Dominance, energetics and stress in female capuchins in Costa Rica

Leakey Foundation Final Report Mackenzie L. Bergstrom, PhD Candidate Department of Anthropology & Archaeology, University of Calgary

OVERVIEW Many primate studies have drawn links between ecology and the formation of social systems, but few have extensively examined the physiological costs associated with seasonality, foraging behavior, resource competition and social behavior. Quantification of physiological processes in wild populations is logistically difficult; therefore, the utilization of recent methodological advances to gain a better understanding of the proximate variables affecting the physical condition of individuals will help to elucidate the relationship among behavior, the environment and reproductive success. The goal of my dissertation research is to better understand how ecological and social variables affect the physical condition of female white-faced capuchins (Cebus capucinus) living in a tropical dry forest in Sector Santa Rosa (SSR) of the Ă rea de ConservaciĎŒn Guanacaste (ACG) in northwest Costa Rica. White-faced capuchins are omnivorous (Fragaszy et al. 2004). The SSR population inhabits a highly seasonal environment and shows considerable variation in the types of foods they consume and their ranging patterns during the annual cycle (Campos et al. 2014; Melin et al. 2014b). Reproductive events coincide with seasonal variation in food availability (Carnegie et al. 2011a; Di Bitetti and Janson 2001; Janson and Verdolin 2005). Capuchins are also highly intelligent and form extensive social networks. Females exhibit linear and stable dominance hierarchies but show both competitive and cooperative social behaviors (Bergstrom and Fedigan 2010; Bergstrom and Fedigan 2013).

These characteristics make capuchins an ideal species in which to investigate how proximate ecological and social variation affects physiological processes and how this may inform us about the differential reproductive success of females.

I conducted 12 months of behavioral observations of 25 adult female capuchins, phenological surveys and nutritional analyses of food items consumed, radio-immunoassays of urinary c-peptide of insulin as a measure of energetic condition, and enzyme-immunoassays of fecal cortisol as a measure of stress. The project objectives were to 1) document the dietary profile of females; 2) measure the extent of the seasonal variation in diet and nutritional intake; 3) determine if seasonal variation in the availability of foods affects the physical condition of females; and 4) determine ecological and social correlates of energy balance and stress using urinary C-peptide and fecal cortisol, respectively. Results suggest that there is seasonal variation in food availability and the types of foods consumed, as well as inter-seasonal and inter-group differences in energy intake and macronutrient intake by females. These seasonal differences

affect the physical condition of females in terms of relative muscle mass and energy balance.

The next step is to include these findings in further analyses to investigate variation in the stress response of females in this long-term study population. This will provide insight into behavioral strategies employed by females at varying reproductive states and social ranks in response to proximate ecological and social pressures.

METHODS Behavioral data During three field seasons spanning the early rainy, late rainy and dry seasons (totaling 12 months), I collected 575 hours of focal data during 2,124 hours of observational contact from 25 adult female capuchins living in three habituated social groups in SSR (10º50ʹ30ʹʹN, 85º37ʹ0ʹʹW).

I collected behavioral data in the form of 10-minute focal animal samples (Altmann 1974), during which I recorded all affiliative, agonistic, sexual, solitary and feeding behaviors as well as five instantaneous samples of general activity, proximity to other individuals ( 5m), and location within the group. I used agonistic behavior collected during focal samples in conjunction with data collected ad libitum to construct dominance hierarchies using the I&SI method in MatMan 1.1 (Noldus Information Technology 1998). I used infant births, known gestation length (Carnegie et al. 2011b) and nursing behavior to determine the reproductive states of females (pre-conception, gestation, lactation). To compare to C-peptide measures, I calculated energy balance using behavioral, nutritional and ranging data as energy intake (kJ) minus energy expenditure (daily travel  expenditure estimated based on basal metabolic rate and additional activity  estimated reproductive state demands) (Altmann 1987; Coelho Jr 1974; Coelho Jr. et al. 1976; Janson 1988; Key and Ross 1999; Kleiber 1961; Taylor et al. 1970).

Ecological and nutritional data To determine the macronutrient and energy content of foods consumed by capuchins, I collected, processed and analyzed (Dairy One Forage Laboratory, NY) 53 plant food items and 10 categories of invertebrate foods. I used these nutritional values in conjunction with behavioral data and previously collected nutritional data from this field site and a neighboring field site

(McCabe 2005; Vogel 2005; Vogel unpublished) to determine energy and macronutrient intake rates. As part of a long-term and on-going collaborative dataset at SSR, I assessed the monthly fruit coverage and maturity of over 300 trees from 30 species important to the capuchin diet.

