Free-living Vertebrates Research Articles

Cardiovascular contributions and energetic costs of thermoregulation in ectothermic vertebrates.

Ectothermic vertebrates use a suite of physiological and behavioral mechanisms to thermoregulate, which result in various thermoregulatory strategies from thermoconformity to thermoregulation. Here, we present a novel synthesis of theoretical and empirical methods to determine cardiovascular contributions to heat transfer in free-living ectothermic vertebrates. We start by identifying the fundamental components of heat transfer and the cardiovascular mechanisms for physiological modulation of heat exchange, and then integrate these components into a single, integrative framework: the cardiovascular heat exchange framework (CHEF). We demonstrate that this framework can identify details of the thermoregulatory strategy in two turtle species, most notably the preponderance of instances where turtles use physiological mechanisms to avoid overheating, suggesting vulnerability to climate change. As modulated physiological contributions to heat flow incur a greater energy demand than relying on unmodulated passive heat transfer, we then asked whether we could characterize the energetic costs of thermoregulation. We measured field metabolic rate (FMR) in free-living turtles and used the CHEF to determine FMR while actively or passively thermoregulating. Comparing an individual's actual FMR to the rate calculated assuming absence of thermoregulation revealed that painted turtles, a partial thermoregulator, elevate their daily energy expenditure (DEE) by about 25%, while box turtles, a thermoconformer, have a DEE that is nearly unchanged as a result of thermoregulation. This integrative framework builds a new paradigm that provides a mechanism to explain correlations between energy demand and thermoregulatory strategy, quantifies the energetic costs of thermoregulation, and identifies the role of cardiovascular contributions to thermoregulation in free-living animals.

A meta-analysis of impacts of immune response and infection on oxidative status in vertebrates.

Inferring from patterns observed in biomedical research, ecoimmunological theory predicts that oxidative stress is a ubiquitous physiological cost that contributes to generating variation in immune function between individuals or species. This prediction is, however, often challenged by empirical studies testing the relationship between immune response or infection and oxidative status markers. This points out the importance of combining ecological immunology and oxidative stress ecology to further our understanding of the proximate causes and fitness consequences of individual variation in health, and adaptability to natural and anthropogenic environmental changes. I reviewed evidence and performed phylogenetic meta-analyses of changes in oxidative status markers owing to either injection of an antigen or infection in captive and free-living vertebrates (141 studies, 1262 effect sizes, 97 species). The dataset was dominated by studies on fish, birds and mammals, which provided 95.8% of effect sizes. Both antigen injection and parasite exposure were associated with changes of oxidative status. There were significant effects of taxonomic class and experimental environment (captivity vs. wild). In contrast with my predictions, age category (young vs. adult), study design (correlational vs. experimental) and proxies of pace of life (clutch size, litter size, and body mass; for birds and mammals only) were negligible in this dataset. Several methodological aspects (type of immunostimulant, laboratory assay, tissue analysed) showed significant effects on both strength and direction of effect. My results suggest that alterations of oxidative status are a widespread consequence of immune function across vertebrates. However, this work also identified heterogeneity in strength and direction of effect sizes, which suggests that immune function does not necessarily result in oxidative stress. Finally, this work identifies methodological caveats that might be relevant for the interpretation and comparability of results and for the application in conservation programs.

The relationship between steroid hormones and seasonal reproductive events in free-living female Pygmy Rattlesnakes, Sistrurus miliarius

