Spider Monkey Taste Responsiveness to Ten Sweet-Tasting Substances

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Linköping University | Department of Physics, Chemistry and Biology Type of thesis, 60 hp | Educational Program: Physics, Chemistry and Biology Spring term 2020 | LITH-IFM-x-EX— 20/3809--SE

Taste responsiveness of black- handed Spider Monkeys (Ateles geoffroyi) to ten substances tasting sweet to humans

Sofia Pereira

Examiner, Lina Roth Supervisor, Matthias Laska

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Department of Physics, Chemistry and Biology Linköping University

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Titel Title

Taste responsiveness of black-handed Spider Monkeys (Ateles geoffroyi) to ten substances tasting sweet to humans

Författare Author

Sofia Pereira

Sammanfattning Abstract Studies on taste perception in nonhuman primates contribute to the understanding of the evolution of the sense of taste. To assess the responsiveness of four adult spider monkeys (Ateles geoffroyi) to a set of substances perceived as sweet by humans, two-bottle preference tests were performed to determine taste preference thresholds, and taste-induced facial responses were analyzed. The spider monkeys displayed a significant preference for concentrations as low as 0.2-1 mM acesulfame K, 0.002-0.5 mM alitame, 10- 20 mM isomalt, 0.002-0.5 mM sodium saccharin, 2-20 mM galactose and 20-50 mM sorbitol over water. The spider monkeys were generally unable to perceive aspartame and, based on their facial responses, probably do not perceive it as sweet. Thaumatin and monellin were not detected, and most likely neither was the sweetness of sodium cyclamate. Sodium saccharine and sodium cyclamate were rejected at high concentrations by at least one monkey, which is congruent with the perception of a bitter side taste as reported in humans. A significant correlation was found between the ranking order of sweetening potency for the different substances of spider monkeys and humans, but not between spider monkeys and chimpanzees. The results suggest that spider monkeys may be generally more sensitive than chimpanzees and at least as sensitive as humans to the tested substances, supporting the notion that high sensitivity to sweet taste may be associated with a frugivorous dietary specialization. The lack of responsiveness to some of the substances supports the notion of a dichotomy in sweet-taste perception between platyrrhine and catarrhine primates.

Nyckelord Keyword

Ateles geoffroyi, spider monkeys, sweet-tasting substances, taste preference thresholds

Contents

1. Abstract ...... 6 2. Introduction ...... 6 3. Materials and Methods ...... 9 3.1. Determination of taste preference thresholds in spider monkeys 3.1.1. Animals and housing ...... 9 3.1.2. Taste stimuli ...... 9 3.1.3. Experimental procedure ...... 13 3.1.4. Data analysis ...... 14 3.2. Determination of taste detection thresholds in humans 3.2.1. Subjects...... 15 3.2.2. Taste stimuli ...... 15 3.2.3. Experimental procedure ...... 15 3.2.4. Data analysis ...... 15 3.3. Analysis of taste-induced facial responses in spider monkeys 3.3.1. Animals and housing ...... 16 3.3.2. Taste stimuli ...... 16 3.3.3. Experimental procedure ...... 17 3.3.4. Data analysis ...... 19 4. Results ...... 19 4.1. Taste preference thresholds of spider monkeys 4.1.1. Artificial sweeteners ...... 19 4.1.2. Sweet-tasting proteins ...... 21 4.1.3. Sweet-tasting saccharides ...... 22 4.1.4. Interindividual variability ...... 23 4.2. Taste detection thresholds of human subjects 4.2.1. Thresholds for alitame and isomalt ...... 24 4.2.2. Interindividual variability ...... 25 4.3. Taste-induced facial responses in spider monkeys 4.3.1. Inter-rater agreement ...... 25 4.3.2. Group-level analysis ...... 25 5. Discussion...... 31 5.1. Within-species comparison of taste preference thresholds between sweeteners ...... 31

5.2. Comparison of sweetening potency order between spider monkeys, humans and chimpanzees...... 34 5.3. Between-species comparisons of taste preference thresholds ...... 35 5.3.1. Artificial sweeteners ...... 35 5.3.2. Sweet-tasting proteins...... 42 5.3.3. Sweet-tasting saccharides...... 45 5.4. Spider monkeys' perception of aspartame as indicated through facial reactivity ...... 47 5.5. Conclusions ...... 51 6. Societal and ethical considerations ...... 52 7. Acknowledgements ...... 53 8. References ...... 54 Appendix ...... 61

1. Abstract

Studies on taste perception in nonhuman primates contribute to the understanding of the evolution of the sense of taste. To assess the responsiveness of four adult spider monkeys (Ateles geoffroyi) to a set of substances perceived as sweet by humans, two-bottle preference tests were performed to determine taste preference thresholds, and taste-induced facial responses were analyzed. The spider monkeys displayed a significant preference for concentrations as low as 0.2-1 mM acesulfame K, 0.002-0.5 mM alitame, 10-20 mM isomalt, 0.002-0.5 mM sodium saccharin, 2-20 mM galactose and 20-50 mM sorbitol over water. The spider monkeys were generally unable to perceive aspartame and, based on their facial responses, probably do not perceive it as sweet. Thaumatin and monellin were not detected, and most likely neither was the sweetness of sodium cyclamate. Sodium saccharine and sodium cyclamate were rejected at high concentrations by at least one monkey, which is congruent with the perception of a bitter side taste as reported in humans. A significant correlation was found between the ranking order of sweetening potency for the different substances of spider monkeys and humans, but not between spider monkeys and chimpanzees. The results suggest that spider monkeys may be generally more sensitive than chimpanzees and at least as sensitive as humans to the tested substances, supporting the notion that high sensitivity to sweet taste may be associated with a frugivorous dietary specialization. The lack of responsiveness to some of the substances supports the notion of a dichotomy in sweet-taste perception between platyrrhine and catarrhine primates.

Keywords: Ateles geoffroyi, spider monkeys, sweet-tasting substances, taste preference thresholds

2. Introduction

Comparative studies of taste perception are an important tool to better understand the mechanisms underlying the evolution of the sense of taste. Taste allows responses to different taste qualities which supply valuable sensory input for an animal’s decision of what to ingest (Nelson et al. 2001). For example, bitter receptors trigger behavioral responses of aversion towards potentially toxic compounds whereas sweet receptors allow for the recognition of highly caloric food resources (Nelson et al. 2001). However, different species have been found

to vary considerably in their taste perception for the same substances, which is thought to reflect evolutionary adaptations to a specific diet (Breslin 2013).

Primates are a particularly suitable order of mammals for the study of taste perception as they comprise a large variety of dietary specializations and the composition of food is commonly thought to affect the taste perception of a given species (Dominy et al. 2001). Black-handed spider monkeys (Ateles geoffroyi) are highly frugivorous New World primates specialized on consuming ripe fruit and their food selection behavior suggests that they may use the sweetness of fruits as a criterion for consumption (Di Fiore et al 2008; Gonzalez-Zamora et al. 2009). Their responsiveness to sweet-tasting carbohydrates (Laska et al. 1996, 1998, 2001) as well as to sour (Laska et al. 2000, 2003), bitter (Laska et al. 2009), salty, and umami (Laska and Hernandez Salazar 2004; Laska et al. 2008) taste stimuli has been assessed in previous studies.

In addition to the carbohydrates sucrose, fructose, glucose, maltose, and lactose which are commonly found in the food of primates (Nagy and Shaw 1980; Kinghorn and Soejarto 1986), a wide variety of substances from diverse chemical classes are perceived as “sweet” by humans (Marie and Piggott 1991; Merillon and Ramawat 2018). Structurally, these sweet-tasting substances range from simple amino acids over peptides and proteins to terpenoids, flavonoids, steroidal saponins, and dihydrochalcones, to name but a few chemical classes. Considering that mammals have only one type of sweet-taste receptor, the TAS1R2+TAS1R3 heterodimer receptor (Bachmanov and Beauchamp 2007), it is intriguing that one type of receptor is capable of binding such a wide array of structurally diverse ligands, though at different affinities. Recent studies have found that both allelic and copy number variation in the sweet-taste receptor gene may explain the marked differences within and between species that have been reported in the ability to perceive and in the sensitivity for sweeteners (Dias et al. 2015).

In addition to their chemical nature, sweeteners can be subdivided into naturally occurring ones which are usually plant-derived, and artificial ones which are not found in nature. This distinction may be interesting to study with regard to taste perception as the former group of substances may share a long evolutionary history with a given species whereas the latter does not. This, in turn, may have implications for the detectability and acceptance of sweet-tasting substances by an animal. The artificial sweeteners aspartame and neotame, for example, have been reported to be detectable by catarrhine primates, but not by prosimian or platyrrhine primates (Glaser et al. 1992).

Furthermore, sweeteners can be classified according to their sweetening potency. So-called high-potency sweeteners are usually perceived by humans as considerably more intensely sweet compared to an isomolar sucrose solution whereas low-potency sweeteners are accordingly perceived as less intensely sweet than sucrose. Very little so far is known about whether nonhuman primates perceive sweeteners in the same manner as humans with regard to their sweetening potency.

Finally, sweet-tasting substances can also be classified based on their caloric values. Ingestion of non-caloric sweeteners provide, as the name implies, no calories at all whereas so-called sugar substitutes provide a lower amount of calories relative to common carbohydrates.

Taste preference thresholds, as a first and widely used approximation of an animal’s taste sensitivity, have been determined in a variety of primate species from all major taxa (e.g. strepsirrhines: Wielbass et al. 2015; platyrrhines: Laska et al. 1996; catarrhines: Laska et al. 1999; Laska 2000). Such thresholds correspond to the lowest concentration of a given substance an animal prefers over water. However, most studies so far only used prototypical representatives of the five basic taste qualities, e.g. quinine as the prototypical representative of bitter taste. With regard to sweet-tasting substances, most studies only assessed the sensitivity of primates for the five food-associated carbohydrates sucrose, fructose, glucose, maltose, and lactose. Surprisingly little, in contrast, is known so far about the sensitivity of the sense of taste in primates at the behavioral level for sweet-tasting substances other than carbohydrates.

Thus, in order to gain more insight into the sweet-taste perception of nonhuman primates, and into the notion that dietary specialization rather than phylogenetic relatedness may account for between-species differences in sweet-taste perception, the present study aimed at assessing the taste responsiveness of spider monkeys to a diverse set of substances perceived as sweet by humans. These include substances from widely different chemical classes, as well as both high- potency and low-potency sweeteners (as perceived by humans), artificial and naturally occurring sweeteners, as well as caloric and non-caloric sweeteners. Thresholds of different primate and non-primate species were compared for phylogenetic relevance. Furthermore, in an attempt to better understand whether the spider monkeys, similar to human subjects, may perceive an unpleasant side taste with some of the artificial sweeteners, taste-induced facial responses were video-recorded and analyzed.

3. Materials and Methods

3.1. Determination of taste preference thresholds in spider monkeys

3.1.1. Animals and housing

The present study included four adult black-handed spider monkeys (Ateles geoffroyi). The monkeys were housed at the Universidad Veracruzana’s research station UMA Doña Hilda Ávila de O’Farrill near Catemaco, Mexico. The group of subjects was composed of one female, Mari, and three males, Cejitas, Gruñon and Lucas. The female was fifteen years old, and the males were fifteen, twelve and fifteen years old, respectively. The four individuals were not genetically related with each other. All individuals were housed in a series of enclosures exposed to natural light and connected to each other through sliding doors. The monkeys were fed a wide variety of fresh seasonal fruits and vegetables once a day.

3.1.2. Taste stimuli

A set of ten substances tasting sweet to humans was used (Figure 1). A sweetener can be described as a ‘high-potency’ sweetener or as a ‘low-potency’ sweetener according to its sweetening potency relative to sucrose. Here, these designations are employed in accordance with the definition by which a high-potency sweetener has a sweetening potency of at least a factor of 10 higher than the one of sucrose and a low-potency sweetener has a sweetening potency lower than sucrose (Yasuura 2014).

