Lee Koren

A novel method using hair for determining hormonal levels in wildlife

ormones influence behaviour, and are also influ-enced by behaviour. Monitoring their levels cantherefore provide insights into the mechanistic aspects ofbehaviour. In male mammals for example, elevated levelsof testosterone are associated with increased aggressionand dominance (Creel et al. 1993, 1997; Mazur & Booth1998) and in social mammals, levels of stress hormones(e.g. corticosterone, glucocorticoid and cortisol) areassociated with rank (Sapolsky 1985; Creel et al. 1996,1997). Research has associated hormone levels with dif-ferent behaviours such as sexual, reproductive, courtship,parental, aggressive and feeding behaviours. Comparativetools for hormonal analysis provide insights into evolu-tionary theories based on behavioural aspects, such asreproductive suppression and the ‘challenge hypothesis’(e.g. Creel et al. 1993).In field studies, hormones are usually extracted fromblood samples, or noninvasively from saliva, urine andfaeces (Creel et al. 1992; Cavigelli 1999; Hirschenhauseret al. 1999; von Engelhardt et al. 2000). Samples derivedfrom trapped or handled animals are problematicalbecause stress may alter blood and urine hormonal levels(Creel et al. 1992). Additional problems with bloodsamples are that they are not always available, theamount that can be taken at a given time is limited, andvarious safety and ethical issues exist. Furthermore, bloodand saliva must be transported cold or frozen, conditionsthat are sometimes difficult to obtain in the field (Yang etal. 1998). Urine and faeces samples are sometimes diffi-cult to obtain from free-ranging animals that cannot becontinuously observed, or from species that deposit incommon latrines.An alternative source for hormones may be found inhair, which can be collected noninvasively, and is alreadyused to extract DNA (Woodruff 1993; Morin et al. 1994),trace metals, naturally occurring compounds and drugs(Wheeler et al. 1998). Hair is safe, readily available, andeasy to store and transport. Hair sampling does notinvolve pain or possible infection, and the analysis isunaffected by the momentary stress of capture (Yang et al.1998). Hair analysis may allow one to monitor hormonalchanges over weeks or months (between moults; Maurelet al. 1986) by shaving off a patch of hair and resamplingthe newly grown hair. Hormonal hair analysis offersonly a long-term profile, however, and is not suitable formonitoring hourly or daily (short-term) fluctuations inhormonal levels. It provides the resolution needed forstudies of main behavioural trends, especially in stablehierarchical social systems. Hair has already been used todiagnose early pregnancy in cows by detection of proges-terone (Liu et al. 1988), to detect oestradiol and testoster-one in cattle (Gleixner & Meyer 1997) and anabolicsteroid and corticosteroid abuse in athletes (Bowers S Hold et al. 1999; Kintz et al. 1999; Cirimeleet al. 2000). In humans, the levels of steroid hormones inhair do not vary significantly between different regions ofthe scalp (Wheeler et al. 1998). Oestradiol, progesteroneand testosterone levels measured in healthy humanadults’ hair correlate significantly with the levelsmeasured in their serum (Yang et al. 1998).As an example of the utility of this method, we usedata from our long-term study on rock hyrax,

Non-breeding feather concentrations of testosterone, corticosterone and cortisol are associated with subsequent survival in wild house sparrows

Potential mechanistic mediators of Darwinian fitness, such as stress hormones or sex hormones, have been the focus of many studies. An inverse relationship between fitness and stress or sex hormone concentrations has been widely assumed, although empirical evidence is scarce. Feathers gradually accumulate hormones during their growth and provide a novel way to measure hormone concentrations integrated over time. Using liquid chromatography–tandem mass spectrometry, we measured testosterone, corticosterone and cortisol in the feathers of house sparrows (Passer domesticus) in a wild population which is the subject of a long-term study. Although corticosterone is considered the dominant avian glucocorticoid, we unambiguously identified cortisol in feathers. In addition, we found that feathers grown during the post-nuptial moult in autumn contained testosterone, corticosterone and cortisol levels that were significantly higher in birds that subsequently died over the following winter than in birds that survived. Thus, feather steroids are candidate prospective biomarkers to predict the future survival of individuals in the wild.

