Spatial and temporal determinants of anxiety in a wild arboreal primate

Spatial and temporal determinants of anxiety in a wild arboreal primate | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Ecology and Evolution This is a preprint and has not been peer reviewed. Data may be preliminary. 13 February 2025 V1 Latest version Share on Spatial and temporal determinants of anxiety in a wild arboreal primate Authors : Edwin Parker 0000-0001-8322-5480 [email protected] , Russell Hill , and Nicola Koyama Authors Info & Affiliations https://doi.org/10.22541/au.173947067.70612322/v1 Published Ecology and Evolution Version of record Peer review timeline 303 views 193 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The fear of being eaten can manifest as anxiety in prey species. In captive primates, anxiety-related behaviours (such as scratching and self-grooming) typically increase when exposed to a predator model. Despite this, it remains largely unknown whether the perception of predation risk can invoke anxiety, particularly in wild primates. We collected focal observations from adult females from two habituated groups of wild samango monkeys over a 12-month period to explore whether scratching increased in areas associated with higher perceived predation risk. To validate scratching as an anxiety-related behaviour, we compared the rates of scratching following an alarm call to a control period. We found scratching occurred significantly more often after an eagle alarm call relative to baseline levels, indicating this may be a reliable anxiety-related behaviour. We then used a generalised linear mixed model to predict the rate of scratching as a function of perceived predation risk and factors potentially associated with risk. Scratching increased in the summer months and towards the end of the day, but was not influenced by perceived predation risk. Our findings suggest that samangos may adopt other behavioural strategies to mitigate anxiety in ‘high-risk’ areas, and that anxiety may be reactive, rather than pre-emptive, in response to predation risk. We propose that scratching may be a useful indicator of reactive anxiety in wild primates and can help to improve knowledge on the environmental factors that induce acute stress in wild populations. Spatial and temporal determinants of anxiety in a wild arboreal primate Predation risk and anxiety in samango monkeys Edwin J. Parker 1,2* , Russell A. Hill 2,3,4 & Nicola F. Koyama 1 1 School of Biological and Environmental Science, Liverpool John Moores University, Liverpool, UK, L3 3AF 2 Primate and Predator Project, Medike Nature Reserve, Makhado (Louis Trichardt), South Africa, 0920 3 Department of Anthropology, Durham University, Durham, UK, DH1 3LE 4 Department of Biological Sciences, University of Venda, Thohoyandou (Venda), South Africa, 0950 * Corresponding author Email: [email protected] Acknowledgements We are grateful to Ian Gaigher and the Lajuma Research Centre for permission to work at the site and to neighboring landowners and managers for granting access to their properties. We also thank the numerous field assistants who have contributed to the data collection across the study period, and particularly Andrew Allan and Caroline Howlett for coordinating the primate side of the project. The authors greatly appreciate the financial support from LJMU for a University PhD Scholarship, Durham University, the Earthwatch Institute and an anonymous donor. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract The fear of being eaten can manifest as anxiety in prey species. In captive primates, anxiety-related behaviours (such as scratching and self-grooming) typically increase when exposed to a predator model. Despite this, it remains largely unknown whether the perception of predation risk can invoke anxiety, particularly in wild primates. We collected focal observations from adult females from two habituated groups of wild samango monkeys over a 12-month period to explore whether scratching increased in areas associated with higher perceived predation risk. To validate scratching as an anxiety-related behaviour, we compared the rates of scratching following an alarm call to a control period. We found scratching occurred significantly more often after an eagle alarm call relative to baseline levels, indicating this may be a reliable anxiety-related behaviour. We then used a generalised linear mixed model to predict the rate of scratching as a function of perceived predation risk and factors potentially associated with risk. Scratching increased in the summer months and towards the end of the day, but was not influenced by perceived predation risk. Our findings suggest