Steroid hormones regulate reproductive investment and play a critical role in coordinating reproductive responses to both short and long-term environmental change. Laboratory studies typically examine the mechanistic relationships among steroids and reproductive processes under controlled conditions and may fail to identify any seasonal patterns in regulatory mechanisms exhibited by free-living organisms. In snakes, field studies describing the seasonal relationships among steroids and male reproduction are prevalent. However, comparatively less work has been conducted on free-living female snakes. To bridge this gap in knowledge, we described the seasonal patterns of estradiol (E2), corticosterone (cort), and plasma constituents (total protein, albumin, phosphorus, and glucose) in relation to the seasonal events of the reproductive cycle in free-living female pygmy rattlesnakes (Sistrurus miliarius). We predicted to observe elevated E2 and plasma constituents during the spring vitellogenenic season, and that E2 would also be elevated during the fall mating season. Additionally, we hypothesized that cort acts to mobilize resources to meet the energetic challenges experienced during reproduction. We, therefore, predicted that elevated cort would be associated with the vitellogenic season and summer pregnancy, and that cort would be positively associated with elevated E2 and plasma constituents. In S. miliarius, plasma estradiol was significantly elevated during both the fall mating and spring vitellogenic seasons. Elevated E2 was associated with significantly higher plasma total protein, albumin, and phosphorous in both seasons, but unrelated to plasma glucose. Corticosterone was not elevated in individuals with elevated E2. Additionally, cort was not related to plasma total protein, albumin, or phosphorus, but was positively related to plasma glucose. Corticosterone was elevated during late pregnancy compared to early pregnancy and postpartum, and also elevated in pregnant compared to non-reproductive females sampled in the field. Our results indicate that the mechanistic links between steroids and reproductive processes may be seasonally uncoupled and highlight the need for the continued study of the hormonal coordination of reproduction in free-living vertebrates with diverse life histories.

Long-term effects of superoxide and DNA repair on lizard telomeres.

Telomeres are the non-coding protein-nucleotide "caps" at chromosome ends that contribute to chromosomal stability by protecting the coding parts of the linear DNA from shortening at cell division, and from erosion by reactive molecules. Recently, there has been some controversy between molecular and cell biologists, on the one hand, and evolutionary ecologists on the other, regarding whether reactive molecules erode telomeres during oxidative stress. Many studies of biochemistry and medicine have verified these relationships in cell culture, but other researchers have failed to find such effects in free-living vertebrates. Here, we use a novel approach to measure free radicals (superoxide), mitochondrial "content" (a combined measure of mitochondrial number and size in cells), telomere length and DNA damage at two primary time points during the mating season of an annual lizard species (Ctenophorus pictus). Superoxide levels early in the mating season vary widely and elevated levels predict shorter telomeres both at that time as well as several months later. These effects are likely driven by mitochondrial content, which significantly impacts late season superoxide (cells with more mitochondria have more superoxide), but superoxide effects on telomeres are counteracted by DNA repair as revealed by 8-hydroxy-2'-deoxyguanosine assays. We conclude that reactive oxygen species and DNA repair are fundamental for both short- and long-term regulation of lizard telomere length with pronounced effects of early season cellular stress detectable on telomere length near lizard death.

Luck in Food Finding Affects Individual Performance and Population Trajectories

Energy harvesting by animals is important because it provides the power needed for all metabolic processes. Beyond this, efficient food finding enhances individual fitness [1] and population viability [2], although rates of energy accumulation are affected by the environment and food distribution. Typically, differences between individuals in the rate of food acquisition are attributed to varying competencies [3], even though food-encounter rates are known to be probabilistic [4]. We used animal-attached technology to quantify food intake in four disparate free-living vertebrates (condors, cheetahs, penguins, and sheep) and found that inter-individual variability depended critically on the probability of food encounter. We modeled this to reveal that animals taking rarer food, such as apex predators and scavengers, are particularly susceptible to breeding failure because this variability results in larger proportions of the population failing to accrue the necessary resources for their young before they starve and because even small changes in food abundance can affect this variability disproportionately. A test of our model on wild animals indicated why Magellanic penguins have a stable population while the congeneric African penguin population has declined for decades. We suggest that such models predicting probabilistic ruin can help predict the fortunes of species operating under globally changing conditions.

