Experimental evolution reveals differences between phenotypic and evolutionary responses to population density

Group living can select for increased immunity, given the heightened risk of parasite transmission. Yet, it also may select for increased male reproductive investment, given the elevated risk of female multiple mating. Trade‐offs between immunity and reproduction are well documented. Phenotypically, population density mediates both reproductive investment and immune function in the Indian meal moth, Plodia interpunctella. However, the evolutionary response of populations to these traits is unknown. We created two replicated populations of P. interpunctella, reared and mated for 14 generations under high or low population densities. These population densities cause plastic responses in immunity and reproduction: at higher numbers, both sexes invest more in one index of immunity [phenoloxidase (PO) activity] and males invest more in sperm. Interestingly, our data revealed divergence in PO and reproduction in a different direction to previously reported phenotypic responses. Males evolving at low population densities transferred more sperm, and both males and females displayed higher PO than individuals at high population densities. These positively correlated responses to selection suggest no apparent evolutionary trade‐off between immunity and reproduction. We speculate that the reduced PO activity and sperm investment when evolving under high population density may be due to the reduced population fitness predicted under increased sexual conflict and/or to trade‐offs between pre‐ and post‐copulatory traits.     

Sex‐specific life history responses to nymphal diet quality and immune status in a field cricket

Individual fitness is expected to benefit from earlier maturation at a larger body size and higher body condition. However, poor nutritional quality or high prevalence of disease make this difficult because individuals either cannot acquire sufficient resources or must divert resources to other fitness‐related traits such as immunity. Under such conditions, individuals are expected to mature later at a smaller body size and in poorer body condition. Moreover, the juvenile environment can also produce longer‐term effects on adult fitness by causing shifts in resource allocation strategies that could alter investment in immune function and affect adult lifespan. We manipulated diet quality and immune status of juvenile Texas field crickets, Gryllus texensis, to investigate how poor developmental conditions affect sex‐specific investment in fitness‐related traits. As predicted, a poor juvenile diet was related to smaller mass and body size at eclosion in both sexes. However, our results also reveal sexually dimorphic responses to different facets of the rearing environment: female life history decisions are affected more by diet quality, whereas males are affected more by immune status. We suggest that females respond to decreased nutritional income because this threatens their ability to achieve a large adult body size, whereas male fitness is more dependent on reaching adulthood and so they invest in immunity and survival to eclosion.     

Natural variation in the contribution of microbial density to inducible immune dynamics

Immune responses evolve to balance the benefits of microbial killing against the costs of autoimmunity and energetic resource use. Models that explore the evolution of optimal immune responses generally include a term for constitutive immunity, or the level of immunological investment prior to microbial exposure, and for inducible immunity, or investment in immune function after microbial challenge. However, studies rarely consider the functional form of inducible immune responses with respect to microbial density, despite the theoretical dependence of immune system evolution on microbe‐ versus immune‐mediated damage to the host. In this study, we analyse antimicrobial peptide (AMP) gene expression from seven wild‐caught flour beetle populations (Tribolium spp.) during acute infection with the virulent bacteria Bacillus thuringiensis (Bt) and Photorhabdus luminescens (P.lum) to demonstrate that inducible immune responses mediated by the humoral IMD pathway exhibit natural variation in both microbe density‐dependent and independent temporal dynamics. Beetle populations that exhibited greater AMP expression sensitivity to Bt density were also more likely to die from infection, while populations that exhibited higher microbe density‐independent AMP expression were more likely to survive P. luminescens infection. Reduction in pathway signalling efficiency through RNAi‐mediated knockdown of the imd gene reduced the magnitude of both microbe‐independent and dependent responses and reduced host resistance to Bt growth, but had no net effect on host survival. This study provides a framework for understanding natural variation in the flexibility of investment in inducible immune responses and should inform theory on the contribution of nonequilibrium host‐microbe dynamics to immune system evolution.     

Immune defense in leaf-cutting ants: a cross-fostering approach

To ameliorate the impact of disease, social insects combine individual innate immune defenses with collective social defenses. This implies that there are different levels of selection acting on investment in immunity, each with their own trade-offs. We present the results of a cross-fostering experiment designed to address the influences of genotype and social rearing environment upon individual and social immune defenses. We used a multiply mating leaf-cutting ant, enabling us to test for patriline effects within a colony, as well as cross-colony matriline effects. The worker's father influenced both individual innate immunity (constitutive antibacterial activity) and the size of the metapleural gland, which secretes antimicrobial compounds and functions in individual and social defense, indicating multiple mating could have important consequences for both defense types. However, the primarily social defense, a Pseudonocardia bacteria that helps to control pathogens in the ants' fungus garden, showed a significant colony of origin by rearing environment interaction, whereby ants that acquired the bacteria of a foster colony obtained a less abundant cover of bacteria: one explanation for this pattern would be co-adaptation between host colonies and their vertically transmitted mutualist. These results illustrate the complexity of the selection pressures that affect the expression of multilevel immune defenses.     

