Christopher P. Johnstone

Interpreting indices of physiological stress in free-living vertebrates

When vertebrate physiological ecologists use the terms ‘stress’ or ‘physiological stress’, they typically mean the level of hypothalamus–pituitary–adrenal (HPA-) axis activation. Measurements of stress hormone concentrations (e.g. glucocorticoids in blood, urine or faeces), leukocytes (e.g. the neutrophil–lymphocyte ratio or heterophil equivalent), immunofunction (e.g. innate, cell-mediated or humoral immunity measures) and regenerative anaemia (e.g. mean erythrocyte volume and red blood cell distribution width) have all been used to estimate HPA-axis activity in free-living vertebrates. Stress metrics have provided insights into aspects of autecology or population regulation that could not have been easily obtained using other indices of population wellbeing, such as body condition or relative abundance. However, short- and long-term stress (often problematically termed acute and chronic stress, respectively) can interact in unpredictable ways. When animals experience trapping and handling stress before blood, faeces and/or urine is sampled, the interaction of short- and long-term stress can confound interpretation of the data, a fact not always acknowledged in studies of stress in free-living vertebrates. This review examines how stress metrics can be confounded when estimates of HPA-axis activation are collected for free-living vertebrates and outlines some approaches that can be used to help circumvent the influence of potentially confounding factors.

Does habitat fragmentation cause stress in the agile antechinus? A haematological approach

Although the vertebrate stress response is essential for survival, frequent or prolonged stress responses can result in chronic physiological stress, which is associated with a suite of conditions that can impair survivorship and reproductive output. Anthropogenic habitat fragmentation and degradation are potential stressors of free-living vertebrates, and in theory could result in chronic stress. To address this issue, we compared haematological indicators of stress and condition in agile antechinus (Antechinus agilis) populations in 30 forest fragments and 30 undisturbed, continuous forest sites (pseudofragments) in south-eastern Australia over 2xa0years. In peripheral blood, the total leucocyte count was lower and the neutrophil/lymphocyte ratio and percentage of eosinophils in the total leucocyte population was higher in fragment than pseudofragment populations, indicating that fragment populations were probably experiencing higher levels of stress hormone-mediated and/or parasite infection-related chronic physiological stress. The total erythrocyte count and haematocrit were higher and mean erythrocyte haemoglobin content was lower in fragment than pseudofragment populations. This suggests that fragment populations showed possible signs of regenerative anaemia, a syndrome associated with elevated hypothalamus–pituitary–adrenal axis mediated stress. However, mean erythrocyte volume was also lower in fragments, and red blood cell distribution width did not differ between the study populations, findings which were not consistent with this diagnosis. Whole blood and mean cell haemoglobin concentrations were similar in fragment and pseudofragment populations. We suggest that where anthropogenic activity results in habitat fragmentation and degradation, chronic stress could contribute to a decline in agile antechinus populations. The broader implication is that chronic stress could be both symptomatic of, and contributing to, decline of some vertebrate populations in anthropogenically fragmented and degraded habitats.

Tolerance of human approaches by Common Mynas along an urban-rural gradient

Abstract Successful urban colonisation by birds requires tolerance of many disturbances and avoidance of chronic stress and major disruption to critical activities. Habituation may be involved in acquiring tolerance of human proximity, in which case tolerance should vary with the degree of opportunity to learn that most humans do not present a threat and should therefore vary with human population density. We studied Common Mynas (Acridotheres tristis) in the urban, suburban, peri-urban and surrounding rural zones of Melbourne, Australia, to determine whether flight-initiation distance (FID) and time allocated to anti-predator vigilance increased along this gradient from the urban to the rural zone. This gradient encompassed a 143-fold variation in average human population density during daylight hours. Urban and suburban Mynas had a 2.3-5.7 times shorter FID, were 1.6-2.5 times less likely to retreat by flying and were 1.9-3.1 times more likely to flee >5 m than their peri-urban and rural conspecifics. Fleeing behaviour was not strongly correlated with estimated human population density, but exhibited an apparent ‘step’ in response at the suburban-peri-urban divide. Vigilance effort did not vary significantly between zones. This suggested that habituation did not occur with respect to surveillance for people, although surveillance presumably encompassed other predators too. Similar foraging effort and rates of food acquisition among zones could suggest that food abundance per individual was similar in urban and rural environments for Common Mynas, although this requires direct measurement.