Higher plasma oxidative damage and lower plasma antioxidant defences in an Arctic seabird exposed to longer perfluoroalkyl acids

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) may cause detrimental effects on physiological function and reproduction of Arctic animals. However, there is a paucity of information on the link between PFASs and oxidative stress, which can have potential detrimental effects on key fitness traits, such as cellular homeostasis or reproduction. We have examined the correlations between multiple blood-based markers of oxidative status and several perfluoroalkyl acids (i.e., with 8 or more carbons) in male Arctic black-legged kittiwakes (Rissa tridactyla) during the pre-laying period. Higher protein oxidative damage was found in those birds having higher concentrations of perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTriA) and perfluorotetradecanoic acid (PFTeA). Lower plasmatic non-enzymatic micro-molecular antioxidants were found in those birds having higher concentrations of perfluoroundecanoic acid (PFUnA), PFDoA and PFTeA. Effect size estimates showed that the significant correlations between PFASs and oxidative status markers were intermediate to strong. The non-enzymatic antioxidant capacity (including antioxidants of protein origin) was significantly lower in those birds having higher plasma concentration of linear perfluorooctanesulfonic acid (PFOSlin). In contrast, the activity of the antioxidant enzyme glutathione peroxidase in erythrocytes was not associated with any PFAS compounds. Our results suggest that increased oxidative stress might be one consequence of long-chain PFAS exposure. Experimental work will be needed to demonstrate whether PFASs cause toxic effects on free-living vertebrates through increased oxidative stress.

Brain-Derived Steroids, Behavior and Endocrine Conflicts Across Life History Stages in Birds: A Perspective

Biological steroids were traditionally thought to be synthesized exclusively by the adrenal glands and gonads. Recent decades have seen the discovery of neurosteroid production that acts locally within the central nervous system to affect physiology and behavior. These actions include, for example, regulation of aggressive behavior, such as territoriality, and locomotor movement associated with migration. Important questions then arose as to how and why neurosteroid production evolved and why similar steroids of peripheral origin do not always fulfill these central roles? Investigations of free-living vertebrates suggest that synthesis and action of bioactive steroids within the brain may have evolved to regulate expression of specific behavior in different life history stages. Synthesis and secretion of these hormones from peripheral glands is broadcast throughout the organism via the blood stream. While widespread, general actions of steroids released into the blood might be relevant for regulation of morphological, physiological, and behavioral traits in one life history stage, such hormonal release may not be appropriate in other stages. Specific and localized production of bioactive steroids in the brain, but not released into the periphery, could be a way to avoid such conflicts. Two examples are highlighted. First, we compare the control of territorial aggression of songbirds in the breeding season under the influence of gonadal steroids with autumnal (non-breeding) territoriality regulated by sex steroid production in the brain either from circulating precursors such as dehydroepiandrosterone or local central production of sex steroids de novo from cholesterol. Second, we outline the production of 7α-hydroxypregnenolone within the brain that appears to affect locomotor behavior in several contexts. Local production of these steroids in the brain may provide specific regulation of behavioral traits throughout the year and independently of life history stage.

The Evolutionary Endocrinology of Circulating Glucocorticoids in Free-Living Vertebrates: Recent Advances and Future Directions across Scales of Study.

Circulating glucocorticoid hormone concentrations are dynamic, flexible, and promote adaptive responses following perturbations in an animal's environment. As a result, circulating glucocorticoid levels are thought to shape fitness and have been suggested to be a key trait for predicting how species will cope with novel environmental change. Nevertheless, the factors that shape variation in glucocorticoid-mediated coping mechanisms remain unclear because the evolutionary underpinnings of the function and regulation of these hormones are poorly understood. Here, I summarize recent advances in our understanding of the evolution of circulating glucocorticoids, which have included (i) longitudinal studies exploring microevolutionary processes that shape within- and between-individual variation in glucocorticoids, (ii) interspecific comparative studies highlighting macro-evolutionary patterns of among-species variation in glucocorticoids, and (iii) intraspecific comparative studies which help to disentangle the relative roles of environment, life-history, and behavior in shaping among-population variation in glucocorticoids. Important avenues for future research will include exploring how natural selection may act on different components of the hypothalamus-pituitary-adrenal axis, characterizing patterns of phenotypic plasticity in circulating glucocorticoids across populations and species, as well as exploring how microevolutionary processes differ across taxa or gradients of environmental conditions.