Sexual segregation in North American elk: the role of density dependence

We investigated how density‐dependent processes and subsequent variation in nutritional condition of individuals influenced both timing and duration of sexual segregation and selection of resources. During 1999–2001, we experimentally created two population densities of North American elk (Cervus elaphus), a high‐density population at 20 elk/km², and a low‐density population at 4 elk/km² to test hypotheses relative to timing and duration of sexual segregation and variation in selection of resources. We used multi‐response permutation procedures to investigate patterns of sexual segregation, and resource selection functions to document differences in selection of resources by individuals in high‐ and low‐density populations during sexual segregation and aggregation. The duration of sexual segregation was 2 months longer in the high‐density population and likely was influenced by individuals in poorer nutritional condition, which corresponded with later conception and parturition, than at low density. Males and females in the high‐density population overlapped in selection of resources to a greater extent than in the low‐density population, probably resulting from density‐dependent effects of increased intraspecific competition and lower availability of resources.     

Captive and free‐living red knots Calidris canutus exhibit differences in non‐induced immunity that suggest different immune strategies in different environments

Experiments on captive animals, in which conditions can be controlled, are useful for examining complex biological phenomena such as immune function. Such experiments have increased our understanding of immune responses in the context of trade‐offs and pathogen pressure. However, few studies have examined how captivity itself affects immune function. We used microbial killing, leukocyte concentrations and complement‐natural antibody assays to examine non‐induced (constitutive) immunity in captive and free‐living red knots Calidris canutus. Univariate and multivariate analyses indicated that captive and free‐living birds differed in their immune strategies. Captive birds showed reduced S. aureus killing, C. albicans killing, heterophils, and eosinophils. In a principal component analysis, the affected variables fell onto a single axis, that reflected phagocytosis and inflammation based immunity. We discuss possible reasons for this result in an immune cost and protective benefit framework.     

Personality and innate immune defenses in a wild bird: Evidence for the pace-of-life hypothesis

We tested the two main evolutionary hypotheses for an association between immunity and personality. The risk-of-parasitism hypothesis predicts that more proactive (bold, exploratory, risk-taking) individuals have more vigorous immune defenses because of increased risk of parasite exposure. In contrast, the pace-of-life hypothesis argues that proactive behavioral styles are associated with shorter lifespans and reduced investment in immune function. Mechanistically, associations between immunity and personality can arise because personality differences are often associated with differences in condition and stress responsiveness, both of which are intricately linked with immunity. Here we investigate the association between personality (measured as proactive exploration of a novel environment) and three indices of innate immune function (the non-specific first line of defense against parasites) in wild superb fairy-wrens Malurus cyaneus. We also quantified body condition, hemoparasites (none detected), chronic stress (heterophil:lymphocyte ratio) and circulating corticosterone levels at the end of the behavioral test (CORT, in a subset of birds). We found that fast explorers had lower titers of natural antibodies. This result is consistent with the pace-of-life hypothesis, and with the previously documented higher mortality of fast explorers in this species. There was no interactive effect of exploration score and duration in captivity on immune indices. This suggests that personality-related differences in stress responsiveness did not underlie differences in immunity, even though behavioral style did modulate the effect of captivity on CORT. Taken together these results suggest reduced constitutive investment in innate immune function in more proactive individuals.     

Exposure to sexual signals during rearing increases immune defence in adult field crickets

Increased investment in immunity is expected to be beneficial under crowded conditions because of the greater risk of pathogen and parasite transmission, but the evolution of this facultative response relies on the ability to accurately assess social cues in the environment and adjust immune defences accordingly. Because of their highly conspicuous nature, long-range sexual signals are prime candidates to be used in evaluating the social conditions likely to be experienced upon adulthood in continuously breeding species; however, their role in mediating immune responses is unknown. We tested whether exposure to acoustic sexual signals in the field cricket Teleogryllus oceanicus affects immunity by manipulating male juvenile experience of acoustic signals, and measuring the effect on adult immunity. Adult males exposed to song during rearing showed stronger immune responses than males reared in silence: they were better able to encapsulate artificial nylon implants and showed higher levels of antimicrobial lysozyme-like activity in their haemolymph. Experience of sexual signals thus translates into increased immunity, which suggests that such signals may play a role in conveying information about population demography and shaping density-dependent responses in unintended receivers.     

