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Among others, the global change involves a worldwide increase in cropland area, with the concomitant rise in nitrogenous fertilizer supplementation and species range alterations, including parasites and pathogens. As most animals rely on their immune systems against these infectious agents, studying the potential effects of nitrogenous compounds on animal immune response is vital to understand their susceptibility to infections under these altered circumstances. Being subjected to an alarming process of global declines, amphibians are the object of particular attention, given their sensitivity to these compounds, especially to ammonium. Moreover, whereas adults can actively avoid polluted patches, larvae are confined within their waterbodies, thus exposed to contaminants in it. In this work, we test whether chronic exposure to a sublethal dose of ammonium during the larval stage of Pelophylax perezi frogs, released from all contamination after metamorphosis, leads to impaired inflammatory response to phytohemagglutinin in adults. We also test whether such a response differs between agrosystem individuals as compared with conspecifics from natural habitats. We found negative carryover effects of chronic exposure of larvae to ammonium on adult inflammatory response, which could imply a greater susceptibility to pathogens and parasites. However, this damage is only true for males, which, according to the immunocompetence handicap hypothesis, could be a consequence of a testosterone-triggered impairment of male immune function. In disagreement with our prediction, however, we detected no differences in the inflammatory response of agrosystem frogs to phytohemagglutinin as compared with natural habitat conspecifics.

Quantitative PCR (qPCR) has been commonly used to measure gene expression ina number of research contexts, but the measured RNA concentrations do not al-ways represent the concentrations of active proteins which they encode. This canbe due to transcriptional regulation or post-translational modifications, or localiza-tion of immune environments, as can occur during infection. However, in studiesusing free-living non-model species, such as in ecoimmunological research, qPCRmay be the only available option to measure a parameter of interest, and so under-standing the quantitative link between gene expression and associated effectorprotein levels is vital. Here, we use qPCR to measure concentrations of RNA from mesenteric lymphnode (MLN) and spleen tissue, and multiplex ELISA of blood serum to measurecirculating cytokine concentrations in a wild population of a model species, Musmusculus domesticus. Few significant correlations were found between gene expression levels and cir-culating cytokines of the same immune genes or proteins, or related functionalgroups. Where significant correlations were observed, these were most fre-quently within the measured tissue (i.e., the expression levels of genes measuredfrom spleen tissue were more likely to correlate with each other rather than withgenes measured from MLN tissue, or with cytokine concentrations measuredfrom blood). Potential reasons for discrepancies between measures including differences indecay rates and transcriptional regulation networks are discussed. We highlightthe relative usefulness of different measures under different research questionsand consider what might be inferred from immune assays., Biotechnology and Biological Sciences Research Council, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

Historically, investigations of how organismal investments in immunity fluctuate in response to environmental and physiological changes have focused on seasonally breeding organisms that confine reproduction to seasons with relatively unchallenging environmental conditions and abundant resources. The red crossbill, Loxia curvirostra, is a songbird that can breed opportunistically if conifer seeds are abundant, on both short, cold, and long, warm days, providing an ideal system to investigate environmental and reproductive effects on immunity. In this study, we measured inter- and intra-annual variation in complement, natural antibodies, PIT54 and leucocytes in crossbills across four summers (2010-2013) and multiple seasons within 1 year (summer 2011-spring 2012). Overall, we observed substantial changes in crossbill immune investment among summers, with interannual variation driven largely by food resources, while variation across multiple seasons within a single cone year was less pronounced and lacked a dominant predictor of immune investment. However, we found weak evidence that physiological processes (e.g. reproductive condition, moult) or abiotic factors (e.g. temperature, precipitation) affect immune investment. Collectively, this study suggests that a reproductively flexible organism may be able to invest in both reproduction and survival-related processes, potentially by exploiting rich patches with abundant resources. More broadly, these results emphasize the need for more longitudinal studies of trade-offs associated with immune investment.