Demographic, endocrine and behavioral responses to mirex in the South polar skua

Population consequences of chronic exposure to multiple pollutants at low environmental doses remain speculative, because of the lack of appropriate long-term monitoring surveys. This study integrates proximate and ultimate aspects of persistent organic pollutants (POP) burden in free-living vertebrates, by coupling hormonal and behavioral endpoints, life-history traits, and population dynamics. Blood samples (N=70) were collected in South polar skuas during two breeding periods, in 2003 and 2005, and individuals were annually monitored until 2011. Multi-state mark recapture models were used to test the effects of POP levels on demographic traits. Survival rate and long-term breeding probability were not related to individual POP levels, whereas long-term breeding success significantly decreased with increasing blood levels of mirex, an organochlorine insecticide. At the proximate level, corticosterone (stress hormone) and prolactin (parental care hormone) levels were not linked to individual POP burden. Nest defense in 2005 was significantly less intensive in chick-rearing skuas bearing higher mirex levels, suggesting reproductive behavioral impairment. Matrix population models were then built to project the rate of population decline according to increasing mirex burden. Although mirex levels were 2.8 times higher in 2003 than in 2005, the population-level effect of mirex was only detected in 2005, the year of higher corticosterone levels. The combination of endocrine traits with demographic analysis thereby enables to provide new support of synergistic interactions between pollutants and stress levels on long-term breeding outputs and population dynamics.

Luck in Food-Finding Affects Individual Performance and Population Trajectories

Differences between individuals in the rate of food acquisition are conventionally attributed to varying competencies even though food encounter rates are known to be probabilistic. We used animal-attached technology to quantify food intake in four disparate free-living vertebrates (condors, cheetahs, penguins and sheep) and found that inter-individual variability depended critically on the probability of food encounter. We modelled this to reveal that animals taking rarer food, such as apex predators and scavengers, are particularly susceptible to breeding failure because this variability results in larger proportions of the population failing to accrue the necessary resources for their young before they starve, and because even small changes in food abundance can affect this variability disproportionately. A test of our model on wild animals explained why Magellanic penguins have a stable population while the congeneric African penguin population has declined for decades. We suggest that such models predicting probabilistic ruin can help predict the fortunes of species operating under globally changing conditions.

Skin penetration in silico modeling-improvement by using in vitro data

Vitamin and carotenoid deficiency may impair development in free-living vertebrates, because of the importance of these micronutrients to growth, antioxidant defense and calcium regulation. Micronutrient and calcium insufficiency can be intensified by metal pollution which can interfere with nutrient homeostasis or indirectly reduce food availability. Furthermore, absorption of dietary heavy metals is dependent on food calcium and vitamin levels. We investigated the effect of calcium on plasma vitamin and carotenoid profiles and how these affected growth and survival in two passerine birds with different calcium turnover living along a metal pollution gradient. Vitamins (A, D3 and E) and carotenoids were quantified from blood plasma of great tit (Parus major) and pied flycatcher (Ficedula hypoleuca) nestlings. Metal concentrations in soil and in feces from the same nestlings were used to assess the exposure to air pollution. Additionally, we examined the vitamin level variation between developmental stages (eggs and nestlings within the same brood). Our results showed that generally higher concentrations of vitamins and carotenoids circulate in blood of great tits than in pied flycatchers. In general, birds inhabiting the polluted zone presented lower concentrations of the studied micronutrients. Calcium supplementation and metal pollution decreased vitamin A concentration in pied flycatcher, but not in great tit, while vitamin A affected growth and survival in great tit and pied flycatcher respectively. Our results suggest that populations under exposure to metal pollution may experience increased vitamin A deficiency, and that the two passerine species, while obtaining similar micronutrients in food, respond differently to environmental disturbance of nutrients.