Variance in Centrality within Rock Hyrax Social Networks Predicts Adult Longevity

Background In communal mammals the levels of social interaction among group members vary considerably. In recent years, biologists have realized that within-group interactions may affect survival of the group members. Several recent studies have demonstrated that the social integration of adult females is positively associated with infant survival, and female longevity is affected by the strength and stability of the individual social bonds. Our aim was to determine the social factors that influence adult longevity in social mammals. Methodology/Principal Findings As a model system, we studied the social rock hyrax (Procavia capensis), a plural breeder with low reproductive skew, whose groups are mainly composed of females. We applied network theory using 11 years of behavioral data to quantify the centrality of individuals within groups, and found adult longevity to be inversely correlated to the variance in centrality. In other words, animals in groups with more equal associations lived longer. Individual centrality was not correlated with longevity, implying that social tension may affect all group members and not only the weakest or less connected ones. Conclusions/Significance Our novel findings support previous studies emphasizing the adaptive value of social associations and the consequences of inequality among adults within social groups. However, contrary to previous studies, we suggest that it is not the number or strength of associations that an adult individual has (i.e. centrality) that is important, but the overall configuration of social relationships within the group (i.e. centrality SD) that is a key factor in influencing longevity.

Complex call in male rock hyrax (Procavia capensis): a multi-information distributing channel

Chemicals such as those used for scent marking, or visual cues such as color badges, can transmit information pertaining to different aspects of individual, group and species recognition and attributes. Here, we show that complex acoustic cues, such as calls also have the capacity for such information transfer. Although songs are usually attributed to birds, rock hyraxes (Procavia capensis) engage in a rich and complex vocalizing behavior that we term ‘singing’. Previous studies on various species have shown that a specific vocalization can closely reflect a specific attribute. Using a series of multiple regressions, we show that a single complex vocalization by the adult male rock hyrax closely reflects numerous individual traits, possibly encoding various types of biologically important information (multiple-messages hypothesis). Our study reveals that hyrax songs provide accurate information regarding body weight, size and condition, social status and hormonal state of the singer. We also show that these independent data are sent in a sequential manner, a pattern that probably allows a better partition of the messages embedded in the song. Our results imply that animals, through complex individual vocalizations, can potentially advertise multiple individual attributes in the same manner as that produced by chemical scent marking.

Sample preparation and liquid chromatography-tandem mass spectrometry for multiple steroids in mammalian and avian circulation.

Blood samples from wild mammals and birds are often limited in volume, allowing researchers to quantify only one or two steroids from a single sample by immunoassays. In addition, wildlife serum or plasma samples are often lipemic, necessitating stringent sample preparation. Here, we validated sample preparation for simultaneous liquid chromatography – tandem mass spectrometry (LC-MS/MS) quantitation of cortisol, corticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), 17β-estradiol, progesterone, 17α-hydroxyprogesterone and testosterone from diverse mammalian (7 species) and avian (5 species) samples. Using 100 µL of serum or plasma, we quantified (signal-to-noise (S/N) ratio ≥10) 4–7 steroids depending on the species and sample, without derivatization. Steroids were extracted from serum or plasma using automated solid-phase extraction where samples were loaded onto C18 columns, washed with water and hexane, and then eluted with ethyl acetate. Quantitation by LC-MS/MS was done in positive ion, multiple reaction-monitoring (MRM) mode with an atmospheric pressure chemical ionization (APCI) source and heated nebulizer (500°C). Deuterated steroids served as internal standards and run time was 15 minutes. Extraction recoveries were 87–101% for the 8 analytes, and all intra- and inter-run CVs were ≤8.25%. This quantitation method yields good recoveries with variable lipid-content samples, avoids antibody cross-reactivity issues, and delivers results for multiple steroids. Thus, this method can enrich datasets by providing simultaneous quantitation of multiple steroids, and allow researchers to reimagine the hypotheses that could be tested with their volume-limited, lipemic, wildlife samples.

Cortisol and corticosterone independence in cortisol-dominant wildlife

Species have traditionally been defined as cortisol-dominant or corticosterone-dominant, depending on the glucocorticoid that is reported. To assess the degree of covariance versus independence between cortisol and corticosterone, 245 serum samples belonging to 219 individuals from 18 cortisol-dominant, non-domesticated species (6 mammalian orders) were compared by mass spectrometry. In these samples, which were elevated above baseline, concentration ranges were overlapping for cortisol and corticosterone although cortisol was dominant in every sample except one of 17 bighorn sheep with a corticosterone-biased cortisol-to-corticosterone ratio of 0.17. As expected, cortisol and corticosterone were strongly associated among species (r(2)=0.8; species with high absolute cortisol tend to have high absolute corticosterone concentrations), with wide variation in the species-average cortisol-to-corticosterone ratio (range 7.5-49) and an even wider ratio range across individuals (0.2-341). However, only 9 out of 13 species with >7 individuals showed a positive association between cortisol and corticosterone among individuals, and repeated measures of the cortisol-to-corticosterone ratio within individuals were weakly associated (CV range 3-136%). We conclude that corticosterone, although at lower concentrations, has the potential to signal independently of cortisol, and should be included in integrated endocrine models of stress responses.