that samangos may adopt other behavioural strategies to mitigate anxiety in ‘high-risk’ areas, and that anxiety may be reactive, rather than pre-emptive, in response to predation risk. We propose that scratching may be a useful indicator of reactive anxiety in wild primates and can help to improve knowledge on the environmental factors that induce acute stress in wild populations. Keywords Anxiety, self-directed behaviour, scratching, predator-induced anxiety, wild primate Significance statement Prey species may become anxious over the possibility of encountering predators. This anxiety varies over space and time, depending on both predator and prey activity patterns and environmental heterogeneity. Self-directed behaviours, such as self-scratching, have previously been used as an indicator of anxiety in captive primates, yet studies on wild primates are lacking. Here, we validated self-scratching as an anxiety-related behaviour in wild samango monkeys ( Cercopithecus albogularis schwarzi ) by comparing scratching from focal individuals before and after a predator alarm call. We then explored the spatial and temporal predictors of anxiety, to determine whether movement into areas/times of increased predator activity induces anxiety in this species. We found that the rate of scratching increased during the summer months, and during the late afternoon. Our findings suggests that anxiety is reactive in this species, increasing in response to direct predator encounters. Introduction Fear is an adaptive emotional response which serves to modify an individual’s behaviour in order to avoid potentially dangerous situations (Nelson et al., 2003). Fear can manifest as anxiety, the apprehension over anticipation of potentially threatening stimuli, which is critical to an individual’s survival (Coleman and Pierre, 2014). Whilst anxiety serves as a short-term coping mechanism, prolonged anxiety can lead to chronic stress which can negatively impact the reproductive success and survival of individuals (Wingfield and Romero, 2000; Cox et al., 2010; Crespi et al., 2013). In wild animals, predators are the most significant stimuli invoking anxiety in prey species (Brown, 1999), due to the direct, lethal effects of predation. However, the indirect, non-consumptive effects, may be just as significant, owing to the influence on nearly all aspects of prey behaviour (Lima, 1998; Brown et al., 1999a; Laundre et al., 2010; Peers et al., 2018; Wirsing et al., 2021; Parker et al., 2022). As such, even in the immediate absence of predators, prey should maintain a baseline level of anxiety due to the constant possibility of predation (Brown, 1999). Yet, predation risk varies across both space and time, such as between safe versus risky habitats or depending on time of day (Brown, 1992, 1999; Brown and Kotler, 2004), and so anxiety levels are expected to reflect this variation in risk. Understanding the spatiotemporal variation in anxiety, particularly in the direct absence of predators, can therefore provide important information on the ecological factors that induce anxiety. Predator-induced anxiety has been extensively explored using predator models in numerous species (Apfelbach et al., 2005), including rodents (Berton et al., 1998; Belzung and Griebel, 2001), zebrafish (Blaser et al., 2010; Maximino et al., 2010) and non-human primates (Barros et al., 2000). In primates, including humans, anxiety or stress is typically assessed using faecal glucocorticoids (Rangel-Negrín et al., 2009; Foerster and Monfort, 2010; Crespi et al., 2013; LaBarge et al., 2022). Yet, elevated levels of glucocorticoids do not necessarily mean an individual is anxious or stressed (Beehner and Bergman, 2017). Consequently, behavioural indicators of anxiety may be more informative and more easily observed in natural environments (Lutz and Baker, 2023). Displacement behaviours such as self-scratching (hereafter, scratching), self-grooming, body-shaking, and yawning (Maestripieri et al., 1992; Lutz and Baker, 2023), typically referred to as self-directed behaviours (SDBs), have long been recognised and validated as manifestations of anxiety in nonhuman primates (hereafter, primates) (Baker and Aureli, 1997; Kutsukake, 2003; Maréchal et al., 2011; Polizzi di Sorrentino et al., 2012; Sclafani et al., 2012; Dell’Anna et al., 2022; Lutz and Baker, 2023). Scratching, in particular, was shown to be the most reliable indicator of anxiety in captive chimpanzees (Baker and Aureli, 1997) compared to other SDBs. Whilst prolonged anxiety can be detrimental to an individual in terms of psychological stress (Maestripieri et al., 1992), it can also be beneficial in the short-term by eliciting the