Perfluorinated substances and telomeres in an Arctic seabird: Cross-sectional and longitudinal approaches

Telomeres are non-coding DNA repeats located at the termini of eukaryotic chromosomes, regulated by dynamic processes balancing shortening and maintenance. Despite a mechanism to slow-down telomere shortening, cell division leads to progressive attrition of chromosomes, leading to the onset of cellular senescence or apoptosis. However, telomere restoration based on telomerase activity is the primary mechanism for telomere maintenance. Telomere length is associated to health and survival and can be impacted by a broad panel of environmental factors. However, the effect of contaminants on telomeres is poorly known for living organisms. The aim of this study was to investigate relationships between some poly- and perfluoroalkyl substances (PFASs), body condition and telomere length by using both a cross-sectional and longitudinal approach in adult breeding Black-legged kittiwakes (Rissa tridactyla) from Svalbard. First, we examined the associations between absolute telomere length and PFASs contamination in a given year (cross-sectional approach). Second, we investigated the relationships between telomere dynamics and PFASs contamination within a two years’ time frame (longitudinal approach). Our results did not show any significant relationships of PFASs and body condition with absolute telomere length in a given year. Surprisingly, we found a positive and significant relationship between PFASs and telomere dynamics in both sexes with elongated telomere in birds bearing the highest concentrations of PFASs. Our study underlines (i) the need to investigate PFAS effects on telomere dynamics with a longitudinal approach and (ii) a potential positive effect of these contaminants on telomere length, with the most contaminated birds showing the slowest rate of telomere shortening or even displaying elongated ones. Our study is the first to report a relationship between PFASs and telomere length in free-living vertebrates. A possible underlying mechanism and other potential confounding factors are discussed.

The Physiology of Exercise in Free-Living Vertebrates: What Can We Learn from Current Model Systems?

Many behaviors crucial for survival and reproductive success in free-living animals, including migration, foraging, and escaping from predators, involve elevated levels of physical activity. However, although there has been considerable interest in the physiological and biomechanical mechanisms that underpin individual variation in exercise performance, to date, much work on the physiology of exercise has been conducted in laboratory settings that are often quite removed from the animal's ecology. Here we review current, laboratory-based model systems for exercise (wind or swim tunnels for migration studies in birds and fishes, manipulation of exercise associated with non-migratory activity in birds, locomotion in lizards, and wheel running in rodents) to identify common physiological markers of individual variation in exercise capacity and/or costs of increased activity. Secondly, we consider how physiological responses to exercise might be influenced by (1) the nature of the activity (i.e., voluntary or involuntary, intensity, and duration), and (2) resource acquisition and food availability, in the context of routine activities in free-living animals. Finally, we consider evidence that the physiological effects of experimentally-elevated activity directly affect components of fitness such as reproduction and survival. We suggest that developing more ecologically realistic laboratory systems, incorporating resource-acquisition, functional studies across multiple physiological systems, and a life-history framework, with reproduction and survival end-points, will help reveal the mechanisms underlying the consequences of exercise, and will complement studies in free-living animals taking advantage of new developments in wildlife-tracking.

Erratum: Use of erythrocyte indicators of health and condition in vertebrate ecophysiology: a review and appraisal.

An error has been identified in the abstract of the above article. The word ‘metricfres’ should be replaced by the word ‘metric's’ so that the sentence reads as follows: We suggest that (i) researchers should validate a metric's utility before use, (ii) multiple metrics should be used to construct an overall erythrocyte profile for an individual or population, (iii) there is a need for researchers to compile reference ranges for free-living species, and (iv) some metrics which are useful under controlled, clinical conditions may not have the same utility or applicability for free-living vertebrates. We apologise for this error.