These data were used in conjunction with tree abundance data from 151 botanical transects covering 3.02 ha within the study groups’ home ranges (Melin et al. 2014a) to calculate fruit abundance (kg/ha) and then ripe fruit energy density (kJ/ha) using species-specific nutritional values. Monthly weather data (maximum and minimum temperature and rainfall) were calculated from daily values collected using a Kestrel weather meter and standard rain gauge.

Physiological data I collected 955 urine samples using a plastic sheet and net structure or from vegetation immediately upon excretion. Upon collection, I tested 562 urine samples (for which sufficient volume was collected for subsequent creatinine and C-peptide analysis) for the presence/absence of ketones using urinalysis test strips (Seimens Multistix 10 SG). In the Hominoid Reproductive Ecology Laboratory at the University of New Mexico, I measured urinary creatinine concentration via the Jaffe reaction (Bonsnes and Taussky 1945) and specific gravity of urine using a refractometer (Atago PAL-10S). I measured urinary C-peptide concentration using commercially available human radioimmunoassay kits (RIA Human C-peptide Kit, Millipore Corporation, Billerica, MA) (Emery Thompson and Knott 2008; Emery Thompson et al. 2009; Sherry and Ellison 2007). I obtained specific gravity values greater than zero from 734 urine samples (range = 1.001 to 1.045). Creatinine analysis yielded 746 samples from which valid results were obtained (range = 0.021 to 1.583). Finally, urinary C-peptide analysis yielded 606 samples that contained sufficient volume for analysis, were free from fecal contamination, and from which valid results were obtained (i.e., values fell within the range of the standard curve and replicate CV was  15% for high samples and  25 for low samples). The intra-assay coefficients of variation were 11.9% for low ( 250 pg/ml) and 7.1% for high (> 250 pg/ml) samples, respectively. The inter-assay coefficients of variation were 10.8% and 9.4% for Controls I and II, respectively. Samples were subsequently standardized for urine concentration using both creatinine (N = 599, range = 96 to 2,370 pg/mg Cr) and specific gravity (N = 535, range = 90 to 1,135 pg/ml) for comparative purposes.

I collected 1010 fecal samples immediately upon excretion. I conducted preliminary processing in the field laboratory following a modified version of the solid phase extraction (SPE) protocol described by Ziegler and Wittwer (2005) and used by Carnegie, Fedigan, & Ziegler (2011b) to extract cortisol from each fecal sample. Cortisol was extracted from each sample and stored in a matrix within Prevail C18 SPE cartridges for later elution, processing and analysis at the University of New Mexico following the hormone assay protocol used at the National Primate Research Center (NPRC) in Wisconsin and developed by Munro and Stabenfeldt (1984), which uses competitive binding enzyme immunoassays with antibodies that were raised in rabbits. Further analysis using the cortisol dataset is underway.

RESULTS Dietary profile Female white-faced capuchins focused foraging efforts on fruit and invertebrate food items.

Fruit contributed the most to the overall energy gain despite the greater proportion of time devoted to searching for and consuming insects. Although the nutritional composition of food types was variable, on a dry matter basis fruits were the most important source of sugar, whereas high proportions of protein intake came from invertebrates, particularly when fruit availability was low. Females were able to consume macronutrients at a much higher rate while foraging fruit due to the larger size of food items compared to invertebrates. There was temporal variation in the types of foods consumed and nutritional intake. While females exceeded estimated nutritional requirements during periods of high fruit availability, energy intake was much lower during periods of low fruit availability, warranting a more detailed investigation into capuchin foraging patterns, variation in the utilization of other food types, and nutritional intake during this time period

Seasonal variation in diet and nutritional intake There was seasonal and inter-group variation in home range quality in terms of ripe fruit availability (energy density, kJ/ha) (Figure 1), and a negative logarithmic relationship between the monthly energy density of ripe fruit and the percentage of monthly energy intake from

invertebrate food items (Figure 2). Many orders of invertebrates complemented nutritional intake in terms of energy and protein consumption throughout the annual cycle. The consumption of lepidopteran larvae (i.e., caterpillars) was statistically strongly seasonal and lepidopterans played a particularly important dietary role, as their seasonal outbreak coincided with a four month period of low fruit availability. Consequently, they comprised a considerable proportion of the overall energy and protein intake. However, females did not always appear to meet monthly estimated energy requirements (Figure 3). Even with high caterpillar consumption, it is likely that females were utilizing metabolic energy stores at this time since they were not meeting estimated energy requirements. The analysis of urinary parameters and the metabolic biomarker C-peptide, provided a more detailed picture of the metabolic consequences associated with this seasonality in resource availability.

