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Exploring Indicators of Food Choice for Chimpanzees at Taï National Park, Côte d’Ivoire: Aroma and Antioxidants

Authored by Chahan Yeretzian

Taï National Park in the southwest of Côte d’Ivoire is the largest remaining tropical rain forest in West Africa and covers 555,000 ha. While it is recognized as a “Biodiversity Hotspot”, with a rich natural flora and fauna, it is also one of the last remaining habitats of many endangered species. The Taï Forest reserve was created in 1926 and promoted to National Park status in 1972. It was recognized as a UNESCO Biosphere Reserve in 1978 and added to the list of Natural World Heritage Sites in 1982. Among the many endangered species living in the Taï National park, one species of particularly concern is the chimpanzee (Pan troglodytes verus Blumenbach 1779), a member of the great ape family. Chimpanzees have already disappeared from four African countries, and are nearing extinction in many others, such as the Côte d’Ivoire where a survey reveals a sharp decline of 90 % (from 8 000-12 000 individuals in 1990 to 800-1200 in 2007) [1]. In the Taï National Park, the situation is currently stable with an estimated chimpanzee population of 480 individuals [2].

Primates living in such natural habitats face various constraints for their nutritional needs. Chimpanzees are regarded ripe fruit specialists [3]. Eating predominantly ripe fruits, chimpanzees obtain a higher dietary quality compared to other frugivorous monkeys, whereas during fruit scarcity also other plant parts are consumed [3]. Besides plant food, also vertebrates and invertebrates are part of their diet [4]. Chimpanzees are able to manage environmental constraints, such as e.g. seasonality of food availability. By adapting their feeding behaviour [5,6], they are able to take nutritional advantage of the temporal abundance of ripe fruits to reach a high supply of carbohydrate in their diet [4-7]. Similarly, they consume the fruit flesh (pulpa) of Sacoglottis gabonensis (Baill.) Urb. (Malpighiales: Humiriaceae) as well as the hard seed by using tools [8], again taking full advantage of the nutritional content of the fresh fruits.

Yet, they still have to deal with structural and chemical aspects of the available plants and fruits. As argued by Janzen [8], flora is not just green, but is colored by compounds such as morphine, caffeine, tannin or terpene. Particularly for fruits, chemical components and physical characteristics are often designed either to repulse or attract animals (or humans), with the objective to favour dispersion of the species. Impact of secondary plant metabolites and fruit colour on food choice are well documented in birds and mammals [9-13]. While colour is qualified as an honest signal of food quality and macronutrient rewards for birds [14,15], it might not be enough for a decisive answer on the maturity stage of the fruit. Therefore, primates were observed to use, in addition to colour, different sensory cues, such as the firmness (haptic) by biting and the smell (volatile aroma compounds) by sniffing the fruits, in order to judge the level of ripeness of a fruit. Hence, the aroma of fruits may be an important indicator of food quality, proving useful information to the animals about availability and presence of beneficial nutrients. Especially for nocturnal monkeys olfactory guided foraging plays an important role at narrow range as visual cues cannot be exploited [16].

In this study, it was observed that chimpanzees from the Taï National Park smell on the fruits of the tree Sacoglottis gabonensis (Baill.) Urb., before deciding whether to eat or reject the fruit (N’Guessan, personal observation). Hence, in this study, we aim at examining the aroma composition of S. gabonensis fruits at different ripeness stages, in order to elucidate aroma compounds that may drive the selection of presumably ripe fruits by the apes. In addition, the antioxidant content of the fruits was measured to assess whether fruits, preferentially selected by the apes, were also characterized by a high antioxidant content.

Methods

Study site and fruit collection

The study was conducted at the Taï National Park in the southwest of Côte d’Ivoire with the aim of identifying clues for food choices of the apes. Researchers have conducted studies on chimpanzees’ communities, fully habituated to the presence of human observers since 1984. It is known that Chimpanzees never consume Sacoglotis fruits in trees. After selection and collection of fruits on the ground, they put several fruits in their mouth, mash and eject the stone.

During a field trip in September 2013 (by three authors of this paper: N’Guessan, Ahoua & Yeretzian), Sacoglottis gabonensis fruits have been collected in the natural habitat of the chimpanzees. Fruits were collected at the ground below the trees and the ripeness stages were judged by their texture, colour and smell. Fruits were separated in three lots of ripeness: unripe, ripe and overripe fruits. They were immediately processed by separating the flesh from the stone and immersing the flesh in liquid nitrogen (each in a small and labelled plastic bag) for storage in the Côte d’Ivoire. Later, for transport from the Côte d’Ivoire to Switzerland, fruits were transferred into dry ice.