appropriate anti-predator response, such as increasing vigilance, increasing group cohesion, or evoking predator-specific alarm calls (Stevenson and Poole, 1976), thereby reducing the risk of predation (Lima and Dill, 1990; McNamara and Houston, 1992; Brown, 1999). Despite some studies citing vigilance as a measure of anxiety (Barros et al., 2000; Coleman and Pierre, 2014), vigilant individuals are not necessarily anxious. Studies administering drugs on captive primates have demonstrated the ambiguity of vigilance as an anxiety-related behaviour. For example, anxiolytics such as lorazepam have been shown to reduce SDBs, such as scratching, in captive cynomolgus macaques ( Macaca fascicularis ), whereas anxiogenic compounds increase these behaviours (Schino et al., 1996). In contrast, whilst some anxiogenics have been shown to increase vigilance (Palit et al., 1998), anxiolytics appear to have mixed or negative effects (Schino et al., 1991, 1996; Barros and Tomaz, 2002; Barros et al., 2007), suggesting vigilance may represent a state of arousal as well as anxiety. Therefore, despite many studies linking vigilance to other behavioural contexts (Allan and Hill, 2018), evidence for vigilance as a measure of anxiety is somewhat mixed and SDBs appear to be a more reliable indicators of anxiety in primates. The majority of work exploring predator-induced anxiety in primates has thus far focussed on captive individuals using predator confrontation tests (Barros et al., 2000, 2004; Barros and Tomaz, 2002). These tests presented a taxidermied predator to the study subject in a randomly encountered manner, recording the response of the subject on each occasion (Barros et al., 2000). Responses in marmosets typically involved an increase in alarm calls, vigilance and SDBs including self-grooming and scratching (Barros et al., 2004), although some responses stopped following habituation to the stimulus. Whilst studies on wild primates are lacking, wild-reared rhesus macaques showed higher levels of anxiety, indicated by behavioural disturbance, when exposed to a snake compared to their laboratory-reared counterparts (Mineka et al., 1980). However, in both sets of studies, habituation to the stimuli showed marked individual differences, demonstrating the importance of considering individual variation in anxiety (Mineka et al., 1980; Nelson et al., 2003; Barros et al., 2004). Prey species are known to adopt a variety of anti-predator behaviours depending on perceived predation risk (Lima and Dill, 1990). For example, individuals may avoid areas they perceive to be higher risk (Suhonen, 1993; Cowlishaw, 1997; Laundré et al., 2001; Heithaus and Dill, 2002; Acebes et al., 2013; Coleman and Hill, 2014b), alter movement or activity patterns (Fortin et al., 2005; Creel et al., 2008; Valeix et al., 2009; Willems and Hill, 2009a; Palmer et al., 2017), increase their vigilance (Brown, 1999; Campos and Fedigan, 2014), or increase the number of nearby individuals (McNamara and Houston, 1992; Parker et al., 2022) when accessing high risk areas. Spatial variation in habitat structure also plays an important role in a prey’s perceived risk (Cowlishaw, 1998; Valeix et al., 2009), as visibility affects the ease of escape of prey species (Lima, 1992), whilst aiding predators through camouflage and ambush opportunities (Hopcraft et al., 2005). Canopy height and height above ground are also important determinants of risk perception in arboreal species, with areas closer to the ground viewed as ‘riskier’ habitats (Emerson et al., 2011; Campos and Fedigan, 2014; Nowak et al., 2014). Similarly, more open, non-forested areas are inherently ‘riskier’, due to the increased likelihood of encountering multiple predator guilds, and also increasing the potential for conflict with humans (Nowak et al., 2017; Parker et al., 2022). Perceived predation risk also varies temporally resulting from variation in predator/prey activity patterns (Suraci et al., 2022). Prey species may therefore seek to adopt behaviours that minimise risk at certain times of the day and, on a larger scale, across seasons (Suraci et al., 2022). Whilst diel risk may vary depending on the species and habitat in question, sleep site selection has consistently been linked with perceived predation risk in primates (Anderson, 1998; Reichard, 1998; Day and Elwood, 1999; Ramakrishnan and Coss, 2001; Fan and Jiang, 2008; Albert et al., 2011; Cheyne et al., 2013; Bidner et al., 2018). Anxiety levels may therefore increase when approaching sleep sites owing to the uncertainty over whether sites remain safe on a given day. Other potential anxiety-inducing factors include grouping behaviour, which has consistently been