Non-invasive monitoring of physiological stress in the Western lowland gorilla (Gorilla gorilla gorilla): validation of a fecal glucocorticoid assay and methods for practical application in the field

Enzymeimmunoassays (EIAs) allow researchers to monitor stress hormone output via measurement of fecal glucocorticoid metabolites (FGCMs) in many vertebrates. They can be powerful tools which allow the acquisition of otherwise unobtainable physiological information from both captive animals and wild animals in remote forest habitats, such as great apes. However, methods for hormone measurement, extraction and preservation need to be adapted and validated for field settings. In preparation for a field study of Western lowland gorillas (Gorilla gorilla gorilla) in the Central African Republic we used samples from captive gorillas collected around opportunistic stressful situations to test whether four different glucocorticoid EIAs reflected adrenocortical activity reliably and to establish the lag-time from the stressor to peak excretion. We also validated a field extraction technique and established a simple, non-freezer-reliant method to preserve FGCMs in extracts long-term. We determined the rate of FGCM change over 28 days when samples cannot be extracted immediately and over 12 h when feces cannot be preserved immediately in alcohol. Finally, we used repeat samples from identified individuals to test for diurnal variation in FGCM output. Two group-specific assays measuring major cortisol metabolites detected the predicted FGCM response to the stressor reliably, whereas more specific cortisol and corticosterone assays were distinctly less responsive and thus less useful. We detected a lag time of 2–3 days from stressor to peak FGCM excretion. Our field extraction method performed as well as an established laboratory extraction method and FGCMs in dried extracts stored at ambient temperatures were as stable as those at −20 °C over 1 yr. Hormones in non-extracted feces in alcohol were stable up to 28 days at ambient temperatures. FGCMs in un-fixed gorilla feces deteriorated to almost 50% of the original values within 6 h under field conditions. We detected no diurnal variation in FGCMs in samples from wild gorillas. Our study highlights the importance of thorough biological and immunological validation of FGCM assays, and presents validated, practical methods for the application of non-invasive adrenocortical monitoring techniques to field conservation contexts where it is crucially needed

Validation of a Novel Collection Device for Non-Invasive Urine Sampling from Free-Ranging Animals.

Recent advances in non-invasively collected samples have opened up new and exciting opportunities for wildlife research. Different types of samples, however, involve different limitations and certain physiological markers (e.g., C-peptide, oxytocin) can only be reliably measured from urine. Common collection methods for urine to date work best for arboreal animals and large volumes of urine. Sufficient recovery of urine is thus still difficult for wildlife biologists, particularly for terrestrial and small bodied animals. We tested three collection devices (two commercially available saliva swabs, Salivette synthetic and cotton, and cotton First aid swabs) against a control to permit the collection of small volumes of urine from the ground. We collected urine samples from captive and wild macaques, and humans, measured volume recovery, and analyzed concentrates of selected physiological markers (creatinine, C-peptide, and neopterin). The Salivette synthetic device was superior to the two alternative devices. Concentrations of creatinine, absolute C-peptide, C-peptide per creatinine, absolute neopterin, and neopterin per creatinine measured in samples collected with this device did not differ significantly from the control and were also strongly correlated to it. Fluid recovery was also best for this device. The least suitable device is the First aid collection device; we found that while absolute C-peptide and C-peptide per creatinine concentrations did not differ significantly from the control, creatinine concentrations were significantly lower than the control. In addition, these concentrations were either not or weakly correlated to the control. The Salivette cotton device provided intermediate results, although these concentrations were strongly correlated to the control. Salivette synthetic swabs seem to be useful devices for the collection of small amounts of urine from the ground destined for the assessment of physiological parameters. They thus provide new opportunities for field studies to incorporate physiological markers, particularly on smaller bodied and terrestrial animals and where urine collection is difficult

HESS Opinions: A planetary boundary on freshwater use is misleading

In 2009, a group of prominent Earth scientists introduced the planetary boundaries (PB) framework: they suggested nine global control variables, and defined corresponding thresholds which, if crossed, could generate unacceptable environmental change. The concept builds on systems theory, and views Earth as a complex adaptive system in which anthropogenic disturbances may trigger non-linear, abrupt, and irreversible changes at the global scale, and push the Earth system outside the stable environmental state of the Holocene. While the idea has been remarkably successful in both science and policy circles, it has also raised fundamental concerns, as the majority of suggested processes and their corresponding planetary boundaries do not operate at the global scale, and thus apparently lack the potential to trigger abrupt planetary changes.This paper picks up the debate with specific regard to the planetary boundary on global freshwater use. While the bio-physical impacts of excessive water consumption are typically confined to the river basin scale, the PB proponents argue that water-induced environmental disasters could build up to planetary-scale feedbacks and system failures. So far, however, no evidence has been presented to corroborate that hypothesis. Furthermore, no coherent approach has been presented to what extent a planetary threshold value could reflect the risk of regional environmental disaster. To be sure, the PB framework was revised in 2015, extending the planetary freshwater boundary with a set of basin-level boundaries inferred from environmental water flow assumptions. Yet, no new evidence was presented, either with respect to the ability of those basin-level boundaries to reflect the risk of regional regime shifts or with respect to a potential mechanism linking river basins to the planetary scale.So while the idea of a planetary boundary on freshwater use appears intriguing, the line of arguments presented so far remains speculative and implicatory. As long as Earth system science does not present compelling evidence, the exercise of assigning actual numbers to such a boundary is arbitrary, premature, and misleading. Taken as a basis for water-related policy and management decisions, though, the idea transforms from misleading to dangerous, as it implies that we can globally offset water-related environmental impacts. A planetary boundary on freshwater use should thus be disapproved and actively refuted by the hydrological and water resources community