Seasonal variation in relative muscle mass I measured urinary creatinine and specific gravity to assess variation in the relative muscle mass of females. Creatinine is derived in muscle and the relationship between urinary creatinine excretion and urine density (specific gravity) has been used as an estimate of relative muscle mass in wild primates (Emery Thompson et al. 2012). Using mixed effects models, I determined that season was a significant predictor of the relationship between creatinine and specific gravity, whereby relative muscle mass was greater during months with high ripe fruit availability versus low fruit availability (Figure 4). Although the effect was smaller, group ID also significantly predicted relative muscle mass, whereby females in CP group, whose home range quality in terms of fruit availability is nearly half that of the other two groups, had significantly lower relative muscle mass than females in GN group, and there was a statistical trend for females in CP group to have lower relative muscle mass than females in LV group.

Figure 1 Ripe fruit energy density (kJ/ha) available in the home ranges of three study groups of white-faced capuchin monkeys at Sector Santa Rosa, Costa Rica. Calculations are based on ranging, transect and phenology data for the 2009-2011 study period. The dotted line represents the group’s annual mean ripe fruit energy density.

Mean Energy Intake (kJ/day)

Figure 2 Monthly ripe fruit energy density (kJ/ha) versus the monthly percentage of energy intake from invertebrates by females from three groups of white-faced capuchins.

3000 2500 2000 1500 1000 500 0 J

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Fruit

Invertebrate

Minimum Energy Requirement

Adjusted Energy Requirement

Figure 3 Mean contribution of fruit and invertebrates to the estimated daily energy intake per metabolic body weight per day. The dotted red line indicates monthly estimated energy intake based on the estimated 1000 kJ/day. The dotted black line is adjusted by multiplying minimum requirements by the corresponding energy coefficients (1.0, 1.25, 1.5) of the mean monthly reproductive state (cycling, gestating, lactating, respectively) (Ausman et al. 1986; Ausman and Hegsted 1980; Key and Ross 1999).

Mean Creatinine Residuals

.800 .700 .600 .500 .400 .300 .200 .100 .000 LV

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Group Fruit Availability:

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Figure 4 Comparison of relative muscle mass (ď‚ą SE) across groups and between months with high (grey bars) versus low (white bars) ripe fruit availability. Shown on the y-axis are the model-fitted estimated mean creatinine residuals, displayed per group by season.

Measuring energy balance I measured the difference between energy intake and energy expenditure to calculate energy balance in females and compared it to two urinary parameters: the presence of ketones and urinary C-peptide. Ketones are produced and expected to be present in urine when individuals are metabolically stressed (i.e., during nutritional deficit) and metabolizing fat stores (Sobti 2008). Therefore, individuals produce ketones when they are experiencing negative energy balance. During the two months with the lowest observed energy intake (June and July), one quarter of the urine samples tested for the presence of ketone bodies yielded positive results.

Forty-four percent of the female study subjects exhibited ketonuria in at least one sample during these months, which suggests that females were catabolizing fat stores during this period of reduced energy intake and estimated energy deficit.

C-peptide is produced in an equimolar relationship to insulin (Rubenstein et al. 1969).

Thus, urinary C-peptide levels correspond to insulin production and the energy balance of individuals. Raw C-peptide assay values are most often corrected for the dilution of urine by

dividing by urinary creatinine concentration. However, variation in creatinine-corrected Cpeptide did not correspond to variation in the physical condition of females as indicated using other measures. This may be because creatinine excretion fluctuates with muscle mass, and this population of capuchins shows substantial variation in energy intake and physical condition as indicated by relative muscle mass estimation and urinary ketone production. Consequently, normalizing C-peptide concentration with creatinine may not accurately reflect energy balance.

However, the specific gravity of urine is not sensitive to changes in muscle mass and has been used as an alternative for normalizing urinary hormone concentrations (Miller et al. 2004).