cited as an antipredator behaviour in many species (da Silva and Terhune, 1988; Fitzgibbon, 1990; Magurran, 1990; Fortin et al., 2009; Makin et al., 2017; Iranzo et al., 2018), including primates (Hill and Dunbar, 1998; Castles et al., 1999; Schmitt and Di Fiore, 2015; LaBarge et al., 2020; Dell’Anna et al., 2022; Parker et al., 2022). Thus, more isolated individuals with fewer nearby neighbours may experience increased anxiety (Tkaczynski et al., 2014; Dell’Anna et al., 2022). Furthermore, social stressors, such as competition during mating season (Sclafani et al., 2012), agonistic interactions (Aureli, 1997; Castles and Whiten, 1998), the proximity of higher-ranking conspecifics (Castles et al., 1999), and intergroup encounters (Brooks et al., 2021), may also increase the display of SDBs. Here, we explored how perceived predation risk influences scratching rate, an anxiety-related behaviour, in the arboreal samango monkey ( Cercopithecus albogularis schwarzi ), living in a highly fragmented, highly seasonal, and multi-predator environment (Parker et al. 2020). Predators of the samango monkey include: eagles (African crowned; Stephanoaetus coronatus, and Verreaux’s; Aquila verreauxii ), leopard ( Panthera pardus ), and African rock python ( Python sebae ). The impact of predation from eagles, in particular, has been shown to be the key driver of samango monkey ranging patterns (Coleman and Hill, 2014b), as well as influencing spatial variation in behaviour (Parker et al., 2022). As a result, samangos are expected to display anxiety-related responses to living in this environment. To validate scratching as an anxiety-related behaviour, we predicted that the rate of self-scratching would increase immediately following a predator encounter, indicated by predator specific alarm calls (Fuller, 2013), relative to a control period. We predicted that areas of high perceived risk from eagle predation would be associated with an increase in scratching, which may indicate an increase in anxiety associated with these high-risk areas. Similarly, we also predicted that scratching would increase as individual height above ground decreased, in more open and non-forested areas, and during periods of low light levels, other factors associated with risk in arboreal primates (Cowlishaw, 1998; Hill and Weingrill, 2007; Emerson et al., 2011; Cheyne et al., 2013; Campos and Fedigan, 2014; Nowak et al., 2014, 2017; Bidner et al., 2018; Parker et al., 2022). Finally, we also assumed monkeys would scratch more in areas of increased intergroup encounter risk, during the hotter, wetter summer months, and in individuals towards the periphery of the group, due to the increased perceived risk associated with increasingly isolated individuals (Tkaczynski et al., 2014; Dell’Anna et al., 2022). Study species and field site The samango monkey is an arboreal, diurnal guenon that inhabits the tall-canopy, evergreen indigenous forests of South Africa (Linden et al., 2016; Wimberger et al., 2017; Parker et al., 2020, 2021, 2022). They live groups averaging around 40 individuals (Lawes et al., 2013; Coleman and Hill, 2014a), and comprising a single adult male, multiple adult females, as well as sub-adults and juveniles (Henzi and Lawes, 1987). The breeding season typically occurs between May and July where other, non-resident, adult males may temporarily form part of the group. Whilst their diet mainly comprises fruits (Lawes et al., 2013; Linden et al., 2016; Nowak et al., 2017; Wimberger et al., 2017; Parker et al., 2020), samangos show considerable dietary flexibility due to living in a highly fragmented and seasonal environment (Nowak et al., 2017; Wimberger et al., 2017; Coleman et al., 2021). Fieldwork was conducted at the Primate and Predator Project, Lajuma Research Centre (23°02’23”S, 29°26’05”E), in the western Soutpansberg Mountains of South Africa. The mountain range experiences warm, wet summers from October to March, and cool, dry winters from April to September. This gives rise to significant diversity in vegetation types (Mostert, 2006), including indigenous mistbelt forest (Mostert, 2006; Mucina and Rutherford, 2006), which is highly fragmented by montane grasslands, shorter secondary forests (Hahn, 2006), residential properties, farmlands and commercial plantations. Data collection We followed two well-habituated groups of samango monkeys (‘Barn’, 30 – 40 individuals, and ‘House’, 60 – 70 individuals) between August 2016 and August 2017. Five-minute focal samples (Altmann, 1974) uwere collected sing the ‘Prim8’ app (McDonald and Johnson, 2014) on a mobile smart phone (Samsung Galaxy S5). Focals were collected on identifiable adult females (through