Large Mammals Have More Powerful Antibacterial Defenses Than Expected from Their Metabolic Rates.

AbstractTerrestrial mammals span seven orders of magnitude in body size, ranging from the <2-g Etruscan pygmy shrew (Suncus etruscus) to the >3,900-kg African elephant (Loxodonta africana). Although body size profoundly affects the behavior, physiology, ecology, and evolution of species, how investment in functional immune defenses changes with body size across species is unknown. Here, we (1) developed a novel 12-point dilution curve approach to describe and compare antibacterial capacity against three bacterial species among >160 terrestrial species of mammals and (2) tested published predictions about the scaling of immune defenses. Our study focused on the safety factor hypothesis, which predicts that broad, early-acting immune defenses should scale hypermetrically with body mass. However, our three statistical approaches demonstrated that antibacterial activity in sera across mammals exhibits isometry; killing capacity did not change with body size across species. Intriguingly, this result indicates that the serum of a large mammal is less hospitable to bacteria than would be predicted by its metabolic rates. In other words, if metabolic rates underlie the rates of physiological reactions as postulated by the metabolic theory of ecology, large species should have disproportionately lower antibacterial capacity than small species, but they do not. These results have direct implications for effectively modeling the evolution of immune defenses and identifying potential reservoir hosts of pathogens.

Snap-freezing in the Field: Effect of Sample Holding Time on Performance of Bactericidal Assays.

Comparative analyses in biology rely on the quality of available data. Methodological differences among studies may introduce variation in results that obscure patterns. In the field of eco-immunology, functional immune assays such as antimicrobial capacity assays are widely used for among-species applications. Sample storage time and animal handling time can influence assay results in some species, but how sample holding time prior to freezing influences assay results is unknown. Sample holding time can vary widely in field studies on wild animals, prompting the need to understand the implications of such variation on assay results. We investigated the hypothesis that sample holding time prior to freezing influences assay results in six species (Leiocephalus carinatus, Iguana iguana, Loxodonta africana, Ceratotherium simum, Columba livia, and Buteo swainsoni) by comparing antibacterial capacity of serum with varying processing times prior to snap-freezing. Blood was collected once from each individual and aliquots were placed on ice and assigned different holding times (0, 30, 60, 180, and 240 min), after which each sample was centrifuged, then serum was separated and snap-frozen on dry ice and stored at -80ºC for 60 days prior to assaying. For each aliquot, we conducted antibacterial capacity assays with serial dilutions of serum inoculated with E. coli and extracted the dilution at 50% antibacterial capacity for analysis. We found a decrease in antibacterial capacity with increased holding time in one of the six species tested (B. swainsoni), driven in part by complete loss of antibacterial capacity in some individuals at the 240-min time point. While the majority of species' antibacterial capacity were not affected, our results demonstrate the need to conduct pilot assays spanning the anticipated variation in sample holding times to develop appropriate field protocols.

Physiological effects of capture and short-term captivity in an invasive snake species, the Burmese python (Python bivittatus) in Florida.

It is important to evaluate the role of captivity as a potential stressor. An understanding of stress responses to capture and transition to captivity may inform the limitations of laboratory studies on wild animals, aid in understanding the consequences of introducing animals into captive environments, and help predict which species may be successful invasives. We investigated physiological effects of captivity by comparing at-capture blood variables in wild Burmese pythons (Python bivittatus) in Florida to pythons recently brought into captivity (1-109 days). We conducted an acute restraint test by collecting samples at baseline (immediately at handling) and one hour post-restraint across wild field-sampled (n = 19) and recently-captive (n = 33) pythons to evaluate fluctuations in plasma corticosterone, bacterial killing ability, antibody response, leukogram, and serpentovirus infection. We observed higher baseline plasma corticosterone and monocytes in recently captive compared to wild snakes, which both subsided in snakes held for a longer time in captivity, and a mild decrease in lymphocytes in the middle of the captivity period. Functional immunity and viral infection were not affected by captivity, and pythons maintained restraint-induced responses in corticosterone, heterophil to lymphocyte ratio, and monocyte counts throughout captivity. Prevalence for serpentovirus was 50%, though infection status was related to sampling date rather than captivity, indicating that viral infection may be seasonal. The history of Burmese python as a common captive animal for research and pet trade, as well as its general resilience to effects of capture and short-term captivity, may contribute to its invasion success in Florida.