Historically, investigations of how organismal investments in immunity fluctuate in response to environmental and physiological changes have focused on seasonally breeding organisms that confine reproduction to seasons with relatively unchallenging environmental conditions and abundant resources. The red crossbill, Loxia curvirostra, is a songbird that can breed opportunistically if conifer seeds are abundant, on both short, cold, and long, warm days, providing an ideal system to investigate environmental and reproductive effects on immunity. In this study, we measured inter- and intra-annual variation in complement, natural antibodies, PIT54 and leucocytes in crossbills across four summers (2010-2013) and multiple seasons within 1 year (summer 2011-spring 2012). Overall, we observed substantial changes in crossbill immune investment among summers, with interannual variation driven largely by food resources, while variation across multiple seasons within a single cone year was less pronounced and lacked a dominant predictor of immune investment. However, we found weak evidence that physiological processes (e.g. reproductive condition, moult) or abiotic factors (e.g. temperature, precipitation) affect immune investment. Collectively, this study suggests that a reproductively flexible organism may be able to invest in both reproduction and survival-related processes, potentially by exploiting rich patches with abundant resources. More broadly, these results emphasize the need for more longitudinal studies of trade-offs associated with immune investment.

The vertebrate innate immune system provides hosts with a rapid, non-specific response to a wide range of invading pathogens. However, the speed and duration of innate responses will be influenced by the co-evolutionary dynamics of specific host-pathogen combinations. Here, we show that low pathogenic avian influenza virus (LPAI) subtype H1N1 elicits a strong but extremely transient innate immune response in its main wildlife reservoir, the mallard (Anas platyrhynchos). Using a series of experimental and methodological improvements over previous studies, we followed the expression of retinoic acid inducible gene 1 (RIG-I) and myxovirus resistance gene (Mx) in mallards semi-naturally infected with low pathogenic H1N1. One day post infection, both RIG-I and Mx were significantly upregulated in all investigated tissues. By two days post infection, the expression of both genes had generally returned to basal levels, and remained so for the remainder of the experiment. This is despite the fact that birds continued to actively shed viral particles throughout the study period. We additionally show that the spleen plays a particularly active role in the innate immune response to LPAI. Waterfowl and avian influenza viruses have a long co-evolutionary history, suggesting that the mallard innate immune response has evolved to provide a minimum effective response to LPAIs such that the viral infection is brought under control while minimising the damaging effects of a sustained immune response.

Many studies have used the avian hemosporidians (Leucocytozoon, Plasmodium, and Hemoproteus) to test hypotheses of host-parasite co-evolution, yet documented health and survival consequences of these blood parasites vary among studies and generalizations about their pathogenicity are debatable. In general, the negative effects of the hemosporidians are likely to be greatest during acute infections of young birds, yet most previous studies in wild passerines have examined chronic effects in adults. Here, we evaluated responses of nestling American crows (Corvus brachyrhynchos) to acute infection (prevalence and burden), as well as its short- and long-term survival consequences. We used panel of nine hematological and biochemical parameters that are regularly used to evaluate the health of domestic animals, including leukocyte profiles, hematocrit, and plasma proteins. We assessed the effects of infection on survival in a mark-recapture framework. Overall, 56% of crows (n = 321 samples) were infected by at least one of the three genera. Infections by all genera were associated with elevated plasma proteins and globulins, which could indicate an adaptive immune response. However, only Plasmodium infections were associated with low hematocrit (anemia) and lower fledging success, possibly mediated by the negative effect of low hematocrit values on body condition. Moreover, early Plasmodium infection (<40 days of age) had long-term survival implications: it was associated with lower apparent survival probability within 3 years after fledging. These results suggest that young crows mounted an adaptive immune response to all three genera. Short- and long-term pathological effects, however, were only apparent with Plasmodium infections.