ear tags and/or distinguishing features; ‘Barn’, n = 3; ‘House’, n = 18) to remove potential age and sex biases in anxiety (Rangel-Negrín et al., 2009). Groups were followed from morning to evening sleep site (range: 06:00 – 18:00), with a minimum of 15 minutes between new focal samples on different, randomly selected individuals. The same individual was not sampled more than twice per day, and only once in the morning and afternoon sessions. Only focal samples lasting the full five minutes were used in analysis, resulting in a total of 252 focals over the study period (range 1 – 22 per individual). Information collected during each focal sample included the date, time, individual ID, group ID, individual height (average height in m over the five minute sample), the number of conspecific individuals within 5 m (continuous counts averaged across the five minute sample), and the number of self-scratching events (quick, repetitive movements of the hand or foot through fur; Barros et al., 2000). Scratching was recorded as separate bouts when separated by a period of five seconds or more (Castles et al., 1999). We also recorded the individual’s location for each focal sample using a handheld GPS (Garmin GPSmap 64S). To determine perceived predation risk across the home range we followed the approach of (Coleman and Hill, 2014b) and (Parker et al., 2022). Both the acoustically distinct alarm calls of adult male samango monkeys and, to a lesser extent, group-wide alarm calls, have been attributed to specific predator guilds (Fuller, 2013). In particular, the adult male ka and katrain calls are associated with raptors (Fuller, 2013), whilst group-wide alarm calls are commonly associated with antipredator behaviour linked to raptors, such as jumping down from the canopy, looking up, and scanning the sky (Fuller, 2013). The location of all ka , katrain and group-wide alarm calls were recorded between August 2016 – August 2017, resulting in a total of 356 alarm calls across both groups for the study period. Eighty-three of these calls were directly associated with a predator (eagle: 74, snake: 9), whilst a further 129 calls were associated with antipredator behaviour from raptors, meaning 60% of alarm calls were directly linked to raptors. We used all alarm calls to create the landscapes of fear, giving an overall spatial map of perceived risk. We also used these alarm calls to validate scratching as an anxiety-related behaviour in this species. To determine intergroup encounter risk, the time, location, and details of all encounters were recorded on an all-occurrence basis. Intergroup encounters were defined as the study group being within visual range of another group, regardless of whether encounters became antagonistic (Coleman and Hill, 2014b). There were 104 intergroup encounters recorded across the study period. We downloaded EVI (Enhanced Vegetation Index) composites from Google Earth Engine (https://earthengine.google.com) from the Landsat 8 databases for the entire western Soutpansberg Mountains at a resolution of 30 m 2 , for each month across the study period (August 2016 – August 2017). EVI (Huete et al., 2002) is a remotely sensed measure of plant productivity (Paruelo et al., 1997), and has frequently been used as a proxy for food availability, particularly in species which consume large proportions of leaves (Leimgruber et al., 2001; Ito et al., 2006; Willems et al., 2009). Data processing All data were imported in QGIS 3.0 (QGIS Development Team, 2018). To create annual landscape of fear maps for each group, we followed the approach of Parker et al., (2022). Similarly, we used the same approach to create annual maps of group encounter risk, albeit using GPS locations of encounters as opposed to alarm call locations. Monthly EVI composites were selected where cloud cover did not obscure the study area by >30% were loaded into QGIS, and EVI values for each focal sample location were calculated using the “zonal statistics” plugin. The start time of each focal sample was grouped into one of four categories: early morning (06:00 – 09:00), late morning (09:00 – 12:00), early afternoon (12:00 – 15:00), and late afternoon (15:00 – 18:00). Grouping time in this way allowed for clearer observation of the effect of time of day on scratching rate. We also grouped focal observations into seasons based on when individuals were sampled (summer, October – March; winter, April – September). Statistical analysis We used the ‘dplyr’ package (Wickham et al., 2017) in R 3.5 (R Core Team, 2018) to calculate scratching as a rate per focal sample (frequency per five minutes), as well as the mean number of near neighbours within 5 m for each focal. To