Maaike A. Versteegh

Effects of immune supplementation and immune challenge on oxidative status and physiology in a model bird: implications for ecologists

SUMMARY One route to gain insight into the causes and consequences of ecological differentiation is to understand the underlying physiological mechanisms. We explored the relationships between immunological and oxidative status and investigated how birds cope physiologically with the effects of immune-derived oxidative damage. We successively implemented two experimental manipulations to alter physiological status in a model bird species: the homing pigeon (Columba livia). The first manipulation, an immune supplementation, was achieved by oral administration of lysozyme, a naturally occurring and non-specific antimicrobial enzyme. The second manipulation, an immune challenge, took the form of an injection with lipopolysaccharide, a bacterial endotoxin. Between groups of lysozyme-treated and control birds, we compared lipopolysaccharide-induced changes in reactive oxygen metabolites, total antioxidant capacity, haptoglobin, oxygen consumption, body mass and cloacal temperature. Lysozyme supplementation intensified the lipopolysaccharide-induced inflammatory response and generated short-term oxidative and metabolic costs. We identified significant interactions between immune supplementation and immune challenge in terms of reactive oxygen metabolites, haptoglobin and oxygen consumption. Our study provides alternative interpretations of differences in oxidative and immunological indices and demonstrates that these indices can also fluctuate and interact across very short time scales, reflecting something akin to current ‘health status’ or ‘physiological condition’. These ephemeral effects highlight the need to broadly consider current physiological condition when drawing conclusions that relate physiology to ecology and evolution.

Baseline haptoglobin concentrations are repeatable and predictive of certain aspects of a subsequent experimentally-induced inflammatory response

Ecologists sometimes assume immunological indices reflect fundamental attributes of individuals-an important assumption if an index is to be interpreted in an evolutionary context since among-individual variation drives natural selection. Yet the extent to which individuals vary over different timescales is poorly understood. Haptoglobin, an acute phase protein, is an interesting parameter for studying variability as it is easily quantified and concentrations vary widely due to the molecules role in inflammation, infection and trauma. We quantified haptoglobin in pigeon plasma samples collected over fourteen months and calculated repeatability to evaluate if haptoglobin concentration is a distinctive trait of individuals. We also explored the capacity of baseline haptoglobin concentrations to predict an array of physiological changes associated with a subsequent experimentally-induced inflammatory response. Maximum repeatability, which occurred over a short mid-winter interval, equaled 0.57. Baseline haptoglobin concentrations predicted response haptoglobin concentrations better than any other endotoxin-induced change. Overall, we identified several strengths and limitations of baseline [Hp] quantification. Acknowledging these qualities should lead to more refined conclusions in studies of the ecology and evolution of immune function.

Repeatability and individual correlates of basal metabolic rate and total evaporative water loss in birds: a case study in European stonechats

Basal metabolic rate (BMR) and total evaporative water loss (TEWL) are thought to have evolved in conjunction with life history traits and are often assumed to be characteristic features of an animal. Physiological traits can show large intraindividual variation at short and long timescales, yet natural selection can only act on a trait if it is a characteristic feature of an individual. The repeatability of a trait, a measure of the portion of variance that is caused by differences among individuals, indicates if it is a characteristic feature of an individual. We measured repeatability of BMR and TEWL of 18 captive European stonechats (Saxicola torquata rubicola) within the winter season. Repeatability was 0.56 for BMR and 0.60 for mass-specific BMR. Age and body mass had a significant effect on variation in BMR. Also after accounting for this variation, BMR remained repeatable. TEWL and mass-specific TEWL showed nonsignificant repeatabilities of 0.11 and 0.12, respectively. We conclude that BMR is a characteristic feature of an individual in our population of European stonechats, whereas TEWL is not. We discuss our results in the context of a review of currently available estimates of repeatability of BMR and TEWL for birds.