Constitutive innate immunity is a component of the pace-of-life syndrome in tropical birds

We studied the relationship between one component of immune function and basal metabolic rate (BMR), an indicator of the 'pace-of-life syndrome', among 12 tropical bird species and among individuals of the tropical house wren (Troglodytes aedon), to gain insights into functional connections between life history and physiology. To assess constitutive innate immunity we introduced a new technique in the field of ecological and evolutionary immunology that quantifies the bactericidal activity of whole blood. This in vitro assay utilises a single blood sample to provide a functional, integrated measure of constitutive innate immunity. We found that the bactericidal activity of whole blood varied considerably among species and among individuals within a species. This variation was not correlated with body mass or whole-organism BMR. However, among species, bacteria killing activity was negatively correlated with mass-adjusted BMR, suggesting that species with a slower pace-of-life have evolved a more robust constitutive innate immune capability. Among individuals of a single species, the house wren, bacteria killing activity was positively correlated with mass-adjusted BMR, pointing to physiological differences in individual quality on which natural selection potentially could act.     

Modeling immune responses with handling time

Simple predator-prey type models have brought much insight into the dynamics of both nonspecific and antigen-specific immune responses. However, until now most attention has been focused on examining how the dynamics of interactions between the parasite and the immune system depends on the nature of the function describing the rate of activation or proliferation of immune cells in response to the parasite. In this paper we focus on the term describing the killing of the parasite by cell-mediated immune responses. This term has previously been assumed to be a simple mass-action term dependent solely on the product of the densities of the parasite and the immune cells and does not take into account a handling time (which we define as the time of interaction between an immune cell and its target, during which the immune cell cannot interact with and/or destroy additional targets). We show how the handling time (i) can be incorporated into simple models of nonspecific and specific immunity and (ii) how it affects the dynamics of both nonspecific and antigen-specific immune responses, and in particular the ability of the immune response to control the infection.     

Relationship between pace of life and immune responses in wild rodents

Life histories of animals tend to vary along a slow to fast continuum. Those with fast life histories have shorter life spans, faster development, and higher reproductive rates relative to animals with slower life histories. These differences in life histories have been linked to differences in investment in immunological defenses. Animals with faster life histories are predicted to invest relatively more in innate immune responses, which include rapidly‐deployed, non‐specific defenses against a broad spectrum of invaders. On the other hand, animals with slower life histories are predicted to invest relatively more in adaptive immune responses, which are more slowly‐deployed and are highly pathogen‐specific. These predictions have been confirmed in some taxa, but other studies have not found this association. We tested this prediction by measuring innate and adaptive immunity of white‐footed mice Peromyscus leucopus, chipmunks Tamias striatus, and gray squirrels Sciurus carolinensis, three species of rodents that inhabit deciduous forests in the northeastern US. These species exhibit a range of life histories, with mice having a relatively fast life history, squirrels a relatively slow one, and chipmunks an intermediate one. We found mice to have the greatest ‘bacterial killing capacity’, a common measure of innate immunity, and squirrels the lowest, consistent with the pace‐of‐life immune‐defense hypothesis. We also found squirrels to mount the most pronounced antibody response when challenged with lipopolysaccharide (LPS), an immunogenic component of bacteria, while mice had the lowest, again consistent with predictions based on their life histories. These results have implications beyond ecoimmunology because the probability that a host species will transmit an infection – its ‘reservoir competence’ – has been linked to its immune strategy. Understanding the relationship between immunology and reservoir competence is a critical frontier in the ecology of infectious diseases.     

Social immunity in honeybees—Density dependence,diet, and body mass trade‐offs

Group living is favorable to pathogen spread due to the increased risk of disease transmission among individuals. Similar to individual immune defenses, social immunity, that is antiparasite defenses mounted for the benefit of individuals other than the actor, is predicted to be altered in social groups. The eusocial honey bee (Apis mellifera) secretes glucose oxidase (GOX), an antiseptic enzyme, throughout its colony, thereby providing immune protection to other individuals in the hive. We conducted a laboratory experiment to investigate the effects of group density on social immunity, specifically GOX activity, body mass and feeding behavior in caged honey bees. Individual honeybees caged in a low group density displayed increased GOX activity relative to those kept at a high group density. In addition, we provided evidence for a trade‐off between GOX activity and body mass: Individuals caged in the low group density had a lower body mass, despite consuming more food overall. Our results provide the first experimental evidence that group density affects a social immune response in a eusocial insect. Moreover, we showed that the previously reported trade‐off between immunity and body mass extends to social immunity. GOX production appears to be costly for individuals, and potentially the colony, given that low body mass is correlated with small foraging ranges in bees. At high group densities, individuals can invest less in social immunity than at low densities, while presumably gaining shared protection from infection. Thus, there is evidence that trade‐offs at the individual level (GOX vs. body mass) can affect colony‐level fitness.     