validate scratching as an anxiety-related behaviour, we compared scratching rate within 10 minutes of an eagle alarm call to scratching rate from focals up to one hour before the recorded alarm. Rate of scratching before and after an alarm was compared within the same individual ( n = 10) using a paired t-test with the t.test function in R. We used a generalised linear mixed model with a negative binomial error structure to predict the rate of scratching as a function of the landscape of fear, intergroup encounter risk, EVI, season, focal individual height, the number of nearby individuals from the focal individual, and time (with early morning as the reference category). We controlled for individual differences in anxiety by including individual ID as a random effect, in addition to month. Group was included as an additional fixed effect to control for any effects of group size, as well as sampling effort between the two groups. Focal samples where alarm calls occurred during sampling, or in the 30 minutes prior to sampling, were excluded from analysis ( n = 4). Models were fitted in R using the glmmTMB function in the ‘glmmTMB’ package (Brooks et al., 2017). We ran glms excluding the random effects and used the vif (Variance Inflation Factor) function from the ‘car’ package to investigate collinearity between our predictors. We found no evidence of collinearity with all values below 2.0 (Hair et al., 2014). We also found no evidence of spatial autocorrelation after running a Moran’s I test on the model residuals, using the ‘spdep’ package (Bivand and Wong, 2018). Results Rate of scratching significantly increased in the 10 minutes after an eagle alarm call relative to the control period ( t (9) = - 4.30, P = 0.002, Figure 1). Anxiety was defined as scratching in the subsequent analysis. Figure 1. Rate of scratching (average frequency per five-minute focal observation) up to one hour before and up to 10 minutes after an adult male predator-specific alarm call, in 10 identified adult female samango monkeys at Lajuma Research Centre, Soutpansberg Mountains, South Africa. Contrary to our predictions, perceived predation risk, indicated by the landscape of fear, did not influence the rate of scratching (Table 1). Similarly, EVI, intergroup encounter risk, individual height, and number of nearest neighbours also had no influence on scratching. We found that the rate of scratching significantly decreased in the winter months, but increased in the late afternoon compared to early morning. Table 1. Coefficient estimates and key statistics of GLMM expressing scratching as a function of landscape of fear, Enhanced Vegetation Index (EVI), season, intergroup encounter risk, individual height, number of nearest neighbours, and time of day. (Intercept) 0.019 0.354 (1) -0.675 0.713 (1) Landscape of fear 0.043 0.079 0.549 -0.111 0.198 0.583 EVI -0.084 0.077 -1.098 -0.234 0.066 0.272 Season (winter) -0.416 0.171 -2.435 -0.751 -0.081 0.015 Intergroup encounter risk 0.108 0.081 1.323 -0.052 0.267 0.186 Individual height 0.064 0.075 0.857 -0.083 0.211 0.391 Nearest neighbours 0.106 0.083 1.274 -0.057 0.269 0.203 Time (late morning) 0.082 0.199 0.415 -0.307 0.472 0.678 Time (early afternoon) 0.166 0.203 0.822 -0.231 0.564 0.411 Time (late afternoon) 0.600 0.280 2.141 0.051 1.149 0.032 Group (House) 0.483 0.339 1.426 -0.181 1.147 0.154 EVI, Enhanced Vegetation Index; Nearest neighbours, number of nearby individuals within 5 m of focal subject. (1) not shown because of having no meaningful interpretation. Discussion In wild animals, the fear of being eaten can evoke anxiety in prey species (Brown, 1999). Despite predator-induced anxiety having been explored extensively in a wide-range of species (Hendrie et al., 1996; Barros et al., 2000; Apfelbach et al., 2005; Blaser et al., 2010), studies on primates (Barros and Tomaz, 2002), particularly in the wild, are lacking (Mineka et al., 1980). We explored how perceived predation risk, and measures of habitat structure associated with risk, influenced anxiety-related behaviour in wild samango monkeys. We found that scratching, a self-directed behaviour commonly linked to anxiety in primates (Baker and Aureli, 1997), significantly increased immediately following alarm calls in response to perceived predation risk from eagles. Nevertheless, spatial perception of risk, indicated by the landscape of fear, did not influence the rate of scratching. Instead, scratching increased during the warm summer months, and in the late afternoon. Self-directed behaviours, such as scratching, have long been linked with anxiety in captive primates (Baker and Aureli, 1997; Barros et al., 2007, 2008). Here, we show that