Many studies have used the avian hemosporidians (Leucocytozoon, Plasmodium, and Hemoproteus) to test hypotheses of host-parasite co-evolution, yet documented health and survival consequences of these blood parasites vary among studies and generalizations about their pathogenicity are debatable. In general, the negative effects of the hemosporidians are likely to be greatest during acute infections of young birds, yet most previous studies in wild passerines have examined chronic effects in adults. Here, we evaluated responses of nestling American crows (Corvus brachyrhynchos) to acute infection (prevalence and burden), as well as its short- and long-term survival consequences. We used panel of nine hematological and biochemical parameters that are regularly used to evaluate the health of domestic animals, including leukocyte profiles, hematocrit, and plasma proteins. We assessed the effects of infection on survival in a mark-recapture framework. Overall, 56% of crows (n = 321 samples) were infected by at least one of the three genera. Infections by all genera were associated with elevated plasma proteins and globulins, which could indicate an adaptive immune response. However, only Plasmodium infections were associated with low hematocrit (anemia) and lower fledging success, possibly mediated by the negative effect of low hematocrit values on body condition. Moreover, early Plasmodium infection (<40 days of age) had long-term survival implications: it was associated with lower apparent survival probability within 3 years after fledging. These results suggest that young crows mounted an adaptive immune response to all three genera. Short- and long-term pathological effects, however, were only apparent with Plasmodium infections.

The vertebrate innate immune system provides hosts with a rapid, non-specific response to a wide range of invading pathogens. However, the speed and duration of innate responses will be influenced by the co-evolutionary dynamics of specific host-pathogen combinations. Here, we show that low pathogenic avian influenza virus (LPAI) subtype H1N1 elicits a strong but extremely transient innate immune response in its main wildlife reservoir, the mallard (Anas platyrhynchos). Using a series of experimental and methodological improvements over previous studies, we followed the expression of retinoic acid inducible gene 1 (RIG-I) and myxovirus resistance gene (Mx) in mallards semi-naturally infected with low pathogenic H1N1. One day post infection, both RIG-I and Mx were significantly upregulated in all investigated tissues. By two days post infection, the expression of both genes had generally returned to basal levels, and remained so for the remainder of the experiment. This is despite the fact that birds continued to actively shed viral particles throughout the study period. We additionally show that the spleen plays a particularly active role in the innate immune response to LPAI. Waterfowl and avian influenza viruses have a long co-evolutionary history, suggesting that the mallard innate immune response has evolved to provide a minimum effective response to LPAIs such that the viral infection is brought under control while minimising the damaging effects of a sustained immune response.

Brought to Australia in 1935 to control agricultural pests (from French Guiana, via Martinique, Barbados, Jamaica, Puerto Rico and Hawai'i), repeated stepwise translocations of small numbers of founders enabled the cane toad (Rhinella marina) to escape many parasites and pathogens from its native range. However, the infective organisms that survived the journey continue to affect the dynamics of the toad in its new environment. In Australia, the native-range lungworm Rhabdias pseudosphaerocephala decreases its host's cardiac capacity, as well as growth and survival, but not rate of dispersal. The lungworm is most prevalent in long-colonised areas within the toads' Australian range, and absent from the invasion front. Several parasites and pathogens of Australian taxa have host-shifted to cane toads in Australia; for example, invasion-front toads are susceptible to spinal arthritis caused by the soil bacterium, Ochrobactrum anthropi. The pentastome Raillietiella frenata has host-shifted to toads and may thereby expand its Australian range due to the continued range expansion of the invasive toads. Spill-over and spill-back of parasites may be detrimental to other host species; however, toads may also reduce parasite loads in native taxa by acting as terminal hosts. We review the impact of the toad's parasites and pathogens on the invasive anuran's biology in Australia, as well as collateral effects of toad-borne parasites and pathogens on other host species in Australia. Both novel and co-evolved pathogens and parasites may have played significant roles in shaping the rapid evolution of immune system responses in cane toads within their invaded range.