Vitamin profiles in two free-living passerine birds under a metal pollution gradient – A calcium supplementation experiment

Vitamin and carotenoid deficiency may impair development in free-living vertebrates, because of the importance of these micronutrients to growth, antioxidant defense and calcium regulation. Micronutrient and calcium insufficiency can be intensified by metal pollution which can interfere with nutrient homeostasis or indirectly reduce food availability. Furthermore, absorption of dietary heavy metals is dependent on food calcium and vitamin levels. We investigated the effect of calcium on plasma vitamin and carotenoid profiles and how these affected growth and survival in two passerine birds with different calcium turnover living along a metal pollution gradient. Vitamins (A, D3 and E) and carotenoids were quantified from blood plasma of great tit (Parus major) and pied flycatcher (Ficedula hypoleuca) nestlings. Metal concentrations in soil and in feces from the same nestlings were used to assess the exposure to air pollution. Additionally, we examined the vitamin level variation between developmental stages (eggs and nestlings within the same brood). Our results showed that generally higher concentrations of vitamins and carotenoids circulate in blood of great tits than in pied flycatchers. In general, birds inhabiting the polluted zone presented lower concentrations of the studied micronutrients. Calcium supplementation and metal pollution decreased vitamin A concentration in pied flycatcher, but not in great tit, while vitamin A affected growth and survival in great tit and pied flycatcher respectively. Our results suggest that populations under exposure to metal pollution may experience increased vitamin A deficiency, and that the two passerine species, while obtaining similar micronutrients in food, respond differently to environmental disturbance of nutrients.

The pathophysiology of survival in harsh environments.

An individual's ability to survive harsh conditions might depend on its available energy, and also on its health, which is expected to decline as conditions deteriorate. Yet, we know little about how health and energy expenditure are shaped by harsh environmental conditions in free-living vertebrates. Here, we studied how African striped mice (Rhabdomys pumilio) that survived summer droughts differed in their energy expenditure and health from non-survivors. Specifically, we tested whether: (1) survivors' and non-survivors' health and energy expenditure differed before environmental conditions declined; (2) non-survivors were in poorer health and had greater energy expenditure than survivors when conditions were harshest; (3) non-survivors' health deteriorated more than that of survivors as conditions deteriorated; and (4) survivors recovered once conditions improved. Survivors and non-survivors' health was assessed using VetsScan ABAXIS, while energy expenditure was measured as resting metabolic rate (RMR). Before conditions declined, non-survivors had lower energy stores and higher globulin levels than survivors. As conditions became harsher, survivors' and non-survivors' health deteriorated but only non-survivors showed signs of permanent pathology (increased glucose and decreased globulin). Once conditions improved, survivors' health improved but was not fully restored (increased alanine aminotransferase and decreased globulin). Furthermore, while survivors and non-survivors had similar RMR before conditions became harsh; their levels diverged considerably when conditions deteriorated, with survivors having a decreased RMR and non-survivors having an increased RMR. Our results show that an individual's health before facing an environmental challenge and the way it regulates its RMR influences its ability to maintain homeostasis when conditions become more taxing.

Predators inhibit brain cell proliferation in natural populations of electric fish, Brachyhypopomus occidentalis.

Compared with laboratory environments, complex natural environments promote brain cell proliferation and neurogenesis. Predators are one important feature of many natural environments, but, in the laboratory, predatory stimuli tend to inhibit brain cell proliferation. Often, laboratory predatory stimuli also elevate plasma glucocorticoids, which can then reduce brain cell proliferation. However, it is unknown how natural predators affect cell proliferation or whether glucocorticoids mediate the neurogenic response to natural predators. We examined brain cell proliferation in six populations of the electric fish, Brachyhypopomus occidentalis, exposed to three forms of predator stimuli: (i) natural variation in the density of predatory catfish; (ii) tail injury, presumably from predation attempts; and (iii) the acute stress of capture. Populations with higher predation pressure had lower density of proliferating (PCNA+) cells, and fish with injured tails had lower proliferating cell density than those with intact tails. However, plasma cortisol did not vary at the population level according to predation pressure or at the individual level according to tail injury. Capture stress significantly increased cortisol, but only marginally decreased cell proliferation. Thus, it appears that the presence of natural predators inhibits brain cell proliferation, but not via mechanisms that depend on changes in basal cortisol levels. This study is the first demonstration of predator-induced alteration of brain cell proliferation in a free-living vertebrate.

Plasma-Glucocorticoids and ACTH Levels During Different Periods of Activity in the European Beaver (Castor fiber L.).