anxiety following perceived presence of a predator also manifests as scratching in wild samango monkeys. This increase in scratching immediately following an alarm call is unsurprising given the direct costs of mortality from successful predation (Barros et al., 2000, 2004). Anxiety in this case should be beneficial to an individual’s survival as it elicits the appropriate antipredator response, such as increasing vigilance and group density, or seeking refuge (Brown et al., 1999b). Interestingly, anxiety-related behaviours did not increase in areas of high perceived predation risk (indicated by the landscape of fear). However, individuals may pre-emptively minimise risk (and subsequently anxiety) by adopting antipredator strategies when entering ‘riskier’ areas. Parker et al. (2022) found that groups became more cohesive, reducing distances between conspecifics, when entering areas perceived as ‘high-risk’ in the same groups of samango monkeys, whilst LaBarge et al. (2020) further found that this response was pre-emptive. A more cohesive group may seek to confuse predators, (Krause and Ruxton, 2002; Scott-Samuel et al., 2015), and/or share the vigilance load between individuals (McNamara and Houston, 1992), thereby minimising risk. Consequently, these behaviours may help to minimise the anxiety associated with ‘high-risk’ areas. Alternatively, anxiety levels in these areas may simply be lowered by the presence of human observers acting as a deterrent towards predators (Emerson et al., 2011; Campos and Fedigan, 2014; Nowak et al., 2017; LaBarge et al., 2022). However, the ‘shield’ effect of human observers may be less pronounced in areas of reduced vegetation (where ease of escape is more difficult, regardless of human presence) or when locating sleep sites during low light levels (Cheyne et al., 2013; Bidner et al., 2018). Despite the absence of an effect of perceived predation risk here, the use of vocalisations as an indicator of spatial behaviour is still valuable (Netoskie et al., 2023). Anxiety in samango monkeys varied temporally, with an increase in scratching rate observed in the late afternoon. This period, particularly the latter part of this period, is consistent with the animals assessing their sleep site locations. Minimising predation risk has long been recognised as a factor influencing sleep site selection in primates (Anderson, 1998; Reichard, 1998; Day and Elwood, 1999; Ramakrishnan and Coss, 2001; Fan and Jiang, 2008; Albert et al., 2011; Cheyne et al., 2013; Bidner et al., 2018), with arboreal species often preferring the tallest trees in the home range as sleep site locations (Chapman, 1990; Barrett and Lowen, 1998; Coleman, 2013) due to the protection afforded from multiple predator guilds. However, sleep sites which are consistently used can still vary in predation risk owning to the spatial and temporal activity patterns of predators. Consequently, choosing the safest sleep site on a given day is likely to be anxiety-inducing. Yet, studies exploring this explicitly appear to be lacking. There are, however, examples of primates being exposed to an increased risk of predation when moving towards sleep sites (Reichard, 1998; Bidner et al., 2018). Individuals often appear to adopt behaviours that seek to minimise predation risk during this time, such as moving quickly towards sleeping trees (Day and Elwood, 1999; Fan and Jiang, 2008; Zhou et al., 2009), adopting low-key, inconspicuous behaviours (Reichard, 1998; Day and Elwood, 1999; Xiang et al., 2010), and increasing vigilance (Reichard, 1998; Zhou et al., 2009; Dayong et al., 2011). A heightened state of anxiety may further prepare individuals for adopting an antipredator response should a predator be encountered, whilst also reflecting the increased risk of locating sleep sites in low light levels when various predators are most active and more difficult to detect (Cheyne et al., 2013; Bidner et al., 2018). Anxiety was also predicted by season, with self-directed scratching occurring more often in the hot, wet summer months compared to the cool, dry winters. The summer months at Lajuma are associated with an increase in fruit availability (Coleman, 2013) and so it may be reasonable to expect an increase in anxiety during this period due to increased intra- and inter-group aggression. However, samangos show very little within-group aggression, particularly over resources (Payne et al., 2003), and we similarly found no influence of intergroup encounter risk on anxiety here, thus suggesting fruit availability is unlikely to influence anxiety in samangos. A more likely explanation may therefore be the influence of weather, and other associated factors, which