Structural balance in the social networks of a wild mammal

The social structure of a population is based on individual social associations, which can be described using network patterns (motifs). Our understanding of the forces stabilizing specific social structures in animals is limited. Structural balance theory was proposed for exploring social alliances and suggested that some network motifs are more stable than others in a society. The theory models the presence of specific triads in the network and their effect on the global population structure, based on the differential stability of specific triad configurations. While structural balance was shown in human social networks, the theory has never been tested in animal societies. Here we use empirical data from an animal social network to determine whether or not structural balance is present in a population of wild rock hyraxes, Procavia capensis. We confirm its presence and show the ability of structural balance to predict social changes resulting from local instability. We present evidence that new individuals entering the population introduce social instability, which counters the tendency of social relationships to seek balanced structures. Our findings imply that structural balance has a role in the evolution of animal social structure.

Individual identity is communicated through multiple pathways in male rock hyrax (Procavia capensis) songs

Communicating individual identity is essential for stable social systems. It is assumed that there are benefits for both senders and receivers to provide and discriminate identity cues. In this study, we investigate the possible routes senders use to acoustically broadcast their individual identity. Using discriminant function analysis of temporal and spectral acoustic measurements and analysis of song-element order, we explore the means male rock hyrax (Procavia capensis) singers utilize vocalization to express individual identity. Despite the fact that males use only three song elements, the pattern of acoustic characteristics, their temporal and frequency attributes vary according to the identity of singer. We show that in hyrax, individuality is expressed by highly variable, complex signals that are not condition dependent and are stable over years in singers that did not alter their spatial position. We also show that individuality signals are not linked to relatedness or to geographic location. The ability to discriminate individuals from vocal signatures needs to be further tested using controlled playback experiments.

Heavily hunted wolves have higher stress and reproductive steroids than wolves with lower hunting pressure

Summary 1. Human-caused harassment and mortality (e.g. hunting) affects many aspects of wildlife population dynamics and social structure. Little is known, however, about the social and physiological effects of hunting, which might provide valuable insights into the mechanisms by which wildlife respond to human-caused mortality. 2. To investigate physiological consequences of hunting, we measured stress and reproductive hormones in hair, which reflect endocrine activity during hair growth. Applying this novel approach, we compared steroid hormone levels in hair of wolves (Canis lupus) living in Canada’s tundra–taiga (n = 103) that experience heavy rates of hunting with those in the northern boreal forest (n = 45) where hunting pressure is substantially lower. 3. The hair samples revealed that progesterone was higher in tundra–taiga wolves, possibly reflecting increased reproductive effort and social disruption in response to human-related mortality. Tundra–taiga wolves also had higher testosterone and cortisol levels, which may reflect social instability. 4. To control for habitat differences, we also measured cortisol in an out-group of boreal forest wolves (n = 30) that were killed as part of a control programme. Cortisol was higher in the boreal out-group than in our study population from the northern boreal forest. 5. Overall, our findings support the social and physiological consequences of human-caused mortality. Long-term implications of altered physiological responses should be considered in management and conservations strategies.

Androgens and social status in female rock hyraxes

Androgens have been linked to social behaviour in males across many vertebrate species. Despite the lack of gender specificity in steroid hormones, the association of androgens with behaviour in females remains obscure. We analysed steroid hormone levels in male and female rock hyraxes, Procavia capensis, and found strong ties between androgens and cortisol in females but not in males, despite the latter being the sex that usually shows this association. In the past we have shown that both female and male hyraxes have similar testosterone levels. In the present work, we found a significant interaction between testosterone levels and social status. Testosterone levels differed only between lower-ranking males and females, with females showing higher levels. Furthermore, dominant females had significantly lower testosterone levels than subordinate females. No association was detected between litter size and female rank, testosterone or cortisol levels. At this stage, we can only hypothesize that the assumed detrimental effects of high circulating androgen levels, reflected in the hair samples, influence the reproductive output or the offspring survival of subordinate females in some other ways, which require further investigation to be revealed.