Life history and environment predict variation in testosterone across vertebrates.

Endocrine systems act as key intermediaries between organisms and their environments. This interaction leads to high variability in hormone levels, but we know little about the ecological factors that influence this variation within and across major vertebrate groups. We study this topic by assessing how various social and environmental dynamics influence testosterone levels across the entire vertebrate tree of life. Our analyses show that breeding season length and mating system are the strongest predictors of average testosterone concentrations, whereas breeding season length, environmental temperature, and variability in precipitation are the strongest predictors of within-population variation in testosterone. Principles from small-scale comparative studies that stress the importance of mating opportunity and competition on the evolution of species differences in testosterone levels, therefore, likely apply to the entire vertebrate lineage. Meanwhile, climatic factors associated with rainfall and ambient temperature appear to influence variability in plasma testosterone, within a given species. These results, therefore, reveal how unique suites of ecological factors differentially explain scales of variation in circulating testosterone across mammals, birds, reptiles, amphibians, and fishes.

Does variation in glucocorticoid concentrations predict fitness? A phylogenetic meta-analysis.

Glucocorticoid hormones (GCs) are central mediators of metabolism and the response to challenges. Because circulating GC levels increase in response to challenges, within-population variation in GCs could reflect among-individual variation in condition or experience. At the same time, individual variation in GC regulation could have causal effects on energetic balance or stress coping capacity in ways that influence fitness. Although a number of studies in vertebrates have tested whether variation in GCs among individuals predicts components of fitness, it is not clear whether there are consistent patterns across taxa. Here we present the first phylogenetic meta-analysis testing whether variation in GCs is associated with survival and reproductive success across vertebrates. At the same time, we introduce and test predictions about a potentially important mediator of GC-fitness relationships: life history context. We suggest that strong context-dependence in the fitness benefit of maintaining elevated GCs could obscure consistent patterns between GCs and fitness across taxa. Meta-analyses revealed that baseline and stress-induced GCs were consistently negatively correlated with reproductive success. This relationship did not differ depending on life history context. In contrast, the relationships between GCs and survival were highly context dependent, differing according to life history strategy. Both baseline and stress-induced GCs were more strongly negatively associated with survival in longer-lived populations and species. Stress-induced GCs were also more positively associated with survival in organisms that engage in relatively more valuable reproductive attempts. Fecal GCs did not correlate with survival or reproductive success. We also found that experimental increases in GCs reduced both survival and reproductive success; however, evidence of publication bias and the small sample size suggest that more data is required to confirm this conclusion. Overall, these results support the prediction that GC-fitness relationships can be strongly context dependent, and suggest that incorporating life history may be particularly important for understanding GC-survival relationships.

Baseline and stress-induced corticosterone levels across birds and reptiles do not reflect urbanization levels.

Rates of human-induced environmental change continue increasing with human population size, potentially altering animal physiology and negatively affecting wildlife. Researchers often use glucocorticoid concentrations (hormones that can be associated with stressors) to gauge the impact of anthropogenic factors (e.g. urbanization, noise and light pollution). Yet, no general relationships between human-induced environmental change and glucocorticoids have emerged. Given the number of recent studies reporting baseline and stress-induced corticosterone (the primary glucocorticoid in birds and reptiles) concentrations worldwide, it is now possible to conduct large-scale comparative analyses to test for general associations between disturbance and baseline and stress-induced corticosterone across species. Additionally, we can control for factors that may influence context, such as life history stage, environmental conditions and urban adaptability of a species. Here, we take a phylogenetically informed approach and use data from HormoneBase to test if baseline and stress-induced corticosterone are valid indicators of exposure to human footprint index, human population density, anthropogenic noise and artificial light at night in birds and reptiles. Our results show a negative relationship between anthropogenic noise and baseline corticosterone for birds characterized as urban avoiders. While our results potentially indicate that urban avoiders are more sensitive to noise than other species, overall our study suggests that the relationship between human-induced environmental change and corticosterone varies across species and contexts; we found no general relationship between human impacts and baseline and stress-induced corticosterone in birds, nor baseline corticosterone in reptiles. Therefore, it should not be assumed that high or low levels of exposure to human-induced environmental change are associated with high or low corticosterone levels, respectively, or that closely related species, or even individuals, will respond similarly. Moving forward, measuring alternative physiological traits alongside reproductive success, health and survival may provide context to better understand the potential negative effects of human-induced environmental change.