Fewer invited talks by women in evolutionary biology symposia

Lower visibility of female scientists, compared to male scientists, is a potential reason for the under‐representation of women among senior academic ranks. Visibility in the scientific community stems partly from presenting research as an invited speaker at organized meetings. We analysed the sex ratio of presenters at the European Society for Evolutionary Biology (ESEB) Congress 2011, where all abstract submissions were accepted for presentation. Women were under‐represented among invited speakers at symposia (15% women) compared to all presenters (46%), regular oral presenters (41%) and plenary speakers (25%). At the ESEB congresses in 2001–2011, 9–23% of invited speakers were women. This under‐representation of women is partly attributable to a larger proportion of women, than men, declining invitations: in 2011, 50% of women declined an invitation to speak compared to 26% of men. We expect invited speakers to be scientists from top ranked institutions or authors of recent papers in high‐impact journals. Considering all invited speakers (including declined invitations), 23% were women. This was lower than the baseline sex ratios of early‐mid career stage scientists, but was similar to senior scientists and authors that have published in high‐impact journals. High‐quality science by women therefore has low exposure at international meetings, which will constrain Evolutionary Biology from reaching its full potential. We wish to highlight the wider implications of turning down invitations to speak, and encourage conference organizers to implement steps to increase acceptance rates of invited talks.

Do immunological, endocrine and metabolic traits fall on a single Pace-of-Life axis? Covariation and constraints among physiological systems

Variation in demographic and physiological attributes of life history is thought to fall on one single axis, a phenomenon termed the Pace‐of‐Life. A slow Pace‐of‐Life is characterized by low annual reproduction, long life span and low metabolic rate, a fast Pace‐of‐Life by the opposite characteristics. The existence of a single axis has been attributed to constraints among physiological mechanisms that are thought to restrict evolutionary potential. In that case, physiological traits should covary in the same fashion at the levels of individual organisms and species. We examined covariation at the levels of individual and subspecies in three physiological systems (metabolic, endocrine and immune) using four stonechat subspecies with distinct life‐history strategies in a common‐garden set‐up. We measured basal metabolic rate, corticosterone as endocrine measure and six measures of constitutive immunity. Metabolic rate covaried with two indices of immunity at the individual level, and with corticosterone concentrations and one index of immunity at the subspecies level, but not with other measures. The different patterns of covariation among individuals and among subspecies demonstrate that links among physiological traits are loose and suggest that these traits can evolve independent of each other.

Wild skylarks seasonally modulate energy budgets but maintain energetically costly inflammatory immune responses throughout the annual cycle.

A central hypothesis of ecological immunology is that immune defences are traded off against competing physiological and behavioural processes. During energetically demanding periods, birds are predicted to switch from expensive inflammatory responses to less costly immune responses. Acute phase responses (APRs) are a particularly costly form of immune defence, and, hence, seasonal modulations in APRs are expected. Yet, hypotheses about APR modulation remain untested in free-living organisms throughout a complete annual cycle. We studied seasonal modulations in the APRs and in the energy budgets of skylarks Alauda arvensis, a partial migrant bird from temperate zones that experiences substantial ecological changes during its annual cycle. We characterized throughout the annual cycle changes in their energy budgets by measuring basal metabolic rate (BMR) and body mass. We quantified APRs by measuring the effects of a lipopolysaccharide injection on metabolic rate, body mass, body temperature, and concentrations of glucose and ketone. Body mass and BMR were lowest during breeding, highest during winter and intermediate during spring migration, moult and autumn migration. Despite this variation in energy budgets, the magnitude of the APR, as measured by all variables, was similar in all annual cycle stages. Thus, while we find evidence that some annual cycle stages are relatively more energetically constrained, we find no support for the hypothesis that during these annual cycle stages birds compromise an immune defence that is itself energetically costly. We suggest that the ability to mount an APR may be so essential to survival in every annual cycle stage that skylarks do not trade off this costly form of defence with other annual cycle demands.

One Problem, Many Solutions: Simple Statistical Approaches Help Unravel the Complexity of the Immune System in an Ecological Context

The immune system is a complex collection of interrelated and overlapping solutions to the problem of disease. To deal with this complexity, researchers have devised multiple ways to measure immune function and to analyze the resulting data. In this way both organisms and researchers employ many tactics to solve a complex problem. One challenge facing ecological immunologists is the question of how these many dimensions of immune function can be synthesized to facilitate meaningful interpretations and conclusions. We tackle this challenge by employing and comparing several statistical methods, which we used to test assumptions about how multiple aspects of immune function are related at different organizational levels. We analyzed three distinct datasets that characterized 1) species, 2) subspecies, and 3) among- and within-individual level differences in the relationships among multiple immune indices. Specifically, we used common principal components analysis (CPCA) and two simpler approaches, pair-wise correlations and correlation circles. We also provide a simple example of how these techniques could be used to analyze data from multiple studies. Our findings lead to several general conclusions. First, relationships among indices of immune function may be consistent among some organizational groups (e.g. months over the annual cycle) but not others (e.g. species); therefore any assumption of consistency requires testing before further analyses. Second, simple statistical techniques used in conjunction with more complex multivariate methods give a clearer and more robust picture of immune function than using complex statistics alone. Moreover, these simpler approaches have potential for analyzing comparable data from multiple studies, especially as the field of ecological immunology moves towards greater methodological standardization.