Sex in troubled waters: Widespread agricultural contaminant disrupts reproductive behaviour in fish.

Chemical pollution is a pervasive and insidious agent of environmental change. One class of chemical pollutant threatening ecosystems globally is the endocrine disrupting chemicals (EDCs). The capacity of EDCs to disrupt development and reproduction is well established, but their effects on behaviour have received far less attention. Here, we investigate the impact of a widespread androgenic EDC on reproductive behaviour in the guppy, Poecilia reticulata. We found that short-term exposure of male guppies to an environmentally relevant concentration of 17β-trenbolone-a common environmental pollutant associated with livestock production-influenced the amount of male courtship and forced copulatory behaviour (sneaking) performed toward females, as well as the receptivity of females toward exposed males. Exposure to 17β-trenbolone was also associated with greater male mass. However, no effect of female exposure to 17β-trenbolone was detected on female reproductive behaviour, indicating sex-specific vulnerability at this dosage. Our study is the first to show altered male reproductive behaviour following exposure to an environmentally realistic concentration of 17β-trenbolone, demonstrating the possibility of widespread disruption of mating systems of aquatic organisms by common agricultural contaminants.

Impact of anthropogenic habitat fragmentation on population health in a small, carnivorous marsupial

Abstract Habitat fragmentation is a major cause of population reduction and loss, and increasing evidence suggests that effects of fragmentation on populations vary as a function of the life history and autecology of species. We investigated the effect of anthropogenic habitat fragmentation on several indicators of population health in the agile antechinus (Antechinus agilis), a small Australian marsupial with an unusual life history. We examined relative abundance, body condition (mass–size residuals [MSR]), and ectoparasite load. Abundance was 2.3-fold higher in continuous than in fragmented Eucalyptus forest, and ectoparasite loads were higher in fragmented than in continuous forest in March, April, May, and July, but not in June or August. Unexpectedly, MSR also was higher in what would usually be considered the less-favorable fragmented habitat than in continuous forest (means: fragmented: +0.6 g; continuous: −0.9 g). Other body-condition indicators did not differ consistently between fragmented and continuous forest populations. Results suggest that an apparently common and secure small mammal species could be declining in anthropogenically fragmented and degraded habitat. Although habitat fragmentation has been associated with nutritional stress in vertebrates, food availability probably was not contributing to the lower abundance of agile antechinus in habitat fragments. The findings indicate that care is needed when generalized expectations about the response of a species to anthropogenic habitat fragmentation are used to inform conservation management.

Impacts of the antidepressant fluoxetine on the anti-predator behaviours of wild guppies (Poecilia reticulata)

Chemical pollution from pharmaceuticals is increasingly recognised as a major threat to aquatic communities. One compound of great concern is fluoxetine, which is one of the most widely prescribed psychoactive drugs in the world and frequently detected in the environment. The aim of this study was to investigate the effects of 28-d fluoxetine exposure at two environmentally relevant levels (measured concentrations: 4ng/L and 16ng/L) on anti-predator behaviour in wild guppies (Poecilia reticulata). This was achieved by subjecting fluoxetine-exposed and unexposed guppies to a simulated bird strike and recording their subsequent behavioural responses. We found that exposure to fluoxetine affected the anti-predator behaviour of guppies, with exposed fish remaining stationary for longer (i.e. freezing behaviour) after the simulated strike and also spending more time under plant cover. By contrast, control fish were significantly more active and explored the tank more, as indicated by the distance covered per minute over the period fish spent swimming. Furthermore, behavioural shifts were sex-dependent, with evidence of a non-monotonic dose-response among the fluoxetine-exposed fish. This is one of the first studies to show that exposure to environmentally relevant concentrations of fluoxetine can alter the anti-predator behaviour of adult fish. In addition to the obvious repercussions for survival, impaired anti-predator behaviour can have direct impacts on fitness and influence the overall population dynamics of species.