Macroevolutionary Patterning in Glucocorticoids Suggests Different Selective Pressures Shape Baseline and Stress-Induced Levels.

Glucocorticoid (GC) hormones are important phenotypic mediators across vertebrates, but their circulating concentrations can vary markedly. Here we investigate macroevolutionary patterning in GC levels across tetrapods by testing seven specific hypotheses about GC variation and evaluating whether the supported hypotheses reveal consistent patterns in GC evolution. If selection generally favors the "supportive" role of GCs in responding effectively to challenges, then baseline and/or stress-induced GCs may be higher in challenging contexts. Alternatively, if selection generally favors "protection" from GC-induced costs, GCs may be lower in environments where challenges are more common or severe. The predictors of baseline GCs were all consistent with supportive effects: levels were higher in smaller organisms and in those inhabiting more energetically demanding environments. During breeding, baseline GCs were also higher in populations and species with fewer lifetime opportunities to reproduce. The predictors of stress-induced GCs were instead more consistent with the protection hypothesis: during breeding, levels were lower in organisms with fewer lifetime reproductive opportunities. Overall, these patterns indicate a surprising degree of consistency in how some selective pressures shape GCs across broad taxonomic scales; at the same time, in challenging environments selection appears to operate on baseline and stress-induced GCs in distinct ways.

Exogenous glucocorticoids amplify the costs of infection by reducing resistance and tolerance, but effects are mitigated by co-infection.

Individual variation in parasite defences, such as resistance and tolerance, can underlie heterogeneity in fitness and could influence disease transmission dynamics. Glucocorticoid hormone concentrations often change in response to fluctuating environmental conditions and mediate changes in immune function, resource allocation and tissue repair. Thus, changes in glucocorticoid hormone concentrations might mediate individual variation in investment in resistance versus tolerance. In this study, we experimentally increased glucocorticoid concentrations in red-winged blackbirds ( Agelaius phoeniceus) that were naturally infected with haemosporidian parasites, and assessed changes in resistance and tolerance of infection. Glucocorticoid treatment increased burdens of Plasmodium, the parasite causing avian malaria, but only in the absence of co-infection with another Haemosporidian, Haemoproteus. Thus, glucocorticoids might reduce resistance to infection, but co-infection can mitigate the negative consequences of increased hormone concentrations. Glucocorticoid treatment also decreased tolerance of infection. We found no evidence that the inflammatory immune response or rate of red blood cell production underlie the effects of glucocorticoids on resistance and tolerance. Our findings suggest that exogenous glucocorticoids can increase the costs of haemosporidian infections by both increasing parasite numbers and reducing an individual's ability to cope with infection. These effects could scale up to impact populations of both host and parasite.

Scaling of Host Competence.

Body size influences many traits including those that affect host competence, the propensity to cause new infections. Here, we employ a new framework to reveal that, for at least two infections, West Nile virus and Lyme disease, large hosts should be more competent than small ones, but their lower abundance could mitigate their impacts on local risk. By contrast, for rabies, small hosts will be disproportionately more competent than large ones, an effect amplified by the higher densities of small species. These outcomes differ quite a bit from previous approaches that incorporate allometries into epidemiological models. Subsequently, we advocate for future integrative work to resolve how interspecific variation in body size influences the emergence and spread of infections.

IUCN Conservation Status Does Not Predict Glucocorticoid Concentrations in Reptiles and Birds.

Circulating glucocorticoids (GCs) are the most commonly used biomarkers of stress in wildlife. However, their utility as a tool for identifying and/or managing at-risk species has varied. Here, we took a very broad approach to conservation physiology, asking whether International Union for the Conservation of Nature (IUCN) listing status (concern versus no obvious concern) and/or location within a geographic range (edge versus non-edge) predicted baseline and post-restraint concentrations of corticosterone (CORT) among many species of birds and reptiles. Even though such an approach can be viewed as coarse, we asked in this analysis whether CORT concentrations might be useful to implicate species at risk. Indeed, our effort, relying on HormoneBase, a repository of data on wildlife steroids, complements several other large-scale efforts in this issue to describe and understand GC variation. Using a phylogenetically informed Bayesian approach, we found little evidence that either IUCN status or edge/non-edge location in a geographic distribution were related to GC levels. However, we did confirm patterns described in previous studies, namely that breeding condition and evolutionary relatedness among species predicted some GC variation. Given the broad scope of our work, we are reluctant to conclude that IUCN status and location within a range are unrelated to GC regulation. We encourage future more targeted efforts on GCs in at-risk populations to reveal how factors leading to IUCN listing or the environmental conditions at range edges impact individual performance and fitness, particularly in the mammals, amphibians, and fish species we could not study here because data are currently unavailable.