Capture stress and the bactericidal competence of blood and plasma in five species of tropical birds

In wild birds, relatively little is known about intra- or interspecific variation in immunological capabilities, and even less is known about the effects of stress on immune function. Immunological assays adaptable to field settings and suitable for a wide variety of taxa will prove most useful for addressing these issues. We describe a novel application of an in vitro technique that measures the intrinsic bacteria-killing abilities of blood. We assessed the capacities of whole blood and plasma from free-living individuals of five tropical bird species to kill a nonpathogenic strain of E. coli before and after the birds experienced an acute stress. Killing invasive bacteria is a fundamental immune function, and the bacteria-killing assay measures constitutive, innate immunity integrated across circulating cell and protein components. Killing ability varied significantly across species, with common ground doves exhibiting the lowest levels and blue-crowned motmots exhibiting the highest levels. Across species, plasma killed bacteria as effectively as whole blood, and higher concentrations of plasma killed significantly better. One hour of acute stress reduced killing ability by up to 40%. This assay is expected to be useful in evolutionary and ecological studies dealing with physiological and immunological differences in birds.     

Variations in elk aggregation patterns across a range of elk population sizes at Wall Creek,Montana

In the Greater Yellowstone Ecosystem, growing concern over increasing rates of brucellosis seroprevalence in wildlife has challenged wildlife managers to develop strategies for minimizing the potential for pathogen exchange within and between wildlife populations. Recent evidence suggests that increases in elk seroprevalence may be associated with increasing elk densities and/or increasing size of elk aggregations. However, the interactions between elk population density, landscape factors, and elk aggregation patterns are not well-understood, making appropriate management responses challenging. Using a unique, long-term elk aggregation dataset collected across a wide range of elk population sizes, we investigated relationships between elk population size, landscape factors, and elk aggregation responses (group size and group density) with goals of clarifying how changes in elk population size may affect elk aggregation patterns. Overall, landscape attributes and weather had a stronger influence on elk aggregation patterns than factors such as elk population size that are within management control. We found little evidence that elk population size affected mean elk group sizes, but we did find evidence that the size and density of the largest elk aggregations increased as elk population size increased. We also found some evidence that group densities increased following the establishment of wolves. However, across the relatively wide range of elk population sizes observed in this study, only modest changes in elk group density were observed, suggesting that dramatic reductions in population sizes would be necessary to produce measureable reductions in elk group density to affect frequency-dependent transmission. Management actions designed to lower disease transmission are likely to negatively affect other objectives related to elk management and conservation. We therefore suggest that a first step in managing disease transmission risk is agreement among stakeholders interested in elk management of all objectives related to elk management, including acknowledgment that disease transmission is undesirable. © 2011 The Wildlife Society.     

Condition‐dependent immune defence in the Magpie: how important is ectoparasitism?

The immune system has high costs of maintenance and production and expression of immune function should be dependent on the condition of the individual. The fitness cost that parasites impose on hosts may then be reflected in the investment that hosts make in immunity. Little is known about sources and consequences of intra- and interspecific variation in the presence and size of organs of the immune system in relation to ectoparasitism. Variation in the presence and size of the bursa of Fabricius and spleen size was measured to assess potential relationships between immune defence, nutritional condition and ectoparasites in the Magpie Pica pica. We found three species of lice (Mallophaga), two species of louse flies (Hippoboscidae), and the larvae of a tick (Ixodidae) infecting magpies. Age explained a significant proportion of variation in prevalence and number of parasite species, first-year birds being more parasitized than adults. Spleen size increased with nutritional condition, and the level of ectoparasitism negatively affected nutritional condition. Only one species of lice (Philopterus picae) was involved; the negative effect of this species on nutritional condition was more pronounced in males than females. These results are consistent with the hypotheses of condition-dependent investment in the immune system and sexual differences in susceptibility to parasite effects. Ectoparasites may thus play a role in modulating the relationship between condition and immune defence. We also found evidence for an interaction between the bursa and the spleen. Magpies with bursa had larger spleens than those where it had atrophied. This interaction did not affect ectoparasitism or nutritional condition.     