Acesulfame K (CAS# 55589-62-3)

An artificial, high-potency sweetener perceived by humans as about 200 times sweeter than sucrose. It is a non-caloric sweetener containing sulphur and nitrogen, and chemically the potassium salt of an oxathiazine. Reported to have a bitter-side taste at high concentrations.

Alitame (CAS# 99016-42-9)

An artificial, high-potency and non-caloric sweetener perceived by humans as about 2,000 times sweeter than sucrose. Chemically a dipeptide composed of L-aspartic acid and L-alanine.

Aspartame (CAS# 22839-47-0)

An artificial, high-potency and non-caloric sweetener perceived by humans as about 200 times sweeter than sucrose. Chemically a methyl ester of the dipeptide built by the amino acids L- aspartic acid and L-phenylalanine. Reported to have a bitter side taste at high concentrations.

Isomalt (CAS# 64519-82-0)

An artificial low-potency sweetener perceived by humans as about 45% as sweet as sucrose. It is a nutritive sweetener, providing 2.0 kcal/g, half the energy value provided by sucrose. Chemically a sugar alcohol manufactured from sucrose.

Sodium cyclamate (CAS# 139-05-9)

An artificial high-potency sweetener perceived by humans as about 30-50 times sweeter than sucrose. Chemically the sodium salt of cyclamic acid. Reported to have a sour side taste at high concentrations.

Sodium saccharine (CAS# 81-07-2)

An artificial, high-potency and non-caloric sweetener perceived by humans as about 300-400 times sweeter than sucrose. Chemically the sodium salt of benzoic sulfimide and thus a sulphur- and nitrogen-containing compound. Reported to have a bitter side taste at high concentrations.

Monellin (CAS# 9062-83-3)

A naturally occurring high-potency sweetener perceived by humans as about 800-2,000 times sweeter than sucrose. Chemically a protein found in the West African serendipity berry (Dioscoreophyllum cumminsii).

Thaumatin (CAS# 53850-34-3)

A naturally occurring high-potency sweetener perceived by humans as about 2,000 times sweeter than sucrose. Chemically a protein found in the West African katemfe fruit (Thaumatococcus daniellii).

Galactose (CAS# 59-23-4)

A naturally occurring low-potency sweetener perceived by humans as about 30% as sweet as sucrose. Chemically a monosaccharide and a constituent of the disaccharide lactose.

Sorbitol (CAS# 50-70-4)

A naturally occurring low-potency sweetener perceived by humans as about 60% as sweet as sucrose. A nutritive sweetener, providing 2.6 kcal/g. Chemically a sugar alcohol found in certain fruits.

Acesulfame K and aspartame were obtained from Ter Hell & Co., Hamburg, Germany, thaumatin was obtained from Xi’an Sgonek Biological Technology Co. Ltd., Xi’an, China and isomalt was obtained from Beneo-Palatinit, Mannheim, Germany. All other substances were obtained from Sigma-Aldrich, Stockholm, Sweden. All tested substances were of the highest available purity (>99%).

Aspartame Acesulfame K Alitame

Sodium cyclamate Sodium saccharin Isomalt

Monellin Galactose Thaumatin

Figure 1: The sweet-tasting substances tested in the present study. For all substances but Monellin and Thaumatin, a 2-D representation of their molecular structure is presented. Monellin and Thaumatin are described in 3-D ribbon diagrams. (National Centre for Biotechnology Information)

Sorbitol

3.1.3. Experimental procedure

Two-bottle preference tests of short duration (Richer & Campbell 1940) were performed to determine the spider monkeys’ taste preference thresholds to the different sweeteners. The animals were simultaneously presented with two 100 mL graduated plastic cylinders with metal drinking spouts, one containing tap water and the other containing an aqueous solution of the taste stimulus at a defined concentration (Figure 2). The individuals were allowed 1 minute to drink from the bottles. In each trial, the experimenter made sure to observe that the animals had tried both bottles. After each trial, the amount of liquid consumed from each bottle was recorded. The testing occurred in the morning before the monkeys were fed, and up to six trials per monkey were performed per day, with inter-trial intervals of at least 10 minutes in order to ensure that no interference from the previous trial occurred. The data collection began in May 2019 and ended in October 2019.

Figure 2: The two-bottle preference test

With each substance, testing started with a presumably perceptible and thus attractive concentration of a given taste stimulus. The starting concentrations were determined as approximately a factor of 10-100 above the known human taste detection threshold value (van

Gemert 2011). The initial concentration was then decreased in 10-fold steps until the animals failed to show a preference. In case the subjects did not show a preference for the initial concentration, gradually higher concentrations were tested in a 10-fold step fashion until a preference was finally shown or until a concentration range covering three 10-fold dilution steps was tested. In order to determine the monkeys’ taste preference thresholds with higher precision, two intermediate concentrations which were between the highest concentration for which an animal did not show a preference and the lowest concentration for which the animal showed a preference were tested. The taste substances were tested sequentially, meaning that testing with a novel substance only started when an animal had finished the testing with a previous substance.

Each concentration of a given taste substance was tested ten times per animal. In order to reduce any potential bias in testing as well as to keep the animals motivated, different concentrations of a given taste substance were presented in a pseudo-randomized order. The same concentration was never consecutively presented to the animals for more than three trials in a row. Additionally, in order to control for any potential side-bias, the side at which the bottle containing the sweet-tasting substance was presented was also pseudo-randomized. This ensured that the sweet-tasting solution was positioned on the left the same number of times as it was positioned on the right, without ever being presented on the same side more than three consecutive times.

3.1.4. Data analysis

The quantities of sweet-tasting solution and tap water consumed over the ten trials were summed and converted to percentages relative to the total amount of liquid consumed. An individual was considered to have preferred the sweet-tasting substance over water if the percentage of consumed sweet-tasting solution was at least 66.7% of the total liquid consumed. If an individual consumed less than 33.3% of the sweet-tasting solution relative to the total amount of liquid, it was considered to have rejected the sweet-tasting substance. Additionally, a one-tailed binomial test was performed. Thus, in addition to the criterion of having reached 66.7% of consumed sweet-tasting substance, an individual should also have drunk more of the sweet-tasting solution than water in at least 8 out of 10 trials (p < 0.05) with a given concentration of a given taste substance to be considered as significantly preferred over water. The taste preference threshold for each substance was considered as the lowest concentration significantly preferred over water by each individual.

The Spearman rank-order correlation test was performed to evaluate the correlation between the ranking order of sweetening potencies of spider monkeys, chimpanzees and humans.

3.2. Determination of taste detection thresholds in humans

As the sensitivity of humans towards alitame and isomalt had never been tested, I decided to determine the human taste detection thresholds for these two substances in order to obtain a means of comparison for the taste preference thresholds determined for the spider monkeys.

3.2.1. Subjects

Ten human subjects (Homo sapiens) between the ages of 23 and 26 years old were tested. Of the ten individuals included, five were women and five were men.

3.2.2. Taste stimuli

The tested taste stimuli were alitame and isomalt. For a detailed description of these substances please consult the one provided in the previous section (3.1.2. Taste stimuli).

3.2.3. Experimental procedure

An up-down, two-alternative forced choice (2-AFC) staircase procedure was performed to determine human taste detection thresholds (Snyder et al. 2015). According to this method, each subject was asked to indicate which of two presented solutions contained the taste stimulus. The testing started at a clearly detectable concentration (0.05 mM for alitame and 160 mM for isomalt), which was then decreased in 2-fold concentration steps until the subject failed to detect the substance. Each concentration was presented twice to the subject. Two correct choices resulted in a subsequent decrease in concentration in the following set of two trials. An incorrect choice, i.e. failing to correctly identify the solution containing the taste stimulus, was followed by an increase in concentration. Once the testing shifted from a decreasing concentration fashion to an increasing concentration fashion, a reversal was registered. The testing terminated once a subject reached seven reversals, i.e. seven turning points in the direction of the concentration staircase.

All subjects were asked not to eat, chew or smoke in the ten minutes preceding the testing session to ensure that no previous taste lingering in the mouth interfered with the subject’s performance. Tap water was provided for the subjects to rinse their mouth in between each moment of tasting the test solutions.

3.2.4. Data analysis

According to the previously described method, each reversal registered corresponds to a tested concentration. Thus, each reversal step was registered and ordered ascendingly, in order to find the median reversal value. The taste detection threshold of a subject corresponds to the concentration value above the one identified as the median value.

3.3. Analysis of taste-induced facial responses in spider monkeys

3.3.1. Animals and housing

The present experiment included six adult black-handed spider monkeys (Ateles geoffroyi) housed at the Universidad Veracruzana’s research station UMA Doña Hilda Ávila de O’Farrill near Catemaco, Mexico. The monkeys were kept in the same conditions previously described in this report. The group of subjects was composed of three females, Frida, Margarita and Mari, and three males, Cejitas, Gruñon and Lucas. The females were twelve, thirteen and fifteen years old, and the males were fifteen, twelve and fifteen years old, respectively. The group of animals included those who had participated in the two-bottle preference tests.

3.3.2. Taste stimuli

A total of five different taste substances was presented to the spider monkeys. Three of the substances represent different taste qualities. Sucrose was used as a sweet taste stimulus, caffeine was used as a bitter taste stimulus and citric acid was used as a sour taste stimulus. Tap water was used as a neutral stimulus and thus, a control. The fifth stimulus, aspartame, was included as a means to better comprehend the monkey’s perception of the substance and thus the results of the previously conducted two-bottle preference tests. In the previously described two-bottle preference tests, two out of the four individuals presented with aspartame showed a preference for highest tested concentration (20 mM). In contrast, the remaining two individuals rejected the same concentration, which is consistent with the notion of a bitter side- taste of aspartame as perceived by humans (Schiffman et al. 1995). Sucrose, caffeine and citric acid were presented at a concentration of 200 mM, 100 mM, and 500 mM, respectively, in order to provide taste stimuli that are clearly detectable for the animals, but not overwhelmingly sweet, bitter, and sour, respectively. Aspartame was used at the concentration of 100 mM, for being well above the previously tested concentration (20 mM), which two of the spider monkeys preferred over water. Table 1 describes the concentrations used for each of the taste stimuli.

Table 1. Concentrations in mM of the different taste stimuli presented to the spider monkeys.

Stimulus Concentration (mM)

Sucrose 200

Caffeine 100

Citric Acid 500

Aspartame 100

3.3.3. Experimental procedure Each substance was presented to the animals using a glass dropper contained in a dark-glass container (Figure 3). Prior to any critical tests, the animals were gradually made familiar with this device through several trials using sucrose as an attractive and thus motivational stimulus. Once the animals were completely familiarized with the device, each substance was presented ten times to each individual. A trial was considered each time a substance was presented to an individual who clearly tasted and/or ingested the taste stimulus. The tasting or ingestion of the taste stimulus was recorded with a video camera (Sony Handycam HDR-CX405) until the dropper (2.5 ml) was emptied or until the animal refused to take in any more substance. In order to prevent the animals from associating any visual cues to the different taste substances, all droppers and containers were of the same model and displayed no visible differences between them. All taste solutions were colorless, ensuring that the animals were not able to pick up on any discriminatory indications between substances. Three to six trials were performed per day with the same substance, depending on the experimenter’s schedule and the animals’ motivation to cooperate The taste substances were tested sequentially, meaning that testing with a novel substance only started when an animal had finished the testing with a previous substance. However, trials with presumably aversive taste stimulus (such as caffeine and citric acid) were sometimes interspersed with sucrose trials to maintain the animals motivated to cooperate. In any case, inter-trial intervals of at least 10 minutes were implemented.

Figure 3: Transparent-glass dropper and respective dark-glass container used to present the different taste stimuli to the spider monkeys.