Species-Specific Means and Within-Species Variance in Glucocorticoid Hormones and Speciation Rates in Birds.

At macroevolutionary scales, stress physiology may have consequences for species diversification and subspecies richness. Populations that exploit new resources or undergo range expansion should cope with new environmental challenges, which could favor higher mean stress responses. Within-species variation in the stress response may also play a role in mediating the speciation process: in species with broad variation, there will always be some individuals that can tolerate an unpredictable environment, whereas in species with narrow variation there will be fewer individuals that are able to thrive in a new ecological niche. We tested for the evolutionary relationship between stress response, speciation rate, and subspecies richness in birds by relying on the HormoneBase repository, from which we calculated within- and among-species variation in baseline (BL) and stress-induced (SI) corticosterone levels. To estimate speciation rates, we applied Bayesian analysis of macroevolutionary mixtures that can account for variation in diversification rate among clades and through time. Contrary to our predictions, lineages with higher diversification rates were not characterized by higher BL or SI levels of corticosterone either at the tips or at the deeper nodes of the phylogeny. We also found no association between mean hormone levels and subspecies richness. Within-species variance in corticosterone levels showed close to zero repeatability, thus it is highly unlikely that this is a species-specific trait that influences diversification rates. These results imply that stress physiology may play a minor, if any, role in determining speciation rates in birds.

Do Seasonal Glucocorticoid Changes Depend on Reproductive Investment? A Comparative Approach in Birds.

Animals go through different life history stages such as reproduction, moult, or migration, of which some are more energy-demanding than others. Baseline concentrations of glucocorticoid hormones increase during moderate, predictable challenges and thus are expected to be higher when seasonal energy demands increase, such as during reproduction. By contrast, stress-induced glucocorticoids prioritize a survival mode that includes reproductive inhibition. Thus, many species down-regulate stress-induced glucocorticoid concentrations during the breeding season. Interspecific variation in glucocorticoid levels during reproduction has been successfully mapped onto reproductive investment, with species investing strongly in current reproduction (fast pace of life) showing higher baseline and lower stress-induced glucocorticoid concentrations than species that prioritize future reproduction over current attempts (slow pace of life). Here we test the "glucocorticoid seasonal plasticity hypothesis", in which we propose that interspecific variation in seasonal changes in glucocorticoid concentrations from the non-breeding to the breeding season will be related to the degree of reproductive investment (and thus pace of life). We extracted population means for baseline (for 54 species) and stress-induced glucocorticoids (for 32 species) for the breeding and the non-breeding seasons from the database "HormoneBase", also calculating seasonal glucocorticoid changes. We focused on birds because this group offered the largest sample size. Using phylogenetic comparative methods, we first showed that species differed consistently in both average glucocorticoid concentrations and their changes between the two seasons, while controlling for sex, latitude, and hemisphere. Second, as predicted seasonal changes in baseline glucocorticoids were explained by clutch size (our proxy for reproductive investment), with species laying larger clutches showing a greater increase during the breeding season-especially in passerine species. In contrast, changes in seasonal stress-induced levels were not explained by clutch size, but sample sizes were more limited. Our findings highlight that seasonal changes in baseline glucocorticoids are associated with a species' reproductive investment, representing an overlooked physiological trait that may underlie the pace of life.

Metabolic Scaling of Stress Hormones in Vertebrates.