Glucocorticoids (GCs) and adrenocorticotropic hormone (ACTH) are major components of the classic endocrine stress response. Free-living vertebrates are characterized by circannual changes in the baseline and/or stress-induced secretion of GCs and ACTH. In mammalian species, GC and ACTH levels vary seasonally but there is no consensus to the season in which animals have elevated GC and ACTH levels. The aim of our study was to determine, for the first time, the type and amount of glucocorticoids produced in free-living beaver (Castor fiber L.)--the largest rodent in Eurasia, and to find out whether stress-induced plasma GC and ACTH levels show seasonal variations. Blood samples were obtained from animals under general anesthesia in April (pregnancy in females), July (offspring rearing) and November (preparing for the winter). The adrenals of beavers produce both cortisol and corticosterone, and plasma cortisol levels were higher than corticosterone. In the current experiment, plasma cortisol concentrations in beavers were affected by the season. The highest stress-associated cortisol levels were noted in males in July during offspring rearing. Corticosterone and ACTH concentrations in beavers remained generally constant, regardless of the season and sex. In conclusion, seasonal changes were observed only in relation to stress-induced plasma cortisol levels in the beaver.

Use of erythrocyte indicators of health and condition in vertebrate ecophysiology: a review and appraisal.

We review evidence for and against the use of erythrocyte indicators of health status and condition, parasite infection level and physiological stress in free-living vertebrates. The use of indicators that are measured directly from the blood, such as haemoglobin concentration, haematocrit and erythrocyte sedimentation rate, and parameters that are calculated from multiple measured metrics, such as mean cell volume, mean cell haemoglobin content or mean cell haemoglobin concentration is evaluated. The evidence for or against the use of any given metric is equivocal when the relevant research is considered in total, although there is sometimes strong support for using a particular metric in a particular taxon. Possibly the usefulness of these metrics is taxon, environment or condition specific. Alternatively, in an uncontrolled environment where multiple factors are influencing a metric, its response to environmental change will sometimes, but not always, be predictable. We suggest that (i) researchers should validate a metric's utility before use, (ii) multiple metrics should be used to construct an overall erythrocyte profile for an individual or population, (iii) there is a need for researchers to compile reference ranges for free-living species, and (iv) some metrics which are useful under controlled, clinical conditions may not have the same utility or applicability for free-living vertebrates. Erythrocyte metrics provide useful information about health and condition that can be meaningfully interpreted in free-living vertebrates, but their use requires careful forethought about confounding factors.

Motivation but not body size influences territorial contest dynamics in a wild cichlid fish

Contests over resources are a key facet of social behaviour and have received extensive theoretical attention. However, the assumptions and predictions of the theoretical models have rarely been tested experimentally in wild free-living vertebrates. Here, we look at resource contests in wild Neolamprologus pulcher, a cooperatively breeding cichlid fish that lives in permanent territories in Lake Tanganyika, Africa. To elicit a contest, we removed a dominant breeding male from his territory and held him for either a short (4.5 h) or a long (20 h) period. The original resident male was then returned to his territory, which typically resulted in an aggressive contest with a usurping male that had taken over in the original resident's absence. We found that contests were shorter than those previously observed in a laboratory setting, with more physically aggressive attacks. Contrary to our predictions, the relative size difference between the males had no effect on contest dynamics or outcome, probably because all of the males involved in these contests were similar in body size. Instead, motivational factors influenced contest dynamics. Longer original resident removal times increased usurper male aggression, as well as the duration and intensity of the contests. Original residents were more aggressive when contesting over their sole territory (socially monogamous) than when contesting over one of their multiple territories (socially polygynous). Usurpers won more contests overall, and more aggressive fish were more likely to win contests. To our knowledge, this is the first study to examine resource contests in wild free-swimming cichlids, and our results reveal that subjective resource value is a primary driver of the dynamics and outcome of territorial conflicts among dominant N. pulcher males. Our results further suggest that respect for ownership may reduce conflict in N. pulcher and be important in governing colony structure.