Once all trials were completed, two coders experienced in working with spider monkeys were asked to analyze the frequency of a list of previously selected facial expressions and facial motor patterns considered relevant for the study. Both coders were unaware of which substance was being presented to the animal in each video. The ethogram compiling the list of behaviors recorded in each trial and its respective description is displayed in the Appendix section of this report. The selected facial expressions had been previously found to be indicative of the pleasantness or unpleasantness of taste stimuli as perceived by several non-human primate species including great apes, Old World monkeys, New World monkeys and prosimians (Steiner et al. 2001). Even though only behavioral frequency was analyzed, the behaviors were separated into two categories. Behaviors which could be analyzed by both frequency and duration were categorized as state behaviors. Behaviors which could only be analyzed by frequency were categorized as behavioral events.

3.3.4. Data analysis

As a preliminary validation of the data and in order to verify a sufficient level of agreement between the two coders’ independent analysis of the video footage, an Intraclass Correlation Analysis (ICC) was run. In order for the data to be considered reliable and included in the study, a correlation coefficient (α) of at least 0.7 was necessary.

The data points which achieved sufficient inter-rater agreement were further analyzed for significant differences in facial responses between the different tested substances. This was done by performing the Related-Samples Wilcoxon Signed Rank test for all pairwise comparisons between the different stimuli for each of the selected behaviors at the group level, pulling all individuals together.

4. Results

4.1. Taste preference thresholds of spider monkeys

4.1.1. Artificial sweeteners

For acesulfame K the four spider monkeys displayed taste preference thresholds between 0.2 mM and 1 mM. The taste preference threshold was 0.2 mM for Lucas, 0.5 mM for Cejitas and Mari, and 1mM for Gruñon (p<0.05, binomial test) (Figure 4). The taste preference thresholds for alitame ranged from 0.002 mM to 0.005 mM. Lucas and Cejitas displayed a taste preference threshold of 0.002 mM and Mari and Gruñon displayed a taste preference threshold of 0.005 mM (p<0.05, binomial test) (Figure 4). The taste preference threshold for aspartame was 20 mM for Lucas and Mari (p<0.05, binomial test) (Figure 4). Cejitas and Gruñon did not show a preference for this substance at any of the concentrations tested. Rather, Cejitas rejected the concentrations 20, 2 and 0,02 mM, and Gruñon rejected the concentrations 20, 2 and 0,2 mM. For isomalt, the taste preference threshold was 10 mM for Mari and 20 mM for Cejitas, Gruñon and Lucas (p<0.05, binomial test) (Figure 4). The taste preference threshold for sodium cyclamate was 1 mM for Cejitas (p<0.05, binomial test) (Figure 4). None of the three remaining individuals displayed a preference for this substance at any of the concentrations tested. Rather, Mari and Lucas rejected the substance at the concentrations 10 and 1 mM, and Gruñon rejected the substance at the concentration 10 mM. For sodium saccharine, the taste preference thresholds ranged from 0.002 mM to 0.5 mM. The taste preference thresholds for

this substance were 0.002 mM for Lucas, 0.02 mM for Cejitas, 0.2 mM for Mari and 0.5 mM for Gruñon (p<0.05, binomial test) (Figure 4).

Acesulfame K Alitame

100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20

10 (%) for Preference alitame 10 Preference for (%) K for Preference acesulfame 0 0 0.1 1 10 0.001 0.01 0.1 1 10 Concentration (mM) Concentration (mM)

Aspartame Isomalt

100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30

20 20 Preference for (%) for Preference isomalt

Preference for (%) for Preference aspartame 10 10 0 0 0.02 0.2 2 20 5 50 500 Concentration (mM) Concentration (mM)

Sodium cyclamate Saccharin 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20

10 (%) for Preference saccharin 10

0 0 Preference for (%) for cyclamate Preference sodium 0.1 1 10 0.001 0.01 0.1 1 10 Concentration (mM) Concentration (mM)

Figure 4: The taste responsiveness of the four spider monkeys when presented simultaneously with water and the artificial sweeteners acesulfame K, alitame, aspartame, isomalt, sodium cyclamate and sodium saccharin. The blue triangles, orange squares, yellow rhombi and green circles represent data points for Mari, Lucas, Cejitas and Gruñon, respectively. The horizontal dashed lines at 33.3%, 50% and 66.7% represent the criterion of the rejection, the level of chance and the criterion of preference, respectively.

4.1.2. Sweet-tasting proteins

For monellin, the taste preference threshold was 0.001 mM for Mari (p<0.05, binomial test) (Figure 5). None of the other individuals showed a preference for this substance. Cejitas and Gruñon displayed a rejection for the substance at the concentrations 0.01, 0.001 and 0.0001 mM, and Lucas displayed a rejection at 0.01 and 0.001 mM. For thaumatin, only Mari displayed a preference, with a taste preference threshold of 0.1 mM (p<0.05, binomial test) (Figure 5). Cejitas rejected the concentrations 0.1 and 0.01 mM, and Gruñon rejected 0.1, 0.01, 0.001 and 0.0001 mM. Lucas did not display a preference for thaumatin nor rejected the substance.

Monellin Thaumatin 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30

20 20 Preference for monellin for (%) Preference monellin 10 (%) for Preference thaumatin 10 0 0 0.00001 0.0001 0.001 0.01 0.0001 0.001 0.01 0.1 Concentration (mM) Concentration (mM)

Figure 5: The taste preference thresholds of the four spider monkeys when presented simultaneously with water and the sweet-tasting proteins monellin and thaumatin. The blue triangles, orange squares, yellow rhombi and green circles represent data points for Mari, Lucas, Cejitas and Gruñon, respectively. The horizontal dashed lines at 33.3%, 50% and 66.7% represent the criterion of the rejection, the level of chance and the criterion of preference, respectively.

4.1.3. Sweet-tasting saccharides

The taste preference thresholds for galactose ranged between 2 mM and 20 mM in the group of spider monkeys. The taste preference threshold was 2 mM for Cejitas and 20 mM for Gruñon, Lucas and Mari (p<0.05, binomial test) (Figure 6). For sorbitol, the taste preference thresholds were 20 mM for Cejitas, Lucas and Mari, and 50 mM for Gruñon (p<0.05, binomial test) (Figure 6).

Galactose Sorbitol 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30

20 20 Preference for sorbitol (%) for sorbitol Preference Preference for (%) for Preference galactose 10 10 0 0 1 10 100 10 100 Concentration (mM) Concentration (mM)

Figure 6: The taste preference thresholds of the four spider monkeys when presented simultaneously with water and the saccharides galactose and sorbitol. The blue triangles, orange squares, yellow rhombi and green circles represent data points for Mari, Lucas, Cejitas and Gruñon, respectively. The horizontal dashed lines at 33.3%, 50% and 66.7% represent the criterion of the rejection, the level of chance and the criterion of preference, respectively.

4.1.4. Interindividual variability

With four of the ten taste substances, inter-individual variability in taste thresholds was low. The difference between the most- and least sensitive animal ranged from a dilution factor of only 2 with isomalt, a dilution factor of 2.5 with sorbitol and alitame, to a dilution factor of 5 with acesulfame K. For galactose, the difference between the lowest and highest taste preference thresholds determined was a dilution factor of 10, with only one monkey differing from the others. The highest interindividual variability was observed with sodium saccharin for which each monkey displayed a different taste preference threshold. For the latter substance the difference between the most- and least-sensitive animal was a dilution factor of 250. For aspartame, the only two individuals who showed a preference for the substance, Mari and Lucas, displayed the same taste preference threshold. For monellin and thaumatin, only Mari showed a preference for the substances. For sodium cyclamate, only Cejitas preferred the substance over water. In general, Lucas showed a higher sensitivity than the other monkeys, as he displayed the lowest taste preference thresholds with three out of the six substances that all animals preferred over water.

4.2. Taste detection thresholds of human subjects

4.2.1. Thresholds for alitame and isomalt

For alitame, the human subjects displayed thresholds ranging between 0.0015625 mM and 0.0125 mM (Figure 7). Only one of the subjects displayed a taste detection threshold of 0.0015625 mM. Five subjects displayed a taste detection threshold of 0.00625 mM and for the remaining four subjects the threshold values were of 0.0125 mM. Accordingly, the median value for the human taste detection threshold for alitame was 0.00625 mM.

For isomalt, the human subjects displayed thresholds ranging between 5 mM and 40 mM (Figure 7). Only one of the subjects displayed a taste detection threshold of 5 mM. Similarly, only one of the subjects showed a taste detection threshold of 10 mM. Six subjects displayed a taste detection threshold of 20 mM and for the remaining two individuals displayed a threshold of 40 mM. Accordingly, the median value for the human taste detection threshold for alitame was 20 mM.

Alitame Isomalt 0.014 45 0.012 40 35 0.010 30 0.008 25 0.006 20 15 0.004 10

0.002 5 Taste detection threshold (mM) threshold Tastedetection 0.000 (mM) threshold Tastedetection 0 Subjects Subjects

Figure 7: Taste detection thresholds of the ten human subjects when presented simultaneously with water and the artificial sweeteners alitame and isomalt. Each dot represents a human subject. The horizontal lines represent the median value.

4.2.2. Interindividual variability

For both alitame and isomalt, there was considerable variation in sensitivity across individuals. The difference between the most- and least sensitive subject for both substances was a dilution factor of 8.

4.3. Taste-induced facial responses in spider monkeys

4.3.1. Inter-rater agreement Inter-rater agreement was sufficient (α ≥ 0.7, ICC) for all tested substances with four of the six individuals included in the experiment. These individuals were Frida, Margarita, Gruñon and Lucas, of which only Gruñon and Lucas had also previously participated in the two-bottle preference tests. The data relative to the remaining two individuals, Cejitas and Mari, achieved a sufficient level of inter-rater agreement for only three and four of the tested substances, respectively, and were thus removed from the following analysis.

4.3.2. Group-level analysis For licking, significant differences were found for all pairwise comparisons between the different stimuli (Aspartame VS Caffeine: Z = -4.147, p < 0.001; Aspartame VS Citric Acid: Z = -4.288, p < 0.001; Aspartame VS Water: Z = 3.137, p < 0.01; Aspartame VS Sucrose: Z = 5.321, p < 0.001; Sucrose VS Water: Z = -4.722, p < 0.001 ; Sucrose VS Citric Acid: Z = - 5.523, p < 0.001; Sucrose VS Caffeine: Z = -5.437, p < 0.001; Citric Acid VS Caffeine: Z = - 2.161, p < 0.05; Citric Acid VS Water: Z = -5.016, p < 0.001; Caffeine VS Water: Z = -4.798, p < 0.001; Wilcoxon). Licking occurred more often during sucrose, followed by control and aspartame trials, and only then by caffeine and citric acid trials (Figure 8a).

Sucking frequency was significantly higher during sucrose trials than during caffeine trials (Z = -2.636, p < 0.01, Wilcoxon). No other pairwise comparison between any other of the different tested substances revealed a significant difference (Aspartame VS Caffeine: Z = -1.342, p > 0.05; Aspartame VS Citric Acid: Z = -0.535, p > 0.05; Aspartame VS Water: Z = -0.378, p > 0.05; Aspartame VS Sucrose: Z = 1.345, p > 0.05; Sucrose VS Water: Z = -1.31, p > 0.05; Sucrose VS Citric Acid: Z = -1.653, p > 0.05; Citric Acid VS Caffeine: Z = 1, p > 0.05; Citric Acid VS Water: Z =0.816, p > 0.05; Caffeine VS Water: Z = 1.633, p > 0.05; Wilcoxon).

Regarding sniffing frequency, all substances differed significantly from each other (Aspartame VS Water: Z = 3.592, p < 0.001; Aspartame VS Sucrose: Z = -3.491, p < 0.001; Sucrose VS Water: Z = 4.704, p < 0.001 ; Sucrose VS Caffeine: Z = 3.8, p < 0.001; Citric Acid VS Caffeine:

Z = -2.078, p < 0.05; Citric Acid VS Water: Z = -3.796, p < 0.001; Caffeine VS Water: Z = 3.292, p < 0.01; Wilcoxon) with the exception of the pairwise comparisons between sucrose and citric acid (Z = 1.811, p > 0.05, Wilcoxon), and aspartame and caffeine (Z = 0.068, p > 0.05, Wilcoxon) (Figure 8b). A trend rather than a significant difference was found for the comparison between aspartame and citric acid (Z = -1.908, p = 0.056, Wilcoxon).