correlate with season. In Japanese macaques, self-scratching was positively correlated with ambient temperature and relative humidity (Ventura et al., 2005). In this instance, scratching likely does not indicate anxiety but rather serves as a hygienic function, with the increased need for pelage care possibly associated with variations in piloerection, sweating and ectoparasite load (Ventura et al., 2005). Such examples further evidence the multi-faceted nature of self-directed behaviours and scratching, in particular, may serve as both a mediator of anxiety and to maintain pelage care. It is important to note that another correlate of anxiety is mating (Sclafani et al., 2012). However, we can rule out any relationship with mating here as the samango monkey breeding season in this subspecies is during the cool, dry winter months when scratching was observed to be lowest. As forest specialists, samango monkeys are largely restricted to areas of tall-canopy indigenous forests (Linden et al., 2016; Wimberger et al., 2017; Parker et al., 2020, 2021). Despite this, movement into areas of reduced vegetation cover (indicated by the EVI) did not appear to invoke anxiety. However, samangos have frequently been shown to utilise matrix habitat (Nowak et al., 2017; Wimberger et al., 2017; Parker et al., 2021), and move between forest patches to access specific food resources (Beeson et al., 1996; Coleman and Hill, 2014a; Parker et al., 2020). Although inherently riskier for arboreal species, this behavioural flexibility may explain why more open areas with reduced vegetation do not invoke anxiety in this species. One of the most common stressors in primates is that of fellow group members (Cowlishaw, 1997; Hill and Weingrill, 2007). Both conflict between (Dunbar, 1988) and the dominance rank (Castles and Whiten, 1998; Castles et al., 1999) of nearby individuals can increase anxiety in the focal individual. In addition, individuals at the periphery of a group may experience more anxiety due to the increased perceived risk of being more isolated (Tkaczynski et al., 2014; Dell’Anna et al., 2022). Whilst we found no effect of the number of nearby individuals on anxiety, the low level of aggression in this species likely serves to reduce anxiety resulting from conspecifics (Lawes, 1990; Payne et al., 2003). In conclusion, we show that anxiety in response to perceived predation risk appears to be reactive rather than pre-emptive in samango monkeys. Scratching rate (a self-directed behaviour) significantly increased immediately following a predator specific alarm call, thus validating this behaviour as a proxy for anxiety. We also found that anxiety increased during the summer months and in the late afternoon. We suggest that scratching can be a useful indicator of anxiety in wild primates and can help to improve knowledge on the environmental factors that induce stress in wild populations. Declarations Ethics approval and consent to participate All behavioral data collection followed the Association for the Study of Animal Behavior ( ASAB ) Guidelines for the Treatment of Animals in Behavioral Research and Teaching (ASAB, 2012) and were covered by the Liverpool John Moores University’s use of Live Animals in Unregulated Research Protocol (NK_EP/2016-10). All fieldwork was approved by the Life Sciences Ethical Review Process Committee and the Department of Anthropology Ethics Committee at Durham University, UK and was conducted with approved permits from Limpopo Province Department of Economic Development and Tourism (LEDET). It was not possible to record data blind because our study involved focal animals in the field. No human subjects were involved in this study. Conflict of interest The authors declare that they have no conflict of interest. Funding The authors greatly appreciate the financial support from LJMU for a University PhD Scholarship, Durham University, the Earthwatch Institute and an anonymous donor. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data accessibility The datasets generated and/or analysed during the current study are available in the figshare repository at: 10.6084/m9.figshare.24243430. References Acebes, P., Malo, J.E. and Traba, J., (2013) Trade-offs between food availability and predation risk in desert environments: The case of polygynous monomorphic guanaco ( Lama guanicoe ). Journal of Arid Environments , 97, pp.136–142. Albert, A., Savini, T. and Huynen, M.C., (2011) Sleeping site selection and presleep behavior in wild pigtailed macaques. American Journal of Primatology , 7312, pp.1222–1230. Allan, A.T.L. and Hill, R.A., (2018) What have we been looking at? 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