Glucocorticoids (GCs) are stress hormones that can strongly influence physiology, behavior, and an organism's ability to cope with environmental change. Despite their importance, and the wealth of studies that have sought to understand how and why GC concentrations vary within species, we do not have a clear understanding of how circulating GC levels vary within and across the major vertebrate clades. New research has proposed that much interspecific variation in GC concentrations can be explained by variation in metabolism and body mass. Specifically, GC concentrations should vary proportionally with mass-specific metabolic rates and, given known scaling relationships between body mass and metabolic rate, GC concentrations should scale to the -1/4 power of body mass and to the power of 1 with mass-specific metabolic rate. Here, we use HormoneBase, the newly compiled database that includes plasma GC concentrations from free-living and unmanipulated vertebrates, to evaluate this hypothesis. Specifically, we explored the relationships between body mass or mass-specific metabolic rate and either baseline or stress-induced GC (cortisol or corticosterone) concentrations in tetrapods. Our phylogenetically-informed models suggest that, whereas the relationship between GC concentrations and body mass across tetrapods and among mammals is close to -1/4 power, this relationship does not exist in amphibians, reptiles, and birds. Moreover, with the exception of a positive association between stress-induced GC concentrations and mass-specific metabolic rate in birds, we found little evidence that GC concentrations are linked to metabolic rate, although the number of species sampled was quite limited for amphibians and somewhat so for reptiles and mammals. Nevertheless, these results stand in contrast to the generally accepted association between the two and suggest that our observed positive association between body mass and GC concentrations may not be due to the well-established link between mass and metabolism. Large-scale comparative approaches can come with drawbacks, such as pooling and pairing observations from separate sources. However, these broad analyses provide an important counterbalance to the majority of studies examining variation in GC concentrations at the population or species level, and can be a powerful approach to testing both long-standing and new questions in biology.

Understanding Context Dependence in Glucocorticoid-Fitness Relationships: The Role of the Nature of the Challenge, the Intensity and Frequency of Stressors, and Life History.

It has been well-established that there is variation in the strength and direction of the relationship between circulating glucocorticoids (GCs) and fitness. When studies demonstrate such variation or the direction of the GC-fitness relationship is unexpected, the results are often attributed to context-dependency. However, descriptors of context can be vague (e.g., "environmental context") and few studies explicitly test how the optimal hypothalamic-pituitary-adrenal (HPA) axis response to stressors varies across specific contexts. Although existing hypotheses create a strong foundation for understanding GC-fitness relationships, many do not provide explicit predictions of how, when, and why the relationships will change. Here, we discuss three broad factors which we expect to shape the relationships between HPA axis activity and fitness metrics: (1) whether the HPA axis-mediated response matches the challenge, (2) the intensity and frequency of challenges, and (3) life history. We also make predictions for how these factors might affect GC-fitness relationships and discuss ways to test these predictions. Observational studies, experimental manipulations of context, and large-scale cross-species comparisons will be critical to understanding the observed variation in GC-fitness relationships.

HormoneBase, a population-level database of steroid hormone levels across vertebrates.

Hormones are central regulators of organismal function and flexibility that mediate a diversity of phenotypic traits from early development through senescence. Yet despite these important roles, basic questions about how and why hormone systems vary within and across species remain unanswered. Here we describe HormoneBase, a database of circulating steroid hormone levels and their variation across vertebrates. This database aims to provide all available data on the mean, variation, and range of plasma glucocorticoids (both baseline and stress-induced) and androgens in free-living and un-manipulated adult vertebrates. HormoneBase (www.HormoneBase.org) currently includes >6,580 entries from 476 species, reported in 648 publications from 1967 to 2015, and unpublished datasets. Entries are associated with data on the species and population, sex, year and month of study, geographic coordinates, life history stage, method and latency of hormone sampling, and analysis technique. This novel resource could be used for analyses of the function and evolution of hormone systems, and the relationships between hormonal variation and a variety of processes including phenotypic variation, fitness, and species distributions.

The repeatability of glucocorticoids: A review and meta-analysis.

Glucocorticoids are highly conserved hormones that mediate a suite of responses to changing conditions in vertebrates. Recent work has focused on understanding how selection operates on glucocorticoid secretion in natural populations. Because heritability is rarely estimated and difficult to measure in the wild, many studies report within-individual repeatability as an estimate of stable between individual differences in glucocorticoid secretion. We conducted a systematic review and meta-analysis on estimates of within-individual glucocorticoid repeatability to elucidate general patterns of repeatability, and to test for relationships between covariates and estimates of repeatability. To this end, we collected 203 estimates of within-individual glucocorticoid repeatability drawn from 71 separate studies and 55 species. Overall, we found moderate levels of repeatability (0.29). We also found that repeatability varied by sample type. Long-term measures (e.g., fecal and feather samples) and acute stress-induced plasma glucocorticoids had higher repeatability (long-term: 0.44, stress-induced: 0.38), than baseline glucocorticoid levels (0.18). Repeatability also decreased with increasing time between repeated sampling events. Despite significant overall repeatability, there was substantial heterogeneity in estimates from different studies, suggesting that repeatability of glucocorticoid secretion varies substantially across systems and conditions. We discuss the implications of our results for understanding selection on glucocorticoid traits and suggest that continuing work should focus on evaluating the repeatability of within-individual glucocorticoid reaction norms.