With regard to the state of the eyes, the frequency of eyes open more than 50% differed only in the pairwise comparisons between aspartame and citric acid (Z = -2.214, p < 0.05, Wilcoxon), caffeine and water (Z = 2.674 , p < 0.01, Wilcoxon), and citric acid and water (Z = -3.037, p < 0.01, Wilcoxon). A trend rather than a significant difference was found for the comparison between aspartame and caffeine (Z = -1.768, p = 0.077, Wilcoxon). No significant differences were found for the pairwise comparisons between aspartame and water (Z = 0.939, p > 0.05, Wilcoxon), aspartame and sucrose (Z = -0.228, p > 0.05, Wilcoxon), sucrose and caffeine (Z = -1.12, p > 0.05, Wilcoxon), sucrose and citric acid (Z = -1.238, p > 0.05, Wilcoxon), sucrose and water (Z = 0.986, p > 0.05, Wilcoxon), and caffeine and citric acid (Z = -0.312, p > 0.05, Wilcoxon). Higher frequencies of this behavior were recorded for water, and the lower for caffeine and citric acid.

Frequencies of eyes open less than 50% differed significantly between all substances (Aspartame VS Caffeine: Z = -2.8, p < 0.01; Aspartame VS Citric Acid: Z = -3.438, p < 0.01; Aspartame VS Water: Z = 2.657, p < 0.01; Aspartame VS Sucrose: Z = 3.429, p < 0.01; Sucrose VS Citric Acid: Z = -4.596, p < 0.001; Sucrose VS Caffeine: Z = -4.64, p < 0.001; Citric Acid VS Water: Z = -4.878, p < 0.001; Caffeine VS Water: Z = 4.574, p < 0.001; Wilcoxon) with the exception of the pairwise combinations between caffeine and citric acid (Z = -0.869, p > 0.05, Wilcoxon) and tap water and sucrose (Z = -1.182, p > 0.05, Wilcoxon) (Figure 8c). In general, eyes were open less than 50% the most during sucrose and control ingestions.

The frequency of closed eyes differed significantly between aspartame and caffeine (Z = - 2.419, p < 0.05, Wilcoxon), aspartame and citric acid (Z = -2.247, p < 0.05, Wilcoxon), aspartame and sucrose (Z = 2.965, p < 0.01, Wilcoxon), sucrose and caffeine (Z = -3.767, p < 0.001, Wilcoxon), sucrose and citric acid (Z = -3.864, p < 0.001, Wilcoxon), and sucrose and tap water (Z = -3.05, p < 0.01, Wilcoxon) (Figure 8d). No other significant differences were found (Aspartame VS Water: Z = -0.789, p > 0.05; Caffeine VS Water: Z = 1.375, p > 0.05; Citric Acid VS Water: Z = -1.368, p > 0.05; Citric Acid VS Caffeine: Z = -0.237, p > 0.05;

Wilcoxon). In general, eyes were closed the most during sucrose ingestions, followed by aspartame ingestions. Eyes were closed the least during citric acid ingestions.

The frequency of flat tongue protrusions differed significantly between all substances (Aspartame VS Caffeine: Z = -3.520, p < 0.001; Aspartame VS Citric Acid: Z = -3.468, p < 0.001; Aspartame VS Water: Z = 3.378, p < 0.01; Aspartame VS Sucrose: Z = 5.238, p < 0.001; Sucrose VS Water: Z = -4.323, p < 0.001 ; Sucrose VS Citric Acid: Z = -5.509, p < 0.001; Sucrose VS Caffeine: Z = -5.407, p < 0.001; Citric Acid VS Water: Z = -4.873, p < 0.001; Caffeine VS Water: Z = 4.641, p < 0.001; Wilcoxon) with the exception of the pairwise comparison between caffeine and citric acid, for which a trend rather than a significant difference was found (Z = -1.853, p = 0.064, Wilcoxon) (Figure 8e). In general, flat tongue protruding occurred the most during sucrose ingestions, followed by control and then aspartame ingestions. Flat tongue protruding was less frequent during caffeine and citric acid ingestions.

No significant differences were found between substances regarding the frequency of downward-directed tongue protrusions (Aspartame VS Caffeine: Z = -0.977, p > 0.05; Aspartame VS Citric Acid: Z = -0.447, p > 0.05; Aspartame VS Water: Z = -1.319, p > 0.05; Aspartame VS Sucrose: Z = -0.978, p > 0.05; Sucrose VS Water: Z = -0.542, p > 0.05; Sucrose VS Citric Acid: Z = 0.832, p > 0.05; Sucrose VS Caffeine: Z = 0.306, p > 0.05; Citric Acid VS Caffeine: Z = 0.884, p > 0.05; Citric Acid VS Water: Z = 1.327, p > 0.05; Caffeine VS Water: Z = -0.952, p > 0.05, Wilcoxon) nor upward-directed tongue protrusions (Aspartame VS Caffeine: Z = -1, p > 0.05; Aspartame VS Citric Acid: Z = -1, p > 0.05; Aspartame VS Water: Z = 0.816, p > 0.05; Aspartame VS Sucrose: Z = 1.604, p > 0.05; Sucrose VS Water: Z = - 0,816, p > 0.05; Sucrose VS Citric Acid: Z = -1.604, p > 0.05; Sucrose VS Caffeine: Z = - 1.604, p > 0.05; Citric Acid VS Caffeine: Z = -1, p > 0.05; Citric Acid VS Water: Z = -1.342, p > 0.05; Caffeine VS Water: Z = -1.342, p > 0.05, Wilcoxon).

The frequency of repetitive tongue protruding differed significantly between caffeine and citric acid (Z = 2.309 , p < 0.05, Wilcoxon), aspartame and tap water (Z = -2, p < 0.05, Wilcoxon), sucrose and tap water (Z = -2, p < 0.05, Wilcoxon), and tap water and citric acid (Z = 2.714, p < 0.01, Wilcoxon). No other significant differences were found (Aspartame VS Caffeine: Z = -1.342, p > 0.05; Aspartame VS Citric Acid: Z = 1.890, p > 0.05; Aspartame VS Sucrose: Z < 0.001, p > 0.05; Sucrose VS Citric Acid: Z = 1.291, p > 0.05; Sucrose VS Caffeine: Z = -1.342, p > 0.05; Caffeine VS Water: Z = -1, p > 0.05, Wilcoxon).

The frequency of tongue protrusion gape differed significantly between all tested substances (Aspartame VS Caffeine: Z = -4.063, p < 0.001; Aspartame VS Citric Acid: Z = -4.138, p < 0.001; Aspartame VS Water: Z = 3.226, p < 0.01; Aspartame VS Sucrose: Z = 5.195, p < 0.001; Sucrose VS Water: Z = -4.442, p < 0.001 ; Sucrose VS Citric Acid: Z = -5.520, p < 0.001; Sucrose VS Caffeine: Z = -5.489, p < 0.001; Citric Acid VS Caffeine: Z = -2.154, p < 0.05; Citric Acid VS Water: Z = -5.073, p < 0.001; Caffeine VS Water: Z = 4.782, p < 0.001; Wilcoxon) (Figure 8f). In general, tongue protrusion gapes occurred the most during sucrose ingestions, followed by control and then aspartame ingestions. Tongue protrusion gapes occurred less often during caffeine and citric acid ingestions.

Regarding mouth gapes, only sucrose and caffeine differed significantly in the frequency of this behavior (Z = -2.271, p < 0.05, Wilcoxon). No other significant differences were found in the frequency of mouth gapes across substances (Aspartame VS Caffeine: Z = -1.134, p > 0.05; Aspartame VS Citric Acid: Z = -0.175, p > 0.05; Aspartame VS Water: Z = 0.333, p > 0.05; Aspartame VS Sucrose: Z = 1.095, p > 0.05; Sucrose VS Water: Z = -1.027, p > 0.05; Sucrose VS Citric Acid: Z = -1.098, p > 0.05; Citric Acid VS Caffeine: Z = 0.707, p > 0.05; Citric Acid VS Water: Z = -0.272, p > 0.05; Caffeine VS Water: Z = 1.190, p > 0.05, Wilcoxon).

The frequency of lip stretching differed significantly between all substances (Aspartame VS Caffeine: Z = 3.195, p < 0.01; Aspartame VS Sucrose: Z = -2.484, p < 0.05; Sucrose VS Citric Acid: Z = 2.673, p < 0.01; Sucrose VS Caffeine: Z = 4.135, p < 0.001; Citric Acid VS Caffeine: Z = -3.624, p < 0.001; Caffeine VS Water: Z = -3.996, p < 0.001; Wilcoxon) with the exceptions of the pairwise comparisons between aspartame and citric acid (Z = -0.034, p > 0.05, Wilcoxon). Trends rather than significant differences were found for the pairwise comparisons between aspartame and tap water (Z = -1.807, p = 0.071, Wilcoxon), citric acid and tap water (Z = 1.941, p = 0.052, Wilcoxon), and sucrose and tap water (Z = 1.732, p = 0.083, Wilcoxon) (Figure 8g). In general, lip stretching occurred the most during caffeine ingestions, followed by citric acid ingestions.

The frequency of lip smacking differed significantly between aspartame and caffeine (Z = - 2.085, p < 0.05, Wilcoxon), aspartame and sucrose (Z = 1.968, p < 0.05, Wilcoxon), sucrose and caffeine (Z = -2.612, p < 0.01, Wilcoxon), and sucrose and citric acid (Z = -2.615, p < 0.01, Wilcoxon) (Figure 8h). Trends rather than significant differences were found for the pairwise comparisons between aspartame and citric acid (Z = -1.868, p = 0.062, Wilcoxon), caffeine and tap water (Z =1.899, p = 0.058, Wilcoxon), and citric acid and tap water (Z = -1.657, p =

0.098, Wilcoxon). No significant differences were found for the pairwise comparisons between aspartame and water (Z = 0.422, p > 0.05, Wilcoxon), caffeine and citric acid (Z = 0.846, p > 0.05, Wilcoxon), and sucrose and water (Z = -1.186, p > 0.05, Wilcoxon). In general, lip smacking occurred most often during sucrose trials, followed by control trials and then aspartame trials. Lip smacking occurred least often during citric acid trials, followed by caffeine trials.

No significant difference was found between the frequencies of nose wrinkles across substances (Aspartame VS Caffeine: Z = -1, p > 0.05; Aspartame VS Citric Acid: Z < 0.001, p > 0.05; Aspartame VS Water: Z = 0.816, p > 0.05; Aspartame VS Sucrose: Z = -1, p > 0.05; Sucrose VS Water: Z = 1.342, p > 0.05; Sucrose VS Citric Acid: Z = 1, p > 0.05; Sucrose VS Caffeine: Z = -1, p > 0.05; Citric Acid VS Caffeine: Z = 1, p > 0.05; Citric Acid VS Water: Z = -0.816, p > 0.05; Caffeine VS Water: Z = 1.342, p > 0.05, Wilcoxon).

Regarding withdrawals from the dropper, frequencies differed significantly between all tested substances (Aspartame VS Citric Acid: Z = 2.092, p < 0.05; Aspartame VS Sucrose: Z = -3.804, p < 0.001; Sucrose VS Water: Z = 3.140, p < 0.01; Sucrose VS Citric Acid: Z = 4.964, p < 0.001; Sucrose VS Caffeine: Z = 4.696, p < 0.001; Citric Acid VS Water: Z = 3.634, p < 0.001; Caffeine VS Water: Z = -2.938, p < 0.01; Wilcoxon), with the exception of the pairwise comparisons between caffeine and citric (Z = 0.863, p > 0.05, Wilcoxon) and aspartame and tap water (Z = -1.370, p > 0.05, Wilcoxon). The pairwise comparison between aspartame and caffeine revealed a trend rather than a significant difference (Z = 1.900, p = 0.057, Wilcoxon). In general, monkeys withdrew from dropper most often during citric acid, caffeine and aspartame trials, and withdrew the least during sucrose and control trials.

a) b)

* *

c) d) * * * *

* *

e) f) * * *

g) h) * *

* * *

Figure 8: Behavioral frequencies recorded for the group of four spider monkeys for a) licking, b) sniffing, c) eyes open less than 50%, d) eyes closed, e) flat tongue protrusions, f) tongue protrusion gapes, g) lip stretching and h) lip smacking when presented with different taste stimuli. The numbers 1 through 5 correspond respectively to 1 – aspartame, 2 – caffeine, 3 – citric acid, 4 – tap water and 5 – sucrose. The brackets marked with an asterisk (*) represent significant differences in behavioral frequency between substances (p<0.05). The boxes represent the distribution of behavioral frequencies, with the median value represented by the horizontal lines in bold. The bottom and top whiskers of each box represent, respectively, the lowest and highest recorded frequency for the behavior. The circles (๐) represent the suspected outliers and the asterisks which are not on top of brackets represent the outliers.