When I used this method of normalization, variation in C-peptide followed a similar annual pattern of variation to that of calculated energy balance. I am currently modeling variation in specific gravity-corrected C-peptide to quantify the strength of the relationship between Cpeptide and a number of ecological and social predictors of energy balance.

PUBLICATION PLANS I will soon submit four manuscripts for publication based on my dissertation chapters: 1) Dietary profile, food composition and nutritional intake by female white-faced capuchins 2) The nutritional importance of invertebrates to female Cebus capucinus in a highly seasonal tropical dry forest 3) Using urinary parameters to estimate seasonal variation in the relative muscle mass of female white-faced capuchin monkeys (Cebus capucinus) 4) Energy intake, energy expenditure and the use of urinary C-peptide as a biomarker for measurement of energy balance in female white-faced capuchins I presented the results of (1) at the Canadian Association of Physical Anthropologists (CAPA) annual meeting in Fredericton, NB, Canada in November, 2014.

The modeling analysis of cortisol that was originally described in my proposal to the Leakey Foundation is not complete. I also plan to prepare at least one publication that focuses on seasonal and inter-individual variation in stress levels with the goal of disentangling the effects of metabolic versus psychosocial stress. Examining the differential stress responses of females in this long-term study population will provide insight into behavioral strategies

employed by females at varying social ranks in response to proximate ecological and social pressures. Cross-species comparison of these behavioral and physiological responses will help to better understand variation in socio-ecology and the evolution of female social relationships in human and non-human primates.

ACKNOWLEDGEMENTS I am extremely grateful to The Leakey Foundation for providing funding for the laboratory analysis of the urinary and fecal biomarkers, which made this project possible. I would also like to thank the following organizations for funding and support: Alberta Innovates Technology Futures, International Primatological Society, Animal Behavior Society, Sigma Xi, University of Calgary and the National Sciences and Engineering Research Council of Canada (NSERC) for funding provided through Linda Fedigan’s (supervisor) Discovery Grant. Thank you to Linda Fedigan for supervision and the opportunity to conduct this project, Chelsea Lees, Barb Kowalzik, Bhavisha Thankey, Heidi Clouse, Monica Myers, Teresa Holmes, and Caroline Turner for assistance with data collection in the field, and Erin Vogel for use of unpublished nutritional data. Additional thanks to John Addicott for assistance with database design, and Tak Fung, Michelle Brown and Melissa Emery Thompson for statistical guidance. Finally, thank you to Sr. R. Blanco Segura, the Costa Rican park staff, and the Ministry of the Environment, Energy and Technology (MINAET), for permission to carry out my research in Sector Santa Rosa, Costa Rica.

REFERENCES Altmann J. 1974. Observational study of behavior: sampling methods. Behaviour 49:227-265.

Altmann SA. 1987. The impact of locomotor energetics on mammalian foraging. The Journal of Zoology 211(2):215-225.

Ausman LM, Gallina DL, Hayes KC, and Hegsted DM. 1986. Comparative assessment of soy and milk protein quality in infant cebus monkeys. The American Journal of Clinical Nutrition 43(1):112-127.

Ausman LM, and Hegsted DM. 1980. Protein requirements of adult cebus monkeys (Cebus albifrons). The American Journal of Clinical Nutrition 33(12):2551-2558.

Bergstrom ML, and Fedigan LM. 2010. Dominance among female white-faced capuchin monkeys (Cebus capucinus): hierarchical linearity, nepotism, strength and stability.

Behaviour 147(7):899-931.

Bergstrom ML, and Fedigan LM. 2013. Dominance style of female white-faced capuchins.

American Journal of Physical Anthropology 150(4):591-601.

Bonsnes RW, and Taussky HH. 1945. On the colorimetric determination of creatinine by the Jaffe reaction. The Journal of Biological Chemistry 158:581-591.

Campos FA, Bergstrom ML, Childers A, Hogan JD, Jack KM, Melin AD, Mosdossy KN, Myers MS, Parr NA, Sargeant E, Schoof VAM, and Fedigan LM. 2014. Drivers of home range characteristics across spatiotemporal scales in a Neotropical primate, Cebus capucinus.

Animal Behaviour 91:93-109.

Carnegie SD, Fedigan LM, and Melin AD. 2011a. Reproductive seasonality in female capuchins (Cebus capucinus) in Santa Rosa (Area de ConservaciĂłn Guanacaste), Costa Rica.

International Journal of Primatology 32(5):1076-1090.