The widely-distributed North American species Peromyscus leucopus and P. maniculatus of cricetine rodents are, between them, important natural reservoirs for several zoonotic diseases of humans: Lyme disease, human granulocytic anaplasmosis, babesiosis, erhlichiosis, hard tickborne relapsing fever, Powassan virus encephalitis, hantavirus pulmonary syndrome, and plague. While these infections are frequently disabling and sometimes fatal for humans, the peromyscines display little pathology and apparently suffer few consequences, even when prevalence of persistent infection in a population is high. While these Peromyscus spp. are unable to clear some of the infections, they appear to have partial resistance, which limits the burden of the pathogen. In addition, they display traits of infection tolerance, which reduces the damage of the infection. Research on these complementary resistance and tolerance phenomena in Peromyscus has relevance both for disease control measures targeting natural reservoirs and for understanding the mechanisms of the comparatively greater sickness of many humans with these and other infections.

Anthropogenic disturbances involving land use change, climate disruption, pollution and invasive species have been shown to impact immune function of wild animals. These immune changes have direct impacts on the fitness of impacted animals and, also, potentially indirect effects on other species and on ecological processes, notably involving the spread of infectious disease. Here, we investigate whether the selective loss of large wildlife can also drive changes in immune function of other consumer species. Using a long-standing large-scale exclosure experiment in East Africa, we investigated the effects of selective removal of large wildlife on multiple measures of immune function in the dominant small rodent in the system, the East African pouched mouse, Saccostomus mearnsi. We find support for a general increase in immune function in landscapes where large wildlife has been removed, but with some variation across immune parameters. These changes may be mediated in part by increased pathogen pressure in plots where large wildlife has been removed due to major increases in rodent density in such plots, but other factors such as changes in food resources are also likely involved. Overall, our research reveals that the elimination of large-bodied wildlife - now recognized as another major form of global anthropogenic change - may have cascading effects on immune health, with the potential for these effects to also impact disease dynamics in ecological communities. Lay Summary Functional Ecology

Anthropogenic disturbances involving land use change, climate disruption, pollution and invasive species have been shown to impact immune function of wild animals. These immune changes have direct impacts on the fitness of impacted animals and, also, potentially indirect effects on other species and on ecological processes, notably involving the spread of infectious disease. Here, we investigate whether the selective loss of large wildlife can also drive changes in immune function of other consumer species. Using a long-standing large-scale exclosure experiment in East Africa, we investigated the effects of selective removal of large wildlife on multiple measures of immune function in the dominant small rodent in the system, the East African pouched mouse, Saccostomus mearnsi. We find support for a general increase in immune function in landscapes where large wildlife has been removed, but with some variation across immune parameters. These changes may be mediated in part by increased pathogen pressure in plots where large wildlife has been removed due to major increases in rodent density in such plots, but other factors such as changes in food resources are also likely involved. Overall, our research reveals that the elimination of large-bodied wildlife - now recognized as another major form of global anthropogenic change - may have cascading effects on immune health, with the potential for these effects to also impact disease dynamics in ecological communities. Lay Summary Functional Ecology

In disease dynamics, high immune gene diversity can confer a selective advantage to hosts in the face of a rapidly evolving and diverse pathogen fauna. This is supported empirically for genes involved in pathogen recognition and signalling. In contrast, effector genes involved in pathogen clearance may be more constrained. beta-Defensins are innate immune effector genes; their main mode of action is via disruption of microbial membranes. Here, five beta-defensin genes were characterized in mallards (Anas platyrhynchos) and other waterfowl; key reservoir species for many zoonotic diseases. All five genes showed remarkably low diversity at the individual-, population-, and species-level. Furthermore, there was widespread sharing of identical alleles across species divides. Thus, specific beta-defensin alleles were maintained not only spatially but also over long temporal scales, with many amino acid residues being fixed across all species investigated. Purifying selection to maintain individual, highly efficacious alleles was the primary evolutionary driver of these genes in waterfowl. However, we also found evidence for balancing selection acting on the most recently duplicated beta-defensin gene (AvBD3b). For this gene, we found that amino acid replacements were more likely to be radical changes, suggesting that duplication of beta-defensin genes allows exploration of wider functional space. Structural conservation to maintain function appears to be crucial for avian beta-defensin effector molecules, resulting in low tolerance for new allelic variants. This contrasts with other types of innate immune genes, such as receptor and signalling molecules, where balancing selection to maintain allelic diversity has been shown to be a strong evolutionary force.