5. Discussion

The spider monkeys’ responsiveness to the different taste substances varied considerably. Only six of the ten substances tasting sweet to humans were preferred over water by all individuals. With four of the ten substances only one or two of the animals were able to detect their sweetness and at least two of the monkeys rejected them.

5.1. Within-species comparison of taste preference thresholds between sweeteners

The spider monkeys’ taste preference thresholds are displayed in Figure 9, alongside with those of chimpanzees and humans (see section 5.2. Comparison of ranking of sweetening potency order between spider monkeys, chimpanzees and humans). The lowest taste preference threshold, and thus the highest taste sensitivity, was recorded for the sweet-tasting protein monellin. However, this value was recorded for only one of the four monkeys. The second and third lowest thresholds correspond to alitame and sodium saccharine, respectively, revealing that the monkeys are more sensitive to these substances than to the remainder of the sweet- tasting substances tested in the present study. The fourth lowest taste preference threshold was the one recorded for thaumatin, also displayed by only one of the four monkeys. Following thaumatin, acesulfame K had the fifth lowest taste preference threshold recorded. As alitame, sodium saccharine and acesulfame K were perceived by all spider monkeys and as the animals displayed taste preference thresholds at least one factor of 10 lower than that of sucrose, these sweet-tasting substances can therefore be described as high-potency sweeteners in this species.

The spider monkeys displayed the highest taste preference threshold, and thus the lowest sensitivity, for sorbitol, showing that they are less sensitive to this substance than to the other sweet-tasting substances tested in the current study. Aspartame and isomalt, in order of mention, had the highest taste preference thresholds following sorbitol. As the taste preference thresholds for these three substances are higher than that of sucrose, they can be described as low-potency sweeteners for the spider monkeys.

In the context of discussing the obtained taste preference thresholds of the spider monkeys, it is important to mention any methodological limitations that may have affected the results. In particular, it is relevant to ponder upon the Clever-Hans effect, since the experimenter presenting the bottles to the animals was visible to them during all trials of the two-bottle preference tests. This raises the possibility that the animals may have picked up on any unintended cues that the experimenter may have unconsciously given and thus potentially biasing the animals’ choice of which bottle to preferentially drink from. Although it is unlikely that any potential cues could have been so systematically transmitted from experimenter to monkey to the extent of biasing the obtained results (which require tens of trials for each one of the tested substances), it is not possible to completely rule out this possibility. Nonetheless, the fact that I underwent a training period during which my supervisors observed my posture and behavior when testing the animals and corrected any potential cues, increases my confidence in my results. Additionally, several trials were performed not by me (the main experimenter) but by other experimenters, with no noticeable differences in the animals’ consumptive behavior, further supporting the results.

Figure 9: Taste preference thresholds of spider monkeys ( ), chimpanzees ( ), and humans ( ) to sorbitol, aspartame, isomalt, galactose, cyclamate, acesulfame K, thaumatin, saccharin, alitame and monellin. For spider monkeys ( ), the substances appear in ascending order of sensitivity (corresponding to a descending order of taste preference threshold values). The brackets mark the substances for which only one or two of the monkeys displayed a preference.

5.2. Comparison of sweetening potency order between spider monkeys, chimpanzees and humans

Figure 9 (section 5.1. Within-species comparison of taste preference thresholds between sweeteners) displays the available taste thresholds for the tested taste substances in the present study for chimpanzees and humans. Similar to the findings reported here for spider monkeys, alitame, sodium saccharin and acesulfame K also qualify as high-potency sweeteners in chimpanzees (Henderson 2019). However, in contrast to spider monkeys, aspartame is also described as a high-potency sweetener in chimpanzees (Henderson 2019). For both spider monkeys and chimpanzees, sorbitol is described as a low-potency sweetener. Despite the similarities between the ranking order of sweetening potencies of spider monkeys and chimpanzees, no significant correlation was found when comparing the two (rs = 0.617, p > 0.05). Note that isomalt and sodium cyclamate were not included in the statistical analysis for this comparison, as there are no available taste preference threshold values for these substances in chimpanzees.

Of the substances described as high-potency sweeteners for spider monkeys, only sodium saccharin holds the same description for humans (van Gemert 2011). Similar to spider monkeys, sorbitol is described as a low-potency sweetener in humans (van Gemert 2011). A comparison between the ranking order of sweetening potency of spider monkeys and humans (see Figure 9) showed a significant correlation between the order of ranking for the two species

(rs = 0.782, p < 0.01). Please note that the higher ranking of sucrose for humans is explained by the difference between thresholds reported in different studies, probably reflecting differences in method and threshold criterion.

For both comparisons it is relevant to remark that for two of the higher-ranked substances in terms of sweetening potency for spider monkeys, monellin and thaumatin, a threshold was only determined for one of the individuals. This may limit our ability to extend the conclusions taken from these results to the population level.

The same comparison of ranking order of sweetening potencies for a similar set of substances to the one used in the present study between chimpanzees and humans was reported by Henderson (2019), showing a higher degree of similarity between the rankings of these two catarrhine primate species than between spider monkeys, which are platyrrhine primates, and chimpanzees and humans, respectively. Given the taxonomic position of these three species within the order of primates, these comparisons suggest that phylogenetic relatedness may

affect the degree of similarity or dissimilarity in the relative order of sensitivity for sweet- tasting substances.

5.3. Between-species comparisons of taste preference thresholds

5.3.1. Artificial sweeteners

Table 2 shows a compilation of taste threshold values for the artificial sweeteners acesulfame K, alitame, aspartame, isomalt, sodium cyclamate and sodium saccharin in different species. The spider monkeys’ taste preference threshold for acesulfame K ranges between 0.2 and 1 mM, which is approximately 3 to 25 times higher than that of humans (0.04-0.07 mM) (van Gemert 2011). However, it should be noted that the procedures used to determine taste thresholds in humans allow for the determination of taste detection thresholds rather than taste preference thresholds. Thus, the taste thresholds for humans represent the lowest concentration of a given substance that human subjects are able to detect but not necessarily prefer over water. As it is possible that animals detect lower concentrations of a given taste substance than the lowest one preferred over water (Spector 2003), it is then plausible to assume that spider monkeys may be able to detect acesulfame k concentrations as low as humans do.

For chimpanzees, the reported taste preference threshold for acesulfame K is 0.5-2 mM (Henderson 2019), which overlaps with that of spider monkeys. To the extent of the information I gathered, there is no reported threshold for acesulfame K for any other catarrhine primate species.

Within the Platyrrhini, in addition to spider monkeys, the common marmoset (Callithrix jacchus) was tested for its responsiveness to acesulfame K. However, only a single concentration (13.8 mM) was tested which was preferred over water (Danilova & Hellekant 2004). Further tests would be needed to clarify if this species prefers acesulfame K at concentrations as low as the ones here reported for spider monkeys.

Within the Strepsirrhini, only the grey mouse lemur (Microcebus murinus) has been included in gustatory responsiveness tests with acesulfame K. However, only a single concentration was tested (7.3 mM), which did not elicit a preference for the substance (Schilling et al. 2004). Electrophysiological studies reported responses in nerve fibres triggered by millimolar concentrations of acesulfame K in the grey mouse lemur (Hellekant et al. 1993). However, it is not certain that such responses signify that the animals would prefer such concentrations

over water. A hypothetical generalized lack of responsiveness to acesulfame K in the grey mouse lemur could potentially be explained by its highly plastic generalist diet, in the sense that no specialization towards sweet-tasting food items such as fruits is displayed by this species (Dammhahn & Kappeler 2008).

Non-primate species whose taste responsiveness to acesulfame K have been tested include cows (Bos taurus) and mice (Mus musculus) with reported taste preference threshold ranges of 1-2 mM and 0.3-10 mM, respectively (Hellekant et al. 1994, Bachmanov et al. 2001). Hamsters (Mesocricetus auratus) were reported to prefer a single concentration (4 mM) over water, but no threshold was determined (Danilova et al. 1998). Pigs (Sus scrofa domesticus) displayed a taste preference threshold for acesulfame K of 0.24-1.73 mM (Glaser et al. 2000), which is similar to the one determined for spider monkeys (0.2-1 mM). Thus, the spider monkeys’ sensitivity to acesulfame K appears to be similar to that of non-primate species. This finding is difficult to interpret given the distinct diets and relatively distant taxonomic classifications of the above-mentioned species.

Table 2. Taste preference thresholds (in mM) for the artificial sweeteners acesulfame K, alitame, aspartame, isomalt, sodium cyclamate and sodium saccharin in primates and non- primate mammals.

Species Acesulfame Alitame Aspartame Isomalt Sodium Sodium Ref. K cyclamate saccharin

Hominoid primates Pan troglodytes 0.5 – 2 0.5 – 2.5 0.5 0.2 – 2 [1] verus Homo sapiens 0.04 – 0.07 0.0015625 0.02 – 0.2 5 – 40 0.28 0.00003 – 0.03 [2,3] – 0.0125 Catarrhine primates Cercocebus 0.6 [4] atys atys Cercopithecus 0.5 [4] nictitans Macaca fuscata 0.3 [5] Macaca mulatta 0.5 [4] Platyrrhine primates Ateles geoffroyi 0.2 -1 0.002 – 20 10 – 20 1 0.002 – 0.5 [3] 0.005 Callithrix 13.8* 0.3* No pref. No pref. No pref. [6] jacchus Strepsirrhine primates

Eulemur No pref. [7] mongoz Microcebus No pref. No pref. No pref. No pref. No pref. [8] murinus Varecia No pref. [9] variegate Non-primates Bos Taurus 1 – 2 No pref. 4 [10]

Mesocricetus 4* No pref. No pref. No pref. 1.6* [11] auratus Mus musculus 0.3 – 10 No pref. No pref. 0.43 - 4.3* [12]

Ovis aries No pref. [13]

Rattus 0.08 [14] norvegicus Sus scrofa 0.24 - 1.73 0.3 No pref. No pref. 2.18 - 4.36 [15] domesticus Please note that the values presented for Homo sapiens are taste detection thresholds. Values marked with one asterisk indicate that a preference was shown for the corresponding concentration, but no taste preference threshold has been determined for that species and substance. “No pref.” indicates that no preference was shown for the substance over water in two-bottle preference tests. [1] Henderson (2019); [2] van Gemert (2011); [3] Present study; [4] Glaser (1992); [5] Sato et al. (1977); [6] Danilova & Hellekant (2004); [7] Hellekant et al. (1993); [8] Schilling et al. (2004); [9] Nicklasson (2015); [10] Hellekant et al. (1994); [11] Danilova et al. (1998); [12] Bachmanov et al. (2001); [13] Goatcher & Church (1970); [14] Stumphauzer & Williams (1969); [15] Glaser et al. (2000).

