system and highlight the importance of taking into account ontogeny in ecoimmunological studies.

No short-term effect of handling and capture stress on immune responses of bats assessed by bacterial killing assay

Ecoimmunology of wild animals becomes increasingly important. However, there are methodical limitations, especially when working on small mammals, e.g. small sample volume and acute stress associated with capture, handling and sampling that can influence immune parameters. The plasma bacterial killing assay measures innate humoral immune responses, mainly complement activity. It is a powerful tool with many methodical advantages. To avoid investigation of artefacts in future ecoimmunological studies the influence of acute stress on the bacterial killing activity was assessed.Bats (Nyctalus noctula, n=9) were repeatedly sampled in three time intervals up to 97min after capture. Bacterial killing activity against Escherichia coli was measured using a microplate absorbance reader. Bacterial killing activity was not influenced by capture, handling and sampling. Hence, released stress hormones did not affect circulating complement activity. To conclude, the plasma bacterial killing assay is reliable and efficient ecoimmunological tool in wildlife studies even of small mammals.

Optimization, validation and efficacy of the phytohaemagglutinin inflammation assay for use in ecoimmunological studies of amphibians.

The global amphibian biodiversity crisis is driven by disease, habitat destruction and drastically altered ecosystems. It has given rise to an unprecedented need to understand the link between rapidly changing environments, immunocompetence and wildlife health (the nascent field of ecoimmunology). Increasing our knowledge of the ecoimmunology of amphibians necessitates the development of reliable, field-applicable methods of assessing immunocompetence in non-model species. The phytohaemagglutinin (PHA) inflammation assay uses a lectin to elicit localized inflammation that reflects an organism's capacity to mount an immune response. Although extensively used in birds to assess responses to environmental change, stress and disease, its application in amphibians has been extremely limited. We developed, validated and optimized a practical and effective phytohaemagglutinin inflammation assay in phylogenetically distant amphibians and demonstrated its suitability for use in a wide range of ecoimmunological studies. The protocol was effective for all species tested and worked equally well for both sexes and for adult and sub-adult animals. We determined that using set-force-measuring instruments resulted in a 'compression effect' that countered the inflammatory response, reinforcing the need for internal controls. We developed a novel method to determine peak response times more accurately and thereby improve assay sensitivity. Histological validation demonstrated considerable interspecies variation in the robustness of amphibian immune defences. Importantly, we applied the assay to a real-world scenario of varying environmental conditions and proved that the assay effectively detected differences in immune fitness between groups of animals exposed to ecologically meaningful levels of density stress. This provided strong evidence that one cost of metamorphic plasticity responses by tadpoles to increasing density is a reduction in post-metamorphic immune fitness and that metamorphosis does not prevent phenotypic carry-over of larval stress to the adult phenotype. This assay provides an effective tool for understanding the role of global environmental change in the amphibian extinction crisis.