Aroma profile of Sacoglottis gabonensis fruits measured with HS GC-MS

After arrival in Switzerland, the fruits were stored at -20 °C. For sample preparation, fruits were directly taken out of the freezer, cut into small pieces and immersed in liquid nitrogen for two minutes. Approximately twenty g of fruit was then homogenized in a ball mill (MM400, Retsch, Haan, Germany). Five g of fruit slurry was put into a headspace vial and stored in the fridge for less than 60 minutes until analysis by headspace gas chromatography coupled to mass spectrometry (HS-GC/MS). GC/MS analyses was performed on a 7890/5975N instrument (Agilent Technologies, Santa Clara, USA) equipped with a DB-WAX column (30m × 0.25mm ID, Agilent Technologies, Santa Clara, USA) in electron impact ionization mode. For the headspace equipment (Gerstel, Mühlheim an der Ruhr, Germany) a 2.5 mL headspace syringe with a syringe temperature of 55 °C was used with a flush time of 60 s. The incubation time of the sample was 10 min at 50°C while agitating at 250 rpm. The injection volume was 1 mL injected with an injection speed of 200.00 μL/s, a split of 5:1 and a helium flow of 1 mL/min. The GC run started at 35 °C for 5 min and was then heating with a ramp of 20°C/min to 240°C with a 5 min hold. For data analysis, the software MSD Chemstation (Version G1701 EA E.02.00.493, Agilent Technologies, Santa Clara, USA.) and a mass spectral library (NIST08, National Institute of Standards and Technology 2008) were used. Compounds were identified by comparison of MS spectra and retention times with the mentioned database. The volatile concentration in the headspace of the three ripeness stages was statistically analyzed using Kruskal-Wallis rank sum test, followed by a post-hoc test. For further differentiation between the samples, we performed a principal component analysis (PCA) on the HS GC/MS data, using the software package R (http://cran.rproject. org/, Tinn-R editor version 2.4.1.5, http://sourceforge.net/ projects/tinn-r/). Odour descriptors were taken from Flavornet by Terry Acree & Heinrich Arn (http://www.flavornet.org, © Datu Inc., 2004) and from The Good Scents Company™ (http://www. thegoodscentscompany.com).

Antioxidant capacity of Sacoglottis gabonensis fruits

For the antioxidant measurements, 500 mg of fruit slurry (see preparation for headspace analyses) were extracted three times with 10 mL of 70% aceton / 30% water phase. The extraction process included 10 minutes treatment in the ultrasonic bath and 2 min of mixing in a vortex. After evaporation, the residue was solved in 25 mL of water and filtered before analysis using 0.45 μm PET filters (Machery-Nagel, Düren, Germany). The Folin Ciocalteu (FC) reagent assay is measured on a FIAlab-3200 instrument (FIAlab Instruments Inc., U.S.A.) applying a FIA method [17]. The sample (diluted fruit extract) or antioxidant standard (gallic acid) were injected (injection loop 100 μl) into the flow stream (flow rate 30 μl/sec) of the FC reagent (0.2 M concentration). After mixing with sodium hydroxide (0.25 M concentration, flow rate 30 μL/ sec), to raise the solution pH for higher reactivity, dispersion in the reaction coil (1m tubing length) led to a mixing of the components and the reaction product (blue colored metal complexes) was measured photometrically (λ = 765 nm, slit 10 nm). A calibration curve was produced by analysis of gallic acid (GA) standards (gallic acid monohydrate, purity > 99 %, Sigma-Aldrich, SZBB0130V) at 765 nm. A stock solution was prepared by dissolving 50 mg GA in 100 mL degassed water and diluting with degassed water to provide working standard solutions of 10, 20, 30, 40, 50 and 60 ppm. For comparison with other studies, all results were related to the antioxidant activity of gallic acid and presented as gallic acid equivalent (GAE).

Results and Discussion

Aroma profiles of Sacoglottis gabonensis fruits

Chimpanzees were observed to actively sniff on S. gabonensis fruits prior to eating. Therefore, there have to be volatile cues emitted from the intact fruit that help the apes to judge e.g. its sensory quality and/or ripeness stage. Our analysis was performed on fruit slurry to increase intensities, which possibly takes also into account volatiles that might not be released and perceived from intact fruits (intact exocarp). Further, the protocol used here for sampling and storage of the fruit did only allow analyses of smashed fruits. However, among the detected substances were also typical fruit flavours, which would also be perceived through the intact exocarp.

Analyzing unripe, ripe and overripe fruits 22 volatile organic compounds (VOCs) were identified in the headspace of the smashed fruit flesh and chosen for characterization of the fruit aroma and for comparison of three maturity stages (Table 1). Only the absolute values of the HS-GC/MS headspace signal intensities from the 22 compounds can be presented here since quantification of headspace volatiles is difficult due to unknown partition coefficients of the volatiles between fruit matrix and headspace. Taking this into account, we applied the highest significance level (p<0.001; 99.9 %) for the decision on flavour differences between the three ripeness stages. In terms of hypothesis testing (Ho: there is no difference between the aroma at different ripeness levels) we strongly reduce the risk for Type I error. Regarding odour evaluation, we use descriptions made by humans. To the best of our knowledge, no such information is available for great apes.

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