Are haematocrit and haemoglobin concentration reliable body condition indicators in nestlings: the Welcome Swallow as a case study

Body condition influences activities and processes affecting fitness. Erythrocyte variables, particularly haemoglobin concentration and haematocrit, are increasingly being used as avian condition indicators. However, some potentially confounding factors should be considered when adopting this approach and there are some conflicting findings concerning such variables’ reliability as condition indicators. We examined whether whole blood haemoglobin concentration (Hb), haematocrit (Hct) and four other erythrocyte and one leukocyte variable were significantly associated with body mass and four commonly used body condition indices (BCI) in 12-day old Welcome Swallow Hirundo neoxena nestlings. Haematocrit and Hb were significantly associated with body mass and all four BCIs and had significant repeatabilities (0.426 and 0.433, respectively); total erythrocyte count, mean corpuscular volume, mean cell haemoglobin, mean cell haemoglobin concentration and total leukocyte count were not associated with mass or the BCIs, despite mostly having significant repeatabilities (0.129–0.433) and being reliable condition indicators in some other bird species. The lack of association between four of these other erythrocyte variables and nestling condition indicated that some caution was appropriate in interpreting the functional significance of the Hct/Hb–condition relationships. It seems advisable to measure a suite of erythrocyte variables when trying to determine whether the commonly measured Hct and Hb are associated with body condition in nestling birds.

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 metricfres 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.

Response of the agile antechinus to habitat edge, configuration and condition in fragmented forest.

Habitat fragmentation and degradation seriously threaten native animal communities. We studied the response of a small marsupial, the agile antechinus Antechinus agilis, to several environmental variables in anthropogenically fragmented Eucalyptus forest in south-east Australia. Agile antechinus were captured more in microhabitats dominated by woody debris than in other microhabitats. Relative abundances of both sexes were positively correlated with fragment core area. Male and female mass-size residuals were smaller in larger fragments. A health status indicator, haemoglobin-haematocrit residuals (HHR), did not vary as a function of any environmental variable in females, but male HHR indicated better health where sites microhabitats were dominated by shrubs, woody debris and trees other than Eucalyptus. Females were trapped less often in edge than interior fragment habitat and their physiological stress level, indicated by the neutrophil/lymphocyte ratio in peripheral blood, was higher where fragments had a greater proportion of edge habitat. The latter trend was potentially due to lymphopoenia resulting from stress hormone-mediated leukocyte trafficking. Using multiple indicators of population condition and health status facilitates a comprehensive examination of the effects of anthropogenic disturbances, such as habitat fragmentation and degradation, on native vertebrates. Male agile antechinus health responded negatively to habitat degradation, whilst females responded negatively to the proportion of edge habitat. The health and condition indicators used could be employed to identify conservation strategies that would make habitat fragments less stressful for this or similar native, small mammals.

Are haematocrit and haemoglobin concentration reliable indicators of body condition in adult Welcome Swallows

Abstract Parent birds often have substantial fat reserves that are critical in successful breeding. Erythrocyte variables, particularly haematocrit and haemoglobin concentration in peripheral blood, are increasingly being used as avian body-condition indices (BCIs) but problems can arise if such variables are used and interpreted uncritically. We investigated whether there were significant relationships between three size-adjusted BCIs thought to reflect the magnitude of fuel reserves, and four erythrocyte variables in free-living, adult Welcome Swallows (Hirundo neoxena) in south-eastern Australia over two breeding seasons. Haematocrit was not correlated with body-mass and condition. Whole-blood haemoglobin concentration, mean cell haemoglobin concentration and haematocrit-haemoglobin residuals were significantly related to body-mass and all the BCIs but the correlations were modest and negative (r ≤—0.39). Thus none of the erythrocyte variables was convincing as a reliable indicator of estimated fuel reserves in breeding adults. Currently the utility of haematocrit and haemoglobin concentration as reliable BCIs in adult birds cannot be assumed and needs to be validated for each population or species being investigated.