Independence among physiological traits suggests flexibility in the face of ecological demands on phenotypes

Phenotypic flexibility allows animals to adjust their physiology to diverse environmental conditions encountered over the year. Examining how these varying traits covary gives insights into potential constraints or freedoms that may shape evolutionary trajectories. In this study, we examined relationships among haematocrit, baseline corticosterone concentration, constitutive immune function and basal metabolic rate in red knot Calidris canutus islandica individuals subjected to experimentally manipulated temperature treatments over an entire annual cycle. If covariation among traits is constrained, we predict consistent covariation within and among individuals. We further predict consistent correlations between physiological and metabolic traits if constraints underlie species‐level patterns found along the slow‐fast pace‐of‐life continuum. We found no consistent correlations among haematocrit, baseline corticosterone concentration, immune function and basal metabolic rate either within or among individuals. This provides no evidence for constraints limiting relationships among these measures of the cardiovascular, endocrine, immune and metabolic systems in individual red knots. Rather, our data suggest that knots are free to adjust individual parts of their physiology independently. This makes good sense if one places the animal within its ecological context where different aspects of the environment might put different pressures on different aspects of physiology.

Repeatability and individual correlates of microbicidal capacity of bird blood

With the rapid development of the field of ecological and evolutionary immunology, a series of new techniques to measure different components of immune function is becoming commonplace. An important step for the interpretation of these new measures is to understand the kind of information about the animal that they convey. We showed that the microbicidal capacity of Stonechat (Saxicola torquata) blood, an integrative measure of constitutive immune function, is highly repeatable when tested against Escherichia coli and not significantly repeatable when tested against Candida albicans. The low repeatability against C. albicans results from relatively low variation among individuals, providing only low resolution to identify if this interindividual variation is consistent. In addition, we explored the effect of sex and age on microbicidal capacity, and found that over a range of ages from 1 to 7 years the blood of older birds had a better capacity to kill microbes. We concluded that, over a time period of weeks, microbicidal capacity of avian blood is an individual-bound trait, that shows consistent interindividual variation partly related to age, and unaffected by sex. This knowledge is important when interpreting the possible evolutionary mechanism underlying immunological differences, for example among individuals, environments and seasons.

Immune response to an endotoxin challenge involves multiple immune parameters and is consistent among the annual-cycle stages of a free-living temperate zone bird

SUMMARY Trade-offs between immune function and other physiological and behavioural processes are central in ecoimmunology, but one important problem is how to distinguish a reallocation of resources away from the immune system from a reallocation or redistribution within the immune system. While variation in baseline values of individual immune parameters is well established, studies in wild animals on multiple parameters during an immune response are lacking. It also remains to be tested whether and how immune responses correlate with baseline values that vary, for example, over the course of an annual cycle. We studied immunological responses to an endotoxin challenge in skylarks (Alauda arvensis), a partial migrant bird breeding in temperate zones. We compared birds injected with the endotoxin LPS with un-injected controls, characterizing immunological responses with leukocyte profiles, titres of lytic enzymes and natural antibodies, and concentrations of haptoglobin and heat shock proteins. We did this in five annual-cycle stages to test whether the response varied throughout the year. The endotoxin challenge affected six of 10 measured parameters. Lysis titres and proportions of heterophils increased; haptoglobin concentrations and proportions of lymphocytes, basophils and eosinophils decreased. The variable effects on different immune components demonstrate the complexity of an immune response. We found no evidence that the response differed between annual-cycle stages. The response was independent of baseline measures taken directly upon capture in the field, indicating that birds were facing no immunological ceiling when mounting an immune response. Values of five parameters collected under field conditions were significantly related to values taken under standardized laboratory conditions. We conclude that multiple parts of the immune system are modulated during an immunological response and that responses are not re-organized throughout the annual cycle.