Carnegie SD, Fedigan LM, and Ziegler TE. 2011b. Social and environmental factors affecting fecal glucocorticoids in wild, female white-faced capuchins (Cebus capucinus). American Journal of Primatology 73(9):861-869.

Coelho Jr AM. 1974. Socio-bioenergetics and sexual dimorphism in primates. Primates 15(23):263-269.

Coelho Jr. AM, Bramblett CA, Quick LB, and Bramblett SS. 1976. Resource availability and population density in primates. A sociobioenergetic analysis of energy budgets of Guatemalan howler and spider monkeys. Primates 17(1):63-80.

Di Bitetti MS, and Janson CH. 2001. When will the stork arrive? Patterns of birth seasonality in neotropical primates. American Journal of Primatology 50:109-130.

Emery Thompson M, and Knott CD. 2008. Urinary C-peptide of insulin as a non-invasive marker of energy balance in wild orangutans. Hormones and Behavior 53:526-535.

Emery Thompson M, Muller MN, and Wrangham RW. 2012. Technical note: Variation in muscle mass in wild chimpanzees: Application of a modified urinary creatinine method.

American Journal of Physical Anthropology 149(4):622-627.

Emery Thompson M, Muller MN, Wrangham RW, Lwanga JS, and Potts KB. 2009. Urinary Cpeptide tracks seasonal and individual variation in energy balance in wild chimpanzees.

Hormones and Behavior 55(2):299-305.

Fragaszy D, Visalberghi E, and Fedigan LM. 2004. The complete capuchin: the biology of the genus Cebus. Cambridge: Cambridge University Press.

Janson CH. 1988. Food competition in brown capuchin monkeys (Cebus apella): Quantitative effects of group size and tree productivity. Behaviour 105(1):53-76.

Janson CH, and Verdolin J. 2005. Seasonality of primate births in relation to climate. In: Brockman DK, and van Schaik CP, editors. Seasonality in primates: studies of living and extinct human and non-human primates. Cambridge: Cambridge University Press. p 307350.

Key C, and Ross C. 1999. Sex differences in energy expenditure in non-human primates.

Proceedings of the Royal Society London, Series B 266(1437):2479–2485.

Kleiber M. 1961. The fire of life: an introduction to animal energetics. Huntington, NY: Krieger.

McCabe GM. 2005. Diet and nutrition in white-faced capuchins (Cebus capucinus): effects of group, sex and reproductive state. Calgary: University of Calgary. 119 p.

Melin AD, Hiramatsu C, Parr NA, Matsushita Y, Kawamura S, and Fedigan LM. 2014a. The behavioral ecology of color vision: considering fruit conspicuity, detection distance, and dietary importance. International Journal of Primatology 35(1):258-287.

Melin AD, Young HC, Mosdossy KN, and Fedigan LM. 2014b. Seasonality, extractive foraging and the evolution of primate sensorimotor intelligence. Journal of Human Evolution 71(0):77-86.

Miller RC, Brindle E, Holman DJ, Shofer J, Klein NA, Soules MR, and O'Connor KA. 2004.

Comparison of specific gravity and creatinine for normalizing urinary reproductive hormone concentrations. Clinical Chemistry 50:924-932.

Munro C, and Stabenfeldt G. 1984. Development of microtitle plate enzyme immunoassay for the determination of progesterone. Journal of Endocrinology 101:41-49.

Noldus Information Technology. 1998. Reference manual. MatMan 11.

Rubenstein AH, Clark JL, Melani F, and Steiner DF. 1969. Secretion of proinsulin C-peptide by pancreatic beta cells and its circulation in blood. Nature 224(5220):697-699.

Sherry DS, and Ellison PT. 2007. Potential applications of urinary c-peptide of insulin for comparative energetics research. American Journal of Physical Anthropology 133:771778.

Sobti RC. 2008. Animal Physiology. Oxford: Alpha Science International Ltd. Taylor CR, Schmidt-Nielsen K, and Raab JL. 1970. Scaling of energetic cost of running to body size in mammals. American Journal of Physiology 219(4):1104-1107.

Vogel ER. 2005. Rank differences in energy intake rates in white-faced capuchin monkeys, Cebus capucinus: the effects of contest competition. Behavioral Ecology and Sociobiology 58:333-344.

Ziegler TE, and Wittwer DJ. 2005. Fecal steroid research in the field and laboratory: improved methods for storage, transport, processing, and analysis. American Journal of Primatology 67(159-174).