The spider monkeys’ taste preference threshold for alitame ranged from 0.002 to 0.005 mM. These threshold values are 600-1500 times lower than those for sucrose in spider monkeys, making alitame a high-potency sweetener in this species. The human threshold for alitame ranges between 0.0015625-0.0125 mM, which overlaps with that of spider monkeys. As it is possible that spider monkeys may detect lower concentrations of a given substance than the one determined as their taste preference threshold, it is plausible to assume that spider monkeys may have a similar or even higher sensitivity to alitame compared to humans. For chimpanzees, the taste preference threshold has been reported at 1 mM (Henderson 2019), at least 200 times higher than that of spider monkeys. This suggests a higher sensitivity to alitame in spider monkeys than in chimpanzees, which could be explained in terms of degree of frugivory. Spider monkeys are highly specialized on the consumption of ripe fruits, and have been suggested to use sweetness as a criterion for food selection (Di Fiore et al 2008; Gonzalez- Zamora et al. 2009), which could ultimately account for an increased sensitivity to sweet taste.

Even though chimpanzees have similarly been described as ripe-fruit specialists, the fact that this species has a much broader diet and thus a lower degree of frugivory may explain their lower sensitivity to sweet taste relatively to spider monkeys.

In the common marmoset, a preference for a single concentration (0.3 mM) over water was reported but no threshold was determined (Danilova & Hellekant 2004). The grey mouse lemur did not display a preference for alitame (Schilling et al. 2004), although it was only presented with one concentration (0.21 mM) of the substance. Further comprehensive studies investigating the taste perception of these non-human primate species are necessary to draw clearer conclusions.

Amongst non-primate animals, pigs displayed a taste preference threshold for alitame of 0.3 mM (Glaser et al. 2000), which is 100 times higher than that of spider monkeys. Additionally, hamsters tested for two alitame concentrations (1.5 and 0.15 mM) did not show a preference for the substance (Danilova et al. 1998). Overall, the taste preference threshold values for alitame reported here in spider monkeys seem to be the lowest reported for this substance so far, which is again congruent with the notion that this species’ highly specialized frugivorous diet may have boosted a higher sensitivity towards sweet-taste.

For aspartame, the spider monkeys’ taste preference threshold was 20 mM, the highest threshold value of the ones compiled in Table 4 for this substance. This value was obtained for only two individuals, as the remaining monkeys did not show a preference for the substance at any of the tested concentrations. The human taste detection threshold for aspartame ranges between 0.02-0.2 mM (van Gemert 2011), which makes it up to 1,000 times lower than that of the two spider monkeys. For chimpanzees, the taste preference threshold was found to be 0.5 mM (Henderson 2019), 40 times lower than that of the two spider monkeys.

The taste preference threshold for aspartame has been determined in several catarrhine species, all of which displayed a preference for the substance. Japanese macaques (Macaca fuscata) displayed the lowest threshold of the catarrhine species, preferring concentrations as low as 0.3 mM of aspartame over water (Sato et al. 1977). Greater spot-nosed monkeys (Cercopithecus nictitans) and rhesus macaques (Macaca mulatta) have a taste preference threshold of 0.5 mM, and sooty mangabeys (Cercocebus atys) of 0.6 mM (Glaser 1992).

Amongst platyrrhines and apart from spider monkeys, only the common marmoset was tested for its taste responsiveness to aspartame, not revealing a preference for the one tested concentration (5mM) of the substance (Danilova & Hellekant 2004).

None of the strepsirrhines tested so far, namely the mongoose lemur (Eulemur mongoz) and the grey mouse lemur, showed a preference for aspartame (Hellekant et al. 1993, Schilling et al. 2004).

Up until now, no Platyrrhine or Strepsirrhine primate had displayed a preference for aspartame. Indeed, there is a strong trend of only Catarrhine primates preferring this sweetener over water, which led to the inference that the ability to detect aspartame may be based on phylogeny (Glaser et al. 1995). Accordingly, there should be an underlying structural difference in the sweet-taste receptors of Catarrhines compared to Platyrrhines and Prosimians, allowing all of them to perceive alitame through a specific binding site which all primates may possess, but only allowing Catarrhines to perceive aspartame through a second specific binding site which is exclusive to this taxon (Glaser et al. 1995). The data from the present study seem to conflict with this view as two of the spider monkeys, did, in fact, prefer aspartame over water at the concentration of 20 mM. This concentration is about 6 times higher than that presented to several platyrrhine species by Glaser et al. (1992) (3.3 mM), which could serve as an explanation for the discrepant results between studies. Interestingly, the individuals which did not show a preference for aspartame, rejected the substance at three of the tested concentrations (20, 2 and 0,2 mM). This suggests that aspartame may in fact have an aversive side-taste perceived only by some individuals at the concentrations tested. One could thus hypothesize that, in contrast to what has been suggested by Glaser et al. (1995), the feature in the sweet- taste receptor that enables aspartame tasting might have emerged before the divergence between Platyrrhines and Catarrhines. Nonetheless, as only two out of four individuals preferred aspartame over water, an alternative explanation may be posed in which differences in the sweet-taste receptor of these two individual spider monkeys allow them to perceive the sweetness of this substance, whereas other individuals cannot perceive it. Such differences in the sweet-taste receptor could be caused, for instance, by single-nucleotide polymorphisms (SNPs), which have been demonstrated to affect taste sensitivity to sugars in humans (Dias et al. 2015). Given that neither aspartame nor alitame are naturally occurring sweeteners, the fact that at least some non-human primates and humans are able to perceive them may be puzzling. A potential explanation put forward is that compounds with a similar molecular structure may exist in nature as constituents of plants which these primate taxa adapted to exploit (Li et al. 2011).

Amongst non-primate animals, cows, hamsters, mice and pigs have been tested for their gustatory responsiveness to aspartame, but no preference for the substance was elicited in any

of the species (Hellekant et al. 1994, Danilova et al. 1998, Bachmanov et al. 2001, Glaser et al. 2000). The fact that the diets of these species are not primarily based on sweet-tasting foods such as fruits, could potentially serve as an explanation for their lack of responsiveness to this substance.

For isomalt, the taste preference threshold determined here for spider monkeys was 10-20 mM, making it 3-7 times higher than that for sucrose in this species and thus a low-potency sweetener. For humans, isomalt is reported as a low-potency sweetener about 45% as sweet as sucrose. The taste detection threshold determined for humans ranges from 5 to 40 mM, which overlaps with the taste preference threshold range of spider monkeys. As mentioned previously, as it is possible that spider monkeys may detect lower concentrations of a given substance than the one determined as their taste preference threshold, it is plausible to assume that spider monkeys may be as sensitive as humans to isomalt. No taste preference threshold has yet been reported for chimpanzees or any other animal species. Thus, further comparative studies on the taste perception of isomalt are required in order to draw any conclusions.

For sodium cyclamate, the taste preference threshold of the spider monkeys was 1 mM. However, as only one of the four monkeys displayed a preference for this substance, this value was obtained for only one individual. These results suggest that spider monkeys are probably generally unable to perceive the sweetness of sodium cyclamate. For the higher concentrations tested, 10 mM and 1 mM, respectively 3 and 2 individuals rejected the substance. The rejection responses of the spider monkeys to this sweetener are congruent with the notion that sodium cyclamate may have an unpleasant side-taste at higher concentrations, described for humans as a distinct sweet-sour lingering (Nikoleli & Nikolelis 2012).

Within hominoid primates, only humans have been tested for their taste sensitivity to sodium cyclamate, with a reported threshold value of 0.28 mM (van Gemert 2011). This value is approximately 3.6 times lower than that determined for spider monkeys, making this substance the one with the closest threshold values between spider monkeys and humans here reported. Within the Platyrrhini and apart from spider monkeys, common marmosets have been reported not to show a preference for this substance (Danilova & Hellekant 2004). The latter species, however, was only presented with one sodium cyclamate concentration (9.9 mM). Amongst strepsirrhine primates, the grey mouse lemur did not show a preference for sodium cyclamate at a concentration of 33.5 mM (Schilling et al. 2004).

Non-primate species tested for their responsiveness to sodium cyclamate include hamsters, mice and pigs, none of which displayed a preference for the substance (Danilova et al. 1998, Bachmanov et al. 2001, Glaser et al. 2000). Hamsters were presented with the concentrations 10, 25 and 50 mM, mice with the concentration range 0.15-151 mM and pigs with 24.84, 49.69 and 99.39 mM.

Thus, it appears that humans are, so far, the only species to perceive the sweetness of sodium cyclamate. Although further data with regard to the taste responsiveness of non-human primate and non-primate species to sodium cyclamate are needed, it is reasonable to assume that a variation in the sweet-taste receptor which is exclusive to humans may underlie this phenomenon.

For sodium saccharine, the spider monkeys’ taste preference threshold was 0.002-0.5 mM. This is the first taste preference threshold value reported in the Platyrrhini and thus the first evidence that species within this taxon are able to perceive this sweetener. For humans, the taste threshold ranges between 0.00003 and 0.03 mM (van Gemert 2011), making it approximately 16 to 66 times lower than that of spider monkeys. For chimpanzees, the taste preference threshold for this substance was 0.2-2 mM (Henderson 2019), which is a factor of 4 to 100 times higher than that of spider monkeys. Thus, of the three species mentioned so far, humans are the most sensitive species to sodium saccharin, followed by spider monkeys, and only then chimpanzees. The higher sensitivity to sodium saccharine of the highly frugivorous spider monkeys compared to chimpanzees may, thus, be explained in terms of degree of frugivory.

Interestingly, 25% of human subjects are reported to detect a bitter-side taste at high sodium saccharin concentrations (Nabors & Gelardi 1991). The fact that one of the four individuals presented with sodium saccharin rejected the substance at the highest concentration tested (10 mM) suggests that spider monkeys are also likely to perceive an aversive bitter side-taste at high concentrations. Additionally, individual spider monkeys seem to differ in their tolerance towards bitter taste, as do humans.

Within platyrrhine primates, the common marmoset did not show a preference for this substance (1.6 mM) (Danilova & Hellekant 2004).

Amongst strepsirrhines, both the grey mouse lemur and the black-and-white ruffed lemur (Varecia variegata) did not show a preference for this substance (Schilling et al. 2004, Nicklasson 2015). The grey mouse lemur was presented with the single concentration of 2 mM

and the black-and-white ruffed lemur with a range of concentrations from 0.5 to 50 mM. Although the generalist diet of the grey mouse lemur supports the idea that its lack of responsiveness to saccharine may be due to a lack of dependence on carbohydrate-rich and thus sweet-tasting food items, the black-and-white ruffed lemurs are described as a highly frugivorous species (Britt 2000), which ultimately contradicts the latter line of thought and makes it difficult to draw any conclusions.

Non-primate species tested for their taste sensitivity to sodium saccharine include cows, hamsters, mice, sheep (Ovis aries), rats (Rattus norvegicus) and pigs. The taste threshold for sodium saccharin in cows was 4 mM (Hellekant et al. 1994), in rats 0.08 mM (Stumphauzer & Williams 1969) and in pigs 2.18-4-36 mM (Glaser et al. 2000). Hamsters and mice were reported to show a preference for sodium saccharin at the concentrations of 1.6 mM (Danilova et al. 1998) and 0.43-4.3 mM (Bachmanov et al. 2001), respectively, but no taste preference threshold was determined for these species. Sheep (Ovis aries) presented with concentrations of sodium saccharin from 0.007 to 13.6 mM did not show a preference for the substance (Goatcher & Church 1970). Overall, there appears to be a considerable degree of variation with regard to the sensitivity to sodium saccharin amongst non-primate mammals, as well as amongst primates, making it difficult to draw any unequivocal conclusions regarding the factors influencing the perception of this substance.

5.3.2. Sweet-tasting proteins

Table 3 displays the taste preference thresholds for the sweet-tasting proteins monellin and thaumatin reported in different mammal species. For monellin, only one out of the four spider monkeys displayed a preference for the substance. The taste preference threshold for this spider monkey was 0.001 mM, which corresponds to the only monellin concentration the monkey preferred over water. These results suggest that spider monkeys are probably generally unable to perceive monellin.

The human taste detection threshold for monellin ranges between 0.00002-0.00009 mM (van Gemert 2011), which is up to 50 times lower than that of the spider monkey. The taste preference threshold determined for chimpanzees was 0.0025-0.005 mM (Henderson 2019), which is in the same order of magnitude as the value reported here for one of the spider monkeys. Thus, humans seem to possess a higher sensitivity to monellin than chimpanzees (Henderson 2019) and spider monkeys.