Seasonal fluctuations in infectious disease incidence are common, and have been observed for many infectious agents. Immune condition can also change seasonally due to such pathogen fluctuations, a well as to changes in other stress-inducing and immunomodulatory factors such as reproduction and lactation. In addition, most vertebrate hosts are concurrently infected with multiple pathogens, and such coinfections can interact with each other, with the immune system, and with other physiological factors to affect both the individual hosts and population dynamics. While many studies regarding the effects of coinfections and immune trade-offs have been conducted in laboratory settings, similar studies in wildlife are as yet very rare. Fewer studies have been conducted regarding disease and immune seasonality, as these are difficult to model in laboratory settings and require difficult, longitudinal studies in natural systems. In addition, most research regarding disease seasonality in natural systems that has been done has focused on the impacts of abiotic factors on pathogen and vector survival and abundance, or on population-wide dynamics, rather than on physiological mechanisms of host susceptibility. To fully understand the ecology of infectious diseases and the reasons for disease outbreaks, it is necessary to extend laboratory studies into natural hosts in natural systems, as well as to extend wildlife studies to incorporate the complex interactions between environmental, physiological, and coinfection factors. My dissertation research thus focused on the ecological immunology of infectious disease in a natural system, from physiological, seasonal, and coinfection perspectives. I examined the ability of plains zebra (Equus quagga), springbok (Antidocas marsupialis), and African elephant (Loxodonta africana) to respond immunologically to regular anthrax outbreaks in an endemic anthrax system (Etosha National Park, ENP, Namibia). I also examined how zebra and springb

Seasonal fluctuations in infectious disease incidence are common, and have been observed for many infectious agents. Immune condition can also change seasonally due to such pathogen fluctuations, a well as to changes in other stress-inducing and immunomodulatory factors such as reproduction and lactation. In addition, most vertebrate hosts are concurrently infected with multiple pathogens, and such coinfections can interact with each other, with the immune system, and with other physiological factors to affect both the individual hosts and population dynamics. While many studies regarding the effects of coinfections and immune trade-offs have been conducted in laboratory settings, similar studies in wildlife are as yet very rare. Fewer studies have been conducted regarding disease and immune seasonality, as these are difficult to model in laboratory settings and require difficult, longitudinal studies in natural systems. In addition, most research regarding disease seasonality in natural systems that has been done has focused on the impacts of abiotic factors on pathogen and vector survival and abundance, or on population-wide dynamics, rather than on physiological mechanisms of host susceptibility. To fully understand the ecology of infectious diseases and the reasons for disease outbreaks, it is necessary to extend laboratory studies into natural hosts in natural systems, as well as to extend wildlife studies to incorporate the complex interactions between environmental, physiological, and coinfection factors. My dissertation research thus focused on the ecological immunology of infectious disease in a natural system, from physiological, seasonal, and coinfection perspectives. I examined the ability of plains zebra (Equus quagga), springbok (Antidocas marsupialis), and African elephant (Loxodonta africana) to respond immunologically to regular anthrax outbreaks in an endemic anthrax system (Etosha National Park, ENP, Namibia). I also examined how zebra and springb

