Deborah M. Buehler

How Do Migratory Species Stay Healthy Over the Annual Cycle? A Conceptual Model for Immune Function and For Resistance to Disease

Migration has fascinated researchers for years and many active areas of study exist. However, the question of how migratory species stay healthy within the context of their annual cycle remains relatively unexplored. This article addresses this question using Red Knots (Calidris canutus) as a model migrant species. We review recent research on immune function in Red Knots and integrate this work with the broader eco-immunological literature to introduce a conceptual model. This model synthesizes earlier ideas about resource allocation and the costs of immunity with recent increases in our knowledge about the vertebrate immune system and then puts these concepts into the context of defense against real pathogens in environments where a myriad of factors change in time and space. We also suggest avenues for further research, which will help to test the model and better link measures of immune function to pressure from pathogens and to optimal defense against disease.

Limited Access to Food and Physiological Trade‐Offs in a Long‐Distance Migrant Shorebird. II. Constitutive Immune Function and the Acute‐Phase Response

In response to unbalanced energy budgets, animals must allocate resources among competing physiological systems to maximize fitness. Constraints can be imposed on energy availability or energy expenditure, and adjustments can be made via changes in metabolism or trade‐offs with competing demands such as body‐mass maintenance and immune function. This study investigates changes in constitutive immune function and the acute‐phase response in shorebirds (red knots) faced with limited access time to food. We separated birds into two experimental groups receiving either 6 h or 22 h of food access and measured constitutive immune function. After 3 wk, we induced an acute‐phase response, and after 1 wk of recovery, we switched the groups to the opposite food treatment and measured constitutive immune function again. We found little effect of food treatment on constitutive immune function, which suggests that even under resource limitation, a baseline level of immune function is maintained. However, birds enduring limited access to food suppressed aspects of the acute‐phase response (decreased feeding and mass loss) to maintain energy intake, and they downregulated thermoregulatory adjustments to food treatment to maintain body temperature during simulated infection. Thus, under resource‐limited conditions, birds save energy on the most costly aspects of immune defense.

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.

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.

Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay

ABSTRACT Using 16S rRNA gene sequencing analysis, we examined the bacterial diversity and the presence of opportunistic bacterial pathogens (i.e., Campylobacter and Helicobacter) in red knot (Calidris canutus; n = 40), ruddy turnstone (Arenaria interpres; n = 35), and semipalmated sandpiper (Calidris pusilla; n = 22) fecal samples collected during a migratory stopover in Delaware Bay. Additionally, we studied the occurrence of Campylobacter spp., enterococci, and waterfowl fecal source markers using quantitative PCR (qPCR) assays. Of 3,889 16S rRNA clone sequences analyzed, the bacterial community was mostly composed of Bacilli (63.5%), Fusobacteria (12.7%), Epsilonproteobacteria (6.5%), and Clostridia (5.8%). When epsilonproteobacterium-specific 23S rRNA gene clone libraries (i.e., 1,414 sequences) were analyzed, the sequences were identified as Campylobacter (82.3%) or Helicobacter (17.7%) spp. Specifically, 38.4%, 10.1%, and 26.0% of clone sequences were identified as C. lari (>99% sequence identity) in ruddy turnstone, red knot, and semipalmated sandpiper clone libraries, respectively. Other pathogenic species of Campylobacter, such as C. jejuni and C. coli, were not detected in excreta of any of the three bird species. Most Helicobacter-like sequences identified were closely related to H. pametensis (>99% sequence identity) and H. anseris (92% sequence identity). qPCR results showed that the occurrence and abundance of Campylobacter spp. was relatively high compared to those of fecal indicator bacteria, such as Enterococcus spp., E. faecalis, and Catellicoccus marimammalium. Overall, the results provide insights into the complexity of the shorebird gut microbial community and suggest that these migratory birds are important reservoirs of pathogenic Campylobacter species.

Flight performance of western sandpipers Calidris mauri remains uncompromised when mounting an acute phase immune response

SUMMARY Migratory birds have been implicated in the spread of some zoonotic diseases, but how well infected individuals can fly remains poorly understood. We used western sandpipers, Calidris mauri, to experimentally test whether flight is affected when long-distance migrants are mounting an immune response and whether migrants maintain immune defences during a flight in a wind tunnel. We measured five indicators of innate immunity in ‘flown-healthy’ birds (flying in a wind tunnel without mounting an immune response), ‘flown-sick’ birds (flying while mounting an acute phase response, which is part of induced innate immunity), and a non-flying control group (‘not-flown’). Voluntary flight duration did not differ between flown-healthy and flown-sick birds, indicating that mounting an acute phase response to simulated infection did not hamper an individuals ability to fly for up to 3 h. However, in comparison to not-flown birds, bacterial killing ability of plasma was significantly reduced after flight in flown-sick birds. In flown-healthy birds, voluntary flight duration was positively correlated with bacterial killing ability and baseline haptoglobin concentration of the blood plasma measured 1–3 weeks before experimental flights, suggesting that high quality birds had strong immune systems and greater flight capacity. Our findings indicate that flight performance is not diminished by prior immune challenge, but that flight while mounting an acute phase response negatively affects other aspects of immune function. These findings have important implications for our understanding of the transmission of avian diseases, as they suggest that birds can still migrate while fighting an infection.