For thaumatin, only one of the four spider monkeys displayed a preference for the substance. The taste preference threshold for this individual was 0.1 mM, which corresponds to the only thaumatin concentration the monkey preferred over water. Similar to monellin, it is thus likely that spider monkeys are generally unable to perceive thaumatin.

For humans, the taste detection threshold reported for thaumatin was 0.00006-0.0001 mM (van Gemert 2011), which is approximately 1,600 times lower than that of the single spider monkey. The taste preference threshold for thaumatin in chimpanzees ranges between 0.001-0.2 mM (Henderson 2019), which overlaps with the threshold value here reported for the one spider monkey.

Amongst the Platyrrhini, red-handed tamarins (Saguinus midas) tested for their responsiveness to monellin and thaumatin did not display a preference for either substance at the respective concentrations of 1.9 mM and 0.9 mM (Hellekant et al. 1976). Amongst strepsirrhine primates, the grey mouse lemur also did not display a preference for neither monellin nor thaumatin, at the respective concentrations of 0.003 and 0.0015 mM (Schilling et al. 2004).

Similar to alitame and aspartame, the gustatory perception of monellin and thaumatin seems to be related to phylogeny. A study by Glaser et al. (1978) reporting the responses of several primate species to thaumatin and monellin, suggests a clear dichotomy within the Primate order: all catarrhine primates tested so far preferred thaumatin (0.9 mM) over water, whereas none of the platyrrhine and strepsirrhine primates did. Regarding monellin, the scenario lacked clarity, as a few species other than catarrhine primates (such as the strepsirrhine primate Eulemur mongoz) did display a preference for the substance at the concentration of 1.9 mM. The mentioned study (Glaser et al. 1978) did not aim to determine taste preference thresholds, having only tested the thaumatin solution of 0.9 mM and the monellin solution of 1.9 mM. Although it may be tempting to suggest that non-catarrhine primates may be able to perceive monellin and thaumatin based on the results reported here for spider monkeys, such scenario is not likely. Rather, the one individual who displayed a preference for both sweet-tasting proteins most probably carries a variant of the sweet-taste receptor’s typical alleles, which may enable the perception of monellin and thaumatin. Thus, a phylogeny-based dichotomy remains the most acceptable explanation for primate taste responsiveness to monellin and thaumatin. Nonetheless, further research is needed for greater clarification.

Within non-primate species, pigs tested for their responsiveness to monellin (0.018 mM) and thaumatin (0.009 mM) did not display a preference for either one of the substances (Glaser et

al. 2000). Hamsters and mice presented with thaumatin also did not show a preference for the substance (Danilova et al. 1998, Bachmanov 2001). With hamsters, the thaumatin concentrations tested were 0.0014 and 0.0127 mM, whilst mice were presented with a range of concentrations from 0.0000013 to 0.0045 mM. Given the origin of these sweet-tasting proteins, the general inability of these non-primate species to perceive monellin and thaumatin should not be surprising. As monellin and thaumatin are natural components of west African berries, it is plausible that species who have evolved alongside with and possibly exploited these food items – such as the West African chimpanzees – have developed a keen sensitivity to their sweet taste. Accordingly, lack of exposure to these compounds throughout the evolution of both geographically and taxonomically distant species should not have triggered any necessity for the ability to perceive them.

Table 3. Taste preference thresholds (in mM) for the sweet proteins monellin and thaumatin.

Species Monellin Thaumatin Ref.

Hominoid primates Pan troglodytes verus 0.0025 – 0.005 0.001 – 0.2 [1]

Homo sapiens 0.00002 – 0.00009 0.00006 – 0.0001 [2]

Platyrrhine primates Ateles geoffroyi 0.001 0.1 [3]

Callithrix jacchus No pref. [4]

Saguinus midas No pref. No pref. [5]

Strepsirrhine primates Microcebus murinus No pref. No pref. [6]

Non-primates Mesocricetus auratus No pref. [7]

Mus musculus No pref. [8]

Sus scrofa domesticus No pref. No pref. [9]

The values presented for H. sapiens are taste detection thresholds. “No pref.” indicates that no preference was shown for the substance over water in two-bottle preference tests. [1] Henderson et al. (2019); [2] van Gemert (2011); [3] Present study; [4] Danilova & Hellekant (2004); [5] Hellekant et al. (1976); [6] Schilling et al. (2004); [7] Danilova et al. (1998); [8] Bachmanov et al. (2001); [9] Glaser et al. (2000).

5.3.3. Sweet-tasting saccharides

Table 4 displays the taste preference thresholds for the sweet-tasting saccharides galactose and sorbitol in different mammal species. For galactose, the determined taste preference threshold of the spider monkeys was 2-20 mM. The human taste detection threshold for galactose is 10- 39 mM (van Gemert 2011), values which are in the same order of magnitude as those of spider monkeys. For chimpanzees, the taste preference threshold to this substance ranges between 100-500 mM (Henderson 2019), which is 5 to 250 times higher than that of the spider monkeys. These results suggest that the spider monkeys have the highest taste sensitivity to galactose compared to humans and chimpanzees.

The black-and-white ruffed lemur (Varecia variegata), the only other non-human primate species tested so far for its sensitivity to galactose, displayed a taste preference threshold of 70- 90 mM (Nicklasson 2015).

Amongst non-primate species, there is only a reported taste preference threshold for cows, known to prefer galactose concentrations as low as 80 mM over water (Hellekant et al. 1994). Thus, the taste preference threshold reported here for spider monkeys is, so far, the lowest one reported for galactose.

Galactose is a constituent of lactose, a naturally occurring disaccharide in mammalian breastmilk. The reported spider monkey taste preference threshold for lactose is 10 mM (Laska et al. 1996), which is in the same order of magnitude as that reported here for galactose for three of the spider monkeys (20 mM). The fact that one of the spider monkeys preferred galactose concentrations as low as 2 mM, however, indicates that some individuals may be more sensitive to galactose than lactose. This is the case also for humans, who are able to detect lower concentrations of galactose than of lactose (van Gemert 2011). A hypothesis has been postulated which links the sensitivity of mammals to natural sugars to the experience of the sweet taste of lactose in breastmilk. However, there is a lack of consensus regarding this notion, as humans show a higher sensitivity to natural plant sugars such as fructose, glucose, and sucrose than to lactose (Ramirez 1990). Nonetheless, a correlation between the sensitivity to naturally occurring sugars and breastmilk lactose content was recently reported for strepsirrhine primates (Wielbass et al. 2015).

For sorbitol, the spider monkeys’ taste preference threshold was 20-50 mM. For humans, the reported taste detection threshold was 10.6-36.7 mM (van Gemert 2011), which, although being a slightly lower range, is in the same order of magnitude than that of spider monkeys.

Given the fact that the value reported for humans is a detection threshold, and given the possibility that animals may detect lower concentrations than the lowest one preferred over water (Spector 2003), it is possible that spider monkeys are able to detect sorbitol concentrations as low as humans do. For chimpanzees, the reported taste preference threshold for sorbitol is 250 mM (Henderson 2019), which is 5 - 12.5 times higher than that reported for spider monkeys. This suggests that spider monkeys have a higher sensitivity to sorbitol than chimpanzees.

Within the Strepsirrhini, black-and-white ruffed lemurs preferred sorbitol concentrations as low as 30-110 mM (Nicklasson 2015). The range of taste preference threshold for this species is thus considerably wider than that reported for spider monkeys, possibly reflecting a greater individual variability for the sensitivity to sorbitol. Nonetheless, the most sensitive individual displayed a preference for concentrations as low as 30 mM, which is within the same order of magnitude as the threshold values of the spider monkeys.

Amongst non-primate species, mice have taste preference thresholds for sorbitol of 16.5-55 mM (Bachmanov 2001), which is similar to that of spider monkeys. For pigs, the taste preference threshold ranges between 48.8-58.46 mM (Glaser et al. 2000), which partly overlaps with that of spider monkeys.

Sorbitol is a naturally occurring sugar alcohol present in fruits such as apples and blackberries. Therefore, it may be so that differences in sensitivity to sorbitol amongst different species could be attributable to the amount of fruits consumed by each species. The lower sensitivity of chimpanzees to sorbitol compared with that of spider monkeys and black-and-white ruffed lemurs supports this notion, as chimpanzees display a lower degree of frugivory than the latter two species which are both highly frugivorous. However, the taste thresholds of humans and non-primate species, none of which are fruit specialists, are either lower or rather close to those of spider monkeys, making it difficult to draw any conclusions.

Table 4. Taste preference thresholds (in mM) for the sweet-tasting saccharides galactose and sorbitol.

Species Galactose Sorbitol Ref.

Hominoid primates Pan troglodytes verus 100 – 500 250 [1]

Homo sapiens 10 – 39 10.6 – 36.7 [2]

Platyrrhine primates Ateles geoffroyi 2 – 20 20 – 50 [3]

Strepsirrhine primates Varecia variegate 70 – 90 30 – 110 [4]

Non-primates Bos Taurus 80 [5]

Mus musculus 16.5 - 55 [6]

Sus scrofa domesticus 48.8 – 58.46 [7]

The values presented for H. sapiens are taste detection thresholds. “No pref.” indicates that no preference was shown for the substance over water in two-bottle preference tests. [1] Henderson et al. (2019); [2] van Gemert (2011); [3] Present study; [4] Nicklasson (2015); [5] Hellekant et al. (1994); [6] Bachmanov et al. (2001); [7] Glaser et al. (2000).

5.4. Spider monkeys’ perception of aspartame as indicated through facial responses Thirteen out of sixteen recorded behaviors (licking, sucking, sniffing, eyes open more than 50%, eyes open less than 50%, eyes closed, flat tongue protrusions, repetitive tongue protruding, tongue protrusion gape, mouth gape, lip stretching, lip smacking and withdrawal from dropper) yielded significant differences in the facial reactivity of spider monkeys across substances.

The recorded frequencies for licking support a distinction between attractive and aversive palatable substances, with significantly higher frequencies recorded for sucrose, water and aspartame, compared to those recorded for caffeine and citric acid. High-frequency licking has been previously associated with the response to sweet taste in several non-human primate species, including platyrrhines (Steiner & Glaser 1984). Thus, a significantly higher frequency of licking recorded for aspartame compared to those of the stimuli representing presumably less pleasant taste qualities (such as bitter and sour taste) suggests that the latter substance may either have at least a slight sweet quality, or, at least a neutral profile as perceived by the spider monkeys.

Regarding sucking frequency, the results distinguish only sucrose from caffeine for yielding significantly higher frequencies of this behavior. Similarly to licking, sucking has previously been associated with the response to sweet taste in non-human primates (Steiner & Glaser

1984). Thus, the results allow only to support the notion that the spider monkeys perceive sucrose as more attractive than caffeine, while no particular conclusions can be drawn regarding the perception of aspartame.

For sniffing, as water was the stimulus provoking the highest frequency of this behavior, it is plausible to assume that the monkeys may be more prone to sniff at those substances that do not provide any distinct flavor. The fact that the response to aspartame yielded the second highest frequency of sniffing would thus be in line with the idea that this substance may be perceived by spider monkeys as having more of a neutral taste profile. However, it is difficult to link this behavior to any particular taste quality, as it was performed at comparable frequencies in response to sucrose as it was to citric acid (which represent quite contrasting taste qualities). As all taste solutions were both colorless and odorless, it is unlikely that these results may have been influenced by any unintended cues the monkeys may have picked up on. However, this possibility cannot be fully excluded.

The higher frequencies of eyes open with more than 50% of visible area of the eyeball in response to water relative to caffeine and citric acid suggest an association of this behavior to more neutral-tasting stimuli. Accordingly, the fact that aspartame yielded significantly higher frequencies of this behavior also relative to the aversive stimuli may thus suggest that this substance may be perceived as neutral by the spider monkeys. However, it is difficult to conclusively interpret these results, as no previous studies have associated this state of the eyes to any particular taste quality.