When animals are suffering from an infection, they frequently exhibit symptoms such as reductions in activity, reductions in food and water intake, reductions in libido and in social interactions. Adoption of these "sickness behaviors" is thought to promote immune function by reducing energy expenditure in activities that are not essential for recovery from the infection and investing this energy in mounting an immune response. In other words, during disease, since the body has limited resources, these need to be traded-off between investment in immunity and investment in other activities. My dissertation work was focused on exploiting this trade-off idea by examining how different social contexts affect the expression of sickness behaviors in birds. Social modulation of sickness behaviors should be especially relevant when animals have an opportunity to reproduce. Hence, my work focused as well on how immune challenges affect the reproductive system and how the social environment can determine the extent to which animals invest in reproduction while sick. Finally, it was my purpose to understand whether alterations in sickness behaviors due to social context impact the immune response in ways that are costly for animals.The work in this dissertation emphasizes the plasticity of the sickness behavior response. Here, I demonstrate that birds are able to adjust the expression of sickness behaviors when subjected to social circumstances that promote other adaptive opportunities. While the reproductive system is extensively shut down during an immune challenge, I demonstrate that this effect is reversed within 30 minutes of presentation of a potential mate. In addition, my work indicates that the social modulation of sickness behaviors comes at the cost of reduced immune defenses. In a world where infectious diseases represent one of the major causes of death, an increased understanding of the way behavior during infection is impacted by the social environment and the c

When animals are suffering from an infection, they frequently exhibit symptoms such as reductions in activity, reductions in food and water intake, reductions in libido and in social interactions. Adoption of these "sickness behaviors" is thought to promote immune function by reducing energy expenditure in activities that are not essential for recovery from the infection and investing this energy in mounting an immune response. In other words, during disease, since the body has limited resources, these need to be traded-off between investment in immunity and investment in other activities. My dissertation work was focused on exploiting this trade-off idea by examining how different social contexts affect the expression of sickness behaviors in birds. Social modulation of sickness behaviors should be especially relevant when animals have an opportunity to reproduce. Hence, my work focused as well on how immune challenges affect the reproductive system and how the social environment can determine the extent to which animals invest in reproduction while sick. Finally, it was my purpose to understand whether alterations in sickness behaviors due to social context impact the immune response in ways that are costly for animals.The work in this dissertation emphasizes the plasticity of the sickness behavior response. Here, I demonstrate that birds are able to adjust the expression of sickness behaviors when subjected to social circumstances that promote other adaptive opportunities. While the reproductive system is extensively shut down during an immune challenge, I demonstrate that this effect is reversed within 30 minutes of presentation of a potential mate. In addition, my work indicates that the social modulation of sickness behaviors comes at the cost of reduced immune defenses. In a world where infectious diseases represent one of the major causes of death, an increased understanding of the way behavior during infection is impacted by the social environment and the c

The processes driving and maintaining variable immune responses are poorly understood compared with other aspects of an organism’s ecology. This is particularly true from an evolutionary perspective, as the evolutionary relationships between immune responses and other traits and processes in nature remain inadequately explored. I investigated these associations in this thesis using the three-spined stickleback system as an evolutionary and immunological model. I combined sampling of wild individuals with genomic analyses to demonstrate phenotypic and genomic associations between immune responses and life history evolution across multiple populations. I also observe how experimental changes in daylength, a seasonal cue, modulate immune responses and increase parasite susceptibility in a controlled laboratory experiment. These findings occur independently of natural variation in parasite resistance. Stickleback are also a model for studies of speciation. I used sampling of wild hybrids to assess the significance of immune variation in postzygotic isolating mechanisms between diverging ecotypes; although my findings suggest a minor role. Finally, I demonstrate that genomic responses to parasitism and abiotic environmental variation are repeatable across independent, intercontinental adaptive radiation events in sticklebacks; conferring a repeatability of the evolutionary relationships of immune variation documented in this thesis. The findings within this thesis therefore provide novel insights into poorly explored or open areas of research regarding how variable immune responses evolve in nature.