Group size and visitor number predict faecal glucocorticoid concentrations in zoo meerkats

This is the author accepted manuscript. The final version is freely available from Royal Society via the DOI in this record.Measures of physiological stress in zoo animals can give important insights into how they are affected by aspects of their captive environment. We analysed the factors influencing variation in glucocorticoid metabolites in faeces (fGCs) from zoo meerkats as a proxy for blood cortisol concentration, high levels of which are associated with a stress response. Levels of fGCs in captive meerkats declined with increasing group size. Compared to data from wild meerkats, this contrasts with the patterns seen in large stabile groups but matches the pattern seen in dispersing coalitions. In the wild, very small groups of meerkats are at a higher risk of predation, while in larger groups there is increased competition for resources. Indeed, group sizes in captivity tend to be closer to those seen in unstable coalitions in the wild, which may represent a stressful condition to meerkats in captivity and predispose them to chronic stress, even in absence of natural predators. Individuals in large enclosures showed lower levels of stress, but meerkat density had no effect on the stress measures. In contrast to data from wild meerkats, neither sex, age, nor dominance status predicted physiological stress levels in captivity, which may reflect less food stress owing to more equal access to resources in captivity versus wild. Median number of visitors at the enclosure was positively correlated with fGC concentrations on the following day, with variation in the visitor numbers having the opposite effect. Our results are consistent with the hypothesis that there is an optimum group size which minimises physiological stress in meerkats, and that zoo meerkats at most risk of physiological stress are those kept in small groups and small enclosures and are exposed to consistently high numbers of visitors.Funding was provided by a European Social Fund studentship and a Society for Endocrinology grant awarded to K.S., and by NERC grant awarded to M.A.C. (NE/J010278/1)

Concealed fertility and extended female sexuality in a non-human primate (Macaca assamensis)

In numerous primates living in mixed-sex groups, females display probabilistic cues of fertility to simultaneously concentrate paternity to dominant males while diluting it amongst others as a means to reduce the risk of infanticide and to increase male care for offspring. A few species, however, lack these cues and potentially conceal fertility from males; yet, to date, little is known about mating patterns and their underlying proximate mechanisms in such species. Here, we investigated mating activity and sexual consortships relative to female reproductive state in wild Assamese macaques (Macaca assamensis), a species where females lack prominent anogenital swellings and copulation calls. During two mating seasons (2837 contact hours) we recorded sexual and social behaviors, sexual consortships, and collected 1178 fecal samples (n = 15 females) which were analyzed for progestogen concentrations to assess female reproductive state and to determine the timing of ovulation and conception. Although mostly conceiving in their first ovarian cycle, females were sexually receptive throughout the entire 4-month mating season, and within-cycle mating frequencies were not increased during fertile phases. Dominant males did not monopolize fertile matings, and consortships by high-ranking males lasted for long periods, which were not exclusively linked to female fertile phases. Furthermore, females copulated promiscuously but not randomly, i.e. for almost every female, matings were concentrated to a certain male, irrespective of male rank. Collectively, we demonstrate that fertility is undisclosed to males. The extreme extended female sexuality facilitated by concealed fertility may allow females to create differentiated mating relationships within a promiscuous mating system. Our study provides important new insight into the plasticity of female sexuality in non-human primates

Is social dispersal stressful? A study in male crested macaques (Macaca nigra).

In gregarious species, dispersal events represent one of the most dramatic changes in social life and environment an animal will experience during life due to increased predation risk, aggression from unfamiliar conspecifics and the lack of social support. However, little is known about how individuals respond physiologically to dispersal and whether this process is stressful for the individuals involved. We therefore studied the physiological stress response during dispersal in the crested macaque, a primate species in which males often change groups. Over a period of 14months and 14 dispersal events in 4 groups, we determined faecal glucocorticoid metabolite (FGCM) levels during the process of immigration into a new group and examined a variety of factors (e.g. male age, rank achieved, number of males in the group) potentially affecting FGCM levels during this process. We found that FGCM levels were significantly elevated in the first few days upon immigration, after which levels returned quickly to baseline. FGCM response levels upon immigration were significantly and positively influenced by the number of males in the group. The rank a male achieved upon immigration, aggression received, as well as the proximity to other males did not significantly influence FGCM levels. Our data confirm previous findings on other species demonstrating that in crested macaques immigration into a new social group is associated with an acute endocrine stress response. However, given that stress hormone levels remained elevated only for a short period of time, we do not expect males to experience high physiological costs during immigration. Given our limited knowledge on the physiological responses to dispersal in animals, this study contributes to our understanding of dispersal more generally, and particularly inter-individual differences in the stress response and the potential physiological costs associated with these

Sexual signalling in female crested macaques and the evolution of primate fertility signals.