Higher plasma corticosterone is associated with reduced costs of infection in red-winged blackbirds.

Glucocorticoid hormones allow individuals to rapidly adjust their physiology and behavior to meet the challenges of a variable environment. An individual's baseline concentration of glucocorticoids can reflect shifts in life history stage and resource demands while mediating a suite of physiological and behavioral changes that include immune modulation and resource allocation. Thus, glucocorticoids could facilitate a response to parasites that is optimized for an individual's specific challenges and life history stage. We investigated the relationship between endogenous circulating glucocorticoids and measures of resistance and tolerance to Haemosporidian parasites (including those that cause avian malaria) in red-winged blackbirds (Agelaius phoeniceus). We found that higher endogenous concentrations of circulating glucocorticoids were associated with reduced costs of parasite infection, which is indicative of higher tolerance, but were unrelated to parasite burden in free ranging, breeding male birds. Post-breeding, both males and females with higher glucocorticoid concentrations had higher measures of tolerance to Haemosporidian infection. Our findings suggest a potentially adaptive role for glucocorticoids in shifting the response to parasites to align with an individual's current physiological state and the challenges they face.

An experimental test of the physiological consequences of avian malaria infection.

Chronic, low-intensity parasite infections can reduce host fitness through negative impacts on reproduction and survival, even if they produce few overt symptoms. As a result, these parasites can influence the evolution of host morphology, behaviour and physiology. The physiological consequences of chronic infection can provide insight into the processes underlying parasite-driven natural selection. Here, we evaluate the physiological consequences of natural, low-intensity infection in an avian host-parasite system: adult male red-winged blackbirds (Agelaius phoeniceus) infected with haemosporidian parasites. Chronic haemosporidian infection has previously been shown to reduce both reproductive success and survival in several avian species. We used antimalarial medications to experimentally reduce haemosporidian parasitaemia (the proportion of blood cells infected with haemosporidian parasites) and measured the effect of treatment on body condition, haematology, immune function, physiological stress and oxidative state. Treatment with an antimalarial medication reduced parasitaemia for the most prevalent haemosporidian parasites from the genus Plasmodium. Treatment also increased haemoglobin and haematocrit, and decreased red blood cell production rates. We detected no effect of treatment on body condition, immune metrics, plasma corticosterone concentrations, total antioxidant capacity or reactive oxygen metabolites. Our results suggest that the damage and replacement of red blood cells during infection could be important costs of chronic haemosporidian infection. Strong links between parasitaemia and the physiological consequences of infection indicate that even for relatively low-intensity infections, measuring parasitaemia rather than only presence/absence could be important when evaluating the role of infection in influencing hosts' behaviour, physiology or fitness.

Why do caterpillars whistle at birds? Insect defence sounds startle avian predators.

Many insects produce sounds when attacked by a predator, yet the functions of these signals are poorly understood. It is debated whether such sounds function as startle, warning or alarm signals, or merely serve to augment other defences. Direct evidence is limited owing to difficulties in disentangling the effects of sounds from other defences that often occur simultaneously in live insects. We conducted an experiment to test whether an insect sound can function as a deimatic (i.e. startle) display. Variations of a whistle of the walnut sphinx caterpillar (Amorpha juglandis) were presented to a predator, red-winged blackbirds (Agelaius phoeniceus), when birds activated a sensor while feeding on mealworms (Tenebrio molitor). Birds exposed to whistles played back at natural sound levels exhibited significantly higher startle scores (by flying away, flinching, and hopping) and took longer to return to the feeding dish than during control conditions where no sounds were played. Birds habituated to sounds during a one-hour session, but after two days the startling effects were restored. Our results provide empirical evidence that an insect sound alone can function as a deimatic display against an avian predator. We discuss how whistles might be particularly effective 'acoustic eye spots' on avian predators.