Indices of Immune Function are Lower in Red Knots (Calidris canutus) Recovering Protein than in those Storing Fat During Stopover in Delaware Bay

ABSTRACT. n Each year, hundreds of thousands of shorebirds use Delaware Bay, on the northeast coast of the United States, as a final stopover before migration to breeding areas. The bay provides them with abundant Horseshoe Crab (Limulus polyphemus) eggs, which they use to gain the fat stores necessary for continued migration and subsequent survival on the breeding grounds. However, abundant food attracts dense mixed-species flocks, which may facilitate pathogen transmission, and migration itself may suppress immune defense. Despite the potential importance of disease risk and immune function during migration, little is known about how immune function changes during stopover in migratory shorebirds. To examine this, we measured constitutive immune function in Red Knots (Calidris canutus rufa) during stopover in Delaware Bay. We found lower total leukocyte, lymphocyte, and monocyte concentrations, complement-mediated lysis, and haptoglobin activity in birds recovering protein after migration than in birds storing fat to fuel subsequent flight. We discuss two possible reasons for this result. First, fueling birds may have an increased rate of infection or may be bolstering immune defense in response to high antigen exposure. Second, recovering birds may be immuno-compromised because of the physical strain of migratory flight or as a result of adaptive tradeoffs between immune function and migration.

A novel integrative method for measuring body condition in ecological studies based on physiological dysregulation

1. The body condition of free-ranging animals affects their response to stress, decisions, ability to fulfil vital needs and, ultimately, fitness. However, this key attribute in ecology remains difficult to assess, and there is a clear need for more integrativemeasures than the common univariate proxies. 2. We propose a systems biology approach that positions individuals along a gradient from a ‘normal/optimal’ to ‘abnormal/suboptimal’ physiological state based onMahalanobis distance computed from physiological biomarkers. We previously demonstrated the validity of this approach for studying ageing in humans; here, we illustrate its broad potential for ecological studies. 3. As an example, we used biomarker data on shorebirds and found that birds with an abnormal condition had a lower maximal thermogenic capacity and higher scores of inflammation, with important implications for their ecology and health.Moreover,Mahalanobis distance captured a signal of condition not detected by the individual biomarkers. 4. Overall, our results on birds and humans show that individuals with abnormal physiologies are indeed in worse condition.Moreover, our approach appears not to be particularly sensitive to which set of biomarkers is used to assess condition. Consequently, it could be applied easily to existing ecological data sets. 5. Our approach provides a general, powerful way to measure condition that helps resolve confusion as to how to deal with complex interactions and interdependence among multiple physiological and condition measures. It can be applied directly to topics such as the effect of environmental quality on body condition, risks of health outcomes, mechanisms of adaptive phenotypic plasticity, and mechanisms behind long-term processes such as senescence.

Characterization of MHC class I in a long-distance migrant shorebird suggests multiple transcribed genes and intergenic recombination

The major histocompatibility complex (MHC) includes highly polymorphic gene families encoding proteins crucial to the vertebrate acquired immune system. Classical MHC class I (MHCI) genes code for molecules expressed on the surfaces of most nucleated cells and are associated with defense against intracellular pathogens, such as viruses. These genes have been studied in a few wild bird species, but have not been studied in long-distance migrating shorebirds. Red Knots Calidris canutus are medium-sized, monogamous sandpipers with migratory routes that span the globe. Understanding how such long-distance migrants protect themselves from disease has gained new relevance since the emergence of avian-borne diseases, including intracellular pathogens recognized by MHCI molecules, such as avian influenza. In this study, we characterized MHCI genes in knots and found 36 alleles in eight individuals and evidence for six putatively functional and expressed MHCI genes in a single bird. We also found evidence for recombination and for positive selection at putative peptide binding sites in exons 2 and 3. These results suggest surprisingly high MHC diversity in knots, given their demographic history. This may be a result of selection from diverse pathogens encountered by shorebirds throughout their annual migrations.