BACKGROUND: Female signals of fertility have evolved in diverse taxa. Among the most interesting study systems are those of multimale multifemale group-living primates, where females signal fertility to males through multiple signals, and in which there is substantial inter-specific variation in the composition and reliability of such signals. Among the macaques, some species display reliable behavioural and/or anogenital signals while others do not. One cause of this variation may be differences in male competitive regimes: some species show marked sexual dimorphism and reproductive skew, with males fighting for dominance, while others show low dimorphism and skew, with males queuing for dominance. As such, there is variation in the extent to which rank is a reliable proxy for male competitiveness, which may affect the extent to which it is in females' interest to signal ovulation reliably. However, data on ovulatory signals are absent from species at one end of the macaque continuum, where selection has led to high sexual dimorphism and male reproductive skew. Here we present data from 31 cycles of 19 wild female crested macaques, a highly sexually dimorphic species with strong mating skew. We collected measures of ovarian hormone data from faeces, sexual swelling size from digital images, and male and female behaviour. RESULTS: We show that both sexual swelling size and female proceptivity are graded-signals, but relatively reliable indicators of ovulation, with swelling size largest and female proceptive behaviours most frequent around ovulation. Sexual swelling size was also larger in conceptive cycles. Male mating behaviour was well timed to female ovulation, suggesting that males had accurate information about this. CONCLUSION: Though probabilistic, crested macaque ovulatory signals are relatively reliable. We argue that in species where males fight over dominance, male dominance rank is surrogate for competitiveness. Under these circumstances it is in the interest of females to increase paternity concentration and assurance in dominants beyond levels seen in species where such competition is less marked. As such, we suggest that it may in part be variation in male competitive regimes that leads to the evolution of fertility signalling systems of different reliability

Costs of and investment in mate-guarding in wild long-tailed macaques (Macaca fascicularis): Influences of female characteristics and male–female social bonds

Male primates living in multimale groups tend to direct mate and mate-guarding choices toward females of high reproductive value, i.e., high-ranking, parous females, or females with which they share strong bonds. Little is known, however, about the constraints that may limit male mate-guarding choices (the costs of this behavior) and the influence of the females’ quality on male investment in mate-guarding. We aimed to study the effects of female rank, parity status, and male–female social bond strength on the costs of and investment in mate-guarding by males. We carried out our study during two reproductive seasons on three groups of wild long-tailed macaques in Indonesia. We combined behavioral observations on male locomotion and activity with noninvasive measurements of fecal glucocorticoids (fGC). Males spent less time feeding when mate-guarding nulliparous females than when mate-guarding parous females and tended to have higher fGC levels when mate-guarding low-ranking nulliparous females than when mate-guarding high-ranking nulliparous ones. Evolution should thus favor male choice for high-ranking parous females because such a decision brings benefits at proximate (reduced costs of mate-guarding) and ultimate (higher reproductive value) levels. Further, male investment in mate-guarding was flexible and contingent on female reproductive and social value. Males were more vigilant and more aggressive toward other males when mate-guarding females to which they were strongly bonded and/or high-ranking ones than when mate-guarding other females. Our findings bring a new dimension to the study of mate choice by showing that males not only mate preferentially with high-quality females but may also aim to secure paternity with these females through optimized monopolization

Costs of mate-guarding in wild male long-tailed macaques (Macaca fascicularis): Physiological stress and aggression

AbstractMate-guarding is an important determinant of male reproductive success in a number of species. However, it is known to potentially incur costs. The aim of the present study was to assess the effect of mate-guarding on male physiological stress and aggression in long-tailed macaques, a species in which males mate-guard females to a lesser extent than predicted by the Priority of Access model (PoA). The study was carried out during two mating periods on three groups of wild long-tailed macaques in Indonesia by combining behavioral observations with non-invasive measurements of fecal glucocorticoid (fGC) levels. Mate-guarding was associated with a general rise in male stress hormone levels but, from a certain threshold of mate-guarding onwards, increased vigilance time was associated with a decrease in stress hormone output. Mate-guarding also increased male-male aggression rate and male vigilance time. Overall, alpha males were more physiologically stressed than other males independently of mating competition. Increased glucocorticoid levels during mate-guarding are most likely adaptive since it may help males to mobilize extra-energy required for mate-guarding and ultimately maintain a balanced energetic status. However, repeated exposure to high levels of stress over an extended period is potentially deleterious to the immune system and thus may carry costs. This potential physiological cost together with the cost of increased aggression mate-guarding male face may limit the male’s ability to mate-guard females, explaining the deviance from the PoA model observed in long-tailed macaques. Comparing our results to previous findings we discuss how ecological factors, reproductive seasonality and rank achievement may modulate the extent to which costs of mate-guarding limit male monopolization abilities