Density-dependent effects of prey defenses and predator offenses

Defenses protect prey, while offenses arm predators. Some defenses and offenses are constitutive (e.g. tortoise shells), while others are phenotypically plastic and not always expressed (e.g. neckteeth in water fleas). All of them are costly and only adaptive at certain prey densities. Here, I analyse such density-dependent effects, applying a functional response model to categorize defenses and offenses and qualitatively predict at which prey densities each category should evolve (if it is constitutive) or be expressed (if it is phenotypically plastic). The categories refer to the step of the predation cycle that a defense or offense affects: (1) search, (2) encounter, (3) detection, (4) attack, or (5) meal. For example, prey warning signals such as red coloration prevent predator attacks and are hence step 4 defenses, while sharp predator eyes enhance detection and are step 3 offenses. My theoretical analyses predict that step 1 defenses, which prevent predators from searching for their next meal (e.g. toxic substances), evolve or are expressed at intermediate prey densities. Other defenses, however, should be most beneficial at low prey densities. Regarding predators, step 1 offenses (e.g. immunity against prey toxins) are predicted to evolve or be expressed at high prey densities, other offenses at intermediate densities. I provide evidence from the literature that supports these predictions.     

Exploring the role of the immune response in preventing antibiotic resistance

For many bacterial infections, drug resistant mutants are likely present by the time antibiotic treatment starts. Nevertheless, such infections are often successfully cleared. It is commonly assumed that this is due to the combined action of drug and immune response, the latter facilitating clearance of the resistant population. However, most studies of drug resistance emergence during antibiotic treatment focus almost exclusively on the dynamics of bacteria and the drug and neglect the contribution of immune defenses. Here, we develop and analyze several mathematical models that explicitly include an immune response. We consider different types of immune responses and investigate how each impacts the emergence of resistance. We show that an immune response that retains its strength despite a strong drug-induced decline of bacteria numbers considerably reduces the emergence of resistance, narrows the mutant selection window, and mitigates the effects of non-adherence to treatment. Additionally, we show that compared to an immune response that kills bacteria at a constant rate, one that trades reduced killing at high bacterial load for increased killing at low bacterial load is sometimes preferable. We discuss the predictions and hypotheses derived from this study and how they can be tested experimentally.     

The effects of host plant species and larval density on immune function in the polyphagous moth Spodoptera littoralis

Immune functions are costly, and immune investment is usually dependent on the individual''s condition and resource availability. For phytophagous insects, host plant quality has large effects on performance, for example growth and survival, and may also affect their immune function. Polyphagous insects often experience a large variation in quality among different host plant species, and their immune investment may thus vary depending on which host plant species they develop on. Larvae of the polyphagous moth Spodoptera littoralis have previously been found to exhibit density‐dependent prophylaxis as they invest more in certain immune responses in high population densities. In addition, the immune response of S. littoralis has been shown to depend on nutrient quality in experiments with artificial diet. Here, I studied the effects of natural host plant diet and larval density on a number of immune responses to understand how host plant species affects immune investment in generalist insects, and whether the density‐dependent prophylaxis could be mediated by host plant species. While host plant species in general did not mediate the density‐dependent immune expression, particular host plant species was found to increase larval investment in certain functions of the immune system. Interestingly, these results indicate that different host plants may provide a polyphagous species with protection against different kinds of antagonisms. This insight may contribute to our understanding of the relationship between preference and performance in generalists, as well as having applied consequences for sustainable pest management.     

Carotenoid and protein supplementation have differential effects on pheasant ornamentation and immunity

A currently popular hypothesis states that the expression of carotenoid-dependent sexual ornaments and immune function may be correlated because both traits are positively affected by carotenoids. However, such a correlation may arise for another reason: it is well known that immune function is dependent on nutritional condition. A recent study has suggested that the expression of ornaments may too depend on nutritional condition, as males in good nutritional condition are better at assimilating and/or modulating carotenoids. Thus, carotenoid-dependent ornaments and immune function may be correlated because both are dependent on nutritional condition. To elucidate if, and how, ornamentation and immune function are linked, pheasant diets were supplemented with carotenoid and/or protein in a fully factorial experiment. Carotenoid treatment affected wattle coloration and tail growth, but not cellular or humoral immunity. Immunity was unrelated to males' initial ornamentation including wattle colour. Males in better body condition, measured as residual mass, increased their wattle coloration more when carotenoid supplemented. Protein positively affected humoral but not cellular immunity, but had no effect on ornaments. Cellular, but not humoral, immunity increased with male body condition. Thus, there was no evidence that an immune-stimulatory effect of carotenoids resulted in wattle coloration honestly signalling immune function, but wattle coloration may still signal male body condition.