The highest frequencies of eyes open with less than 50% of visible area of the eyeball were found with sucrose and water. As the presumably aversive caffeine and citric acid yielded the lowest frequency of this behavior, the results suggest that more closed eyes may be associated with the pleasantness of the taste stimuli. The intermediate frequencies recorded for aspartame would, thus, point towards a certain neutrality in this substances’ taste profile as perceived by spider monkeys, at least in comparison with the remaining taste stimuli included in this study. A previous study described “slightly closed eyes” as being part of the typical response to sweet taste in several non-human primates including two platyrrhine species (Steiner & Glaser 1984), which is line with the previous reasoning. However, the fact that water triggered approximately the same frequency of this behavior as did sucrose makes it difficult to draw any unequivocal conclusions.

Closed eyes were recorded significantly more often with sucrose than with all remaining taste stimuli. Although the second highest frequencies of closed eyes were recorded in response to aspartame, no significant difference was found between the latter substance and water. Thus, the results do not allow to link the state of closed eyes to the response to sweet taste. Rather, the results suggest that aspartame may be perceived as a non-sweet, neutral stimulus such as water. However, previous studies have yet to link this state of the eyes to the response to any particular taste quality, making it difficult to draw any conclusions.

Flat tongue protrusions were recorded significantly less often with caffeine and citric acid compared to all remaining stimuli, linking a higher frequency of occurrence of this behavior to more pleasant stimuli. This notion is in line with the fact that a significantly higher frequency of flat tongue protrusions was recorded with sucrose compared to all other substances. Previous studies have reported tongue protrusions to be associated to the response to sweet taste in chimpanzees (Steiner et al. 2001; Ueno et al. 2004) and rhesus macaques (Ueno et al. 2004), which is congruent with the results of the present study. The intermediate position of aspartame between water and caffeine regarding the frequency of flat tongue protrusions could thus be explained by the spider monkeys perceiving it as either neutral or slightly bitter.

The results obtained for tongue protrusion gape were similar to those obtained for flat tongue protrusions, with higher recorded frequencies for the presumably pleasant stimuli (sucrose and water) and lowest recorded frequencies for the presumably aversive stimuli (caffeine and citric acid). Accordingly, aspartame took again an intermediate position which, following the reasoning applied when analysing flat tongue protruding, could be due to the spider monkeys perceiving it as either neutral or slightly bitter. However, these results cannot be compared with those of previous studies on taste-induced facial responses in non-human primates as most studies look into mouth gapes (usually associated to responses to aversive stimuli) strictly separately from tongue protrusions (Steiner & Glaser 1995; Steiner et al. 2001).

The only significant result identified for mouth gape was a significantly higher occurrence of this behavior in response to sucrose than in response to caffeine. This result thus suggests a link between mouth gaping and the response to sweet taste. However, this is not in line with previous studies, as this behavior is most often associated with the response to bitter taste (Steiner & Glaser 1984; Steiner & Glaser 1995; Steiner et al. 2001). Furthermore, the fact that tongue protrusion gape yielded such conspicuous results whereas mouth gape did not should be pointed out, as it emphasizes the importance of a minutely structured ethogram.

Lip stretching occurred significantly more in response to caffeine than it did for any other stimulus, suggesting that this behavior may be associated with the response to bitter taste. Accordingly, the fact that aspartame was not closer to caffeine than it was to the other stimuli regarding the frequency of lip stretching puts doubt on the notion that this substance may be perceived as slightly bitter by the spider monkeys. To the extent of my knowledge, this behavior has not been described in any previous studies on taste-induced facial responses in non-human primates, making it difficult to draw any further conclusions and increasing the need for further investigation.

Even though lip smacking revealed only few significant differences across substances, a comparison of the recorded frequencies for each substance yielded interesting results. The highest frequencies of the behavior were recorded with sucrose, which is in line with the notion that lip smacking occurs typically in response to sweet taste, as reported by previous studies (Steiner & Glaser 1995; Steiner et al. 2001). Accordingly, the lowest frequencies of lip smacking were recorded with caffeine and citric acid, with aspartame and water taking intermediate positions for the frequency of this behavior. The similar responsiveness of the spider monkeys to water and aspartame could thus be interpreted as both substances being perceived as neutral, suggesting that the animals do not perceive the sweetness of aspartame.

The frequency of withdrawals from dropper can be interpreted as a measure of aversiveness of a taste stimulus, as it seems intuitive that an animal would avoid a pleasant stimulus less often compared to an aversive stimulus. Accordingly, previous studies in non-human primates have associated withdrawal from the taste stimulus to presumably aversive taste qualities, such as bitter taste (Steiner & Glaser 1984). In this sense, the present results suggest that the animals find caffeine and citric acid the most aversive taste stimuli, followed by aspartame, which is then followed by water, and finally followed by sucrose. The fact that aspartame yielded a higher frequency of withdrawals than water suggests that the former is not perceived by the spider monkeys as being as neutral as water.

Taking together the results which are in line with those of previous studies, two distinct patterns of responsiveness to taste stimuli could be described, one being associated with presumably pleasant and thus attractive stimuli and the other being associated with presumably unpleasant and thus aversive stimuli. Thus, responses to pleasant stimuli should most likely include high frequencies of licking, sucking and tongue protruding as well as slightly closed eyes. On the other hand, responses to aversive stimuli should reveal lower frequencies of licking, sucking

and tongue protruding, as well as higher frequencies of withdrawal from the taste stimuli. In this context, the results of the present study do not allow for an unequivocal determination of the spider monkeys’ perception of aspartame. However, the results do suggest that aspartame is probably not perceived as sweet by spider monkeys. Rather, it is most likely perceived as either neutral or as slightly bitter, as indicated by the mentioned similarities with, respectively, water and caffeine in the recorded frequencies for some of the analyzed behaviors.

For further investigation, and with the aim of achieving sturdier results, future efforts should focus on including a higher number of individuals. Similarly, performing the two-bottle preference tests with aspartame with a higher number of individuals could allow for the identification of more animals showing a preference for or rejecting this substance. These individuals should then be particularly relevant targets for a data analysis at the individual level, as inter-individual differences in the preference-tests could potentially be supported by respective inter-individual differences in taste-induced facial reactions. Nonetheless, the present study supports the idea of similarity in taste-induced facial reactivity between humans and non-human primates and emphasizes the need for further research in this field.

5.5. Conclusions

Not all spider monkeys were able to perceive all ten taste substances. Acesulfame K, alitame, isomalt, sodium saccharin, galactose and sorbitol were clearly perceived by all the spider monkeys and preferred over water. For aspartame, there were mixed results which may reflect differences in the sweet-taste receptors amongst the individuals. Additionally, aspartame- induced facial responses indicate that this substance is probably not perceived as sweet by the spider monkeys. Sodium cyclamate was generally rejected, suggesting that most spider monkeys perceive an aversive side-taste in this substance. Monellin and thaumatin are most likely not perceived by spider monkeys and thus any exceptional case may be explained by differences in the sweet-taste receptor’s alleles. In general, the spider monkey’s taste preference thresholds were either lower or similar to those of chimpanzees. The higher sensitivity of spider monkeys compared to that of chimpanzees towards sweet-tasting substances may be explained by their higher degree of frugivory, supporting the notion that dietary specializations affect taste perception. Relative to humans, the taste thresholds of spider monkeys were usually higher. Nonetheless, as the determination of taste preference thresholds does not allow for an accurate measurement of an animal’s ability to detect a taste substance,

it is possible that spider monkeys may perceive concentrations as low as humans do. Although further research is necessary for a deeper understanding of the sense of taste in non-human primates, the results of the present study may have important implications for the study of the evolution of the sense of taste within the primate order. Taken together, the results suggest that both dietary specialization and phylogenetic relatedness may contribute to between-species differences in sweet taste perception.

6. Societal and ethical considerations

The experiments performed in the present study abide by the Guide for the Care and Use of Laboratory Animals (8th edition, National Research Council, 2011), the American Society of Primatologists’ Principles for the Ethical Treatment of Primates, as well as the current Swedish and Mexican welfare laws.

The spider monkeys that participated in the present study were not coerced to do so in any way. The animals cooperated with the tester and partook in the proposed tasks on a strictly voluntary basis. As such, all tests were entirely dependent on the monkeys’ willingness to cooperate. The monkeys were always free to move away and abandon the testing sessions, and doing so, the trial was interrupted and repeated only when the animal was interested in cooperating again. Furthermore, no food nor water deprivation schedule was adopted. All taste substances presented to the spider monkeys are approved for human consumption and are considered safe to the animals at the tested concentrations.

The study of taste perception in primates allows for a deeper understanding of the sense of taste within this order, which could yield important implications for the welfare of primates kept in captivity. For instance, the results from the present study show that spider monkeys are generally attracted to non-caloric sweeteners such as acesulfame K and alitame, which could then be incorporated in rewards used for animal training without adding to the animals’ calorie intake. This could help prevent obesity issues, which are recurring amongst captive animals. Furthermore, sorbitol and isomalt can be used as sugar-substitutes as these substances provide a lower amount of calories than common sugars. Attractiveness to sorbitol could also be taken advantage of as a means to fight tooth problems in captive spider monkeys, as this substance does not contribute to tooth-decay in contrast to other dietary carbohydrates.

Further from potentially contributing to the welfare of animals in captivity, the present study could have implications on a larger scale. As an increase in knowledge about the perceptual world of primates may contribute to a deeper understanding of their ethology and ecology, the findings of the present study may, in turn, help to improve conservation measures for endangered species such as neotropical primates.

7. Acknowledgements

I would like to thank my supervisor, Matthias Laska, for constant support and thorough guidance throughout the entire project, having immensely contributed to the quality of this work. I am also grateful to my co-supervisor, Laura Tereza Hernandez, who made herself available to assist with various matters during my stay in Mexico. I would like to express my gratitude to my colleagues at UMA Doña Hilda Ávila de O’Farrill research station, namely Gil, Carlos, Veronica and Ana Cristina for all the assistance during my experiments. A special thank you is due to Eduardo and Lais for all the help and valuable input during my time in Mexico, as well as for having taken their personal time to sample a considerable amount of video footage concerning my taste-induced facial reactivity experiment. Finally, I would like to thank my friend, Tiffany Bosshard, for companionship and stimulating exchanges of opinions, as well as my family for supporting and sharing my enthusiasm for this project.

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Appendix

Ethogram of recorded behaviors in each trial of the taste-induced facial responses experiment. The selected behaviors are separated into state behaviors and behavioral events.

State Behaviors Licking, sucking and sniffing 1. Not licking or sucking The monkey is not licking nor sucking on the dropper. No ingestion can be observed. Code: When the monkey is more than 2.5cm away from the dropper. 2. Licking (ingestive) The monkey is ingesting the liquid from the dropper via licking the tip of the device. The tongue is extended, getting into direct contact with the tip of the dropper, and then retracted into the monkey’s mouth. Code: At the first lick (tongue extension followed by tongue retraction). 3. Sucking (ingestive) The monkey is ingesting the liquid from the dropper by putting its mouth around the device’s end and sucking upon it. Code: When the mouth is around the dropper in a sucking position causing a fast consumption of the liquid. 4. Sniffs dropper The monkey is sniffing the dropper. The head and neck are visibly directed towards the dropper. No ingestion is observed. Code: When the monkey is sniffing and within 2.5cm of the dropper. Eyes

5. Eyes open 50-100% The eyes of the monkey have a rounded shape. More than half of the full area of the eye is visible to the observer. There is either no or merely slight muscular contraction in the area around the eyes. Code: At onset of behaviour. 6. Eyes open <50% The eyes of the monkey are not fully open, thus they do not have a rounded shape. There is considerable muscular contraction around the eyes, narrowing them to a slit. The area of the eyes visible to the observer is less than 50% of the area that can be observed when the monkey has its eyes fully open. Code: At onset of behaviour. 7. Eyes closed The eyes of the monkey are closed and the eyeball is not visible. Only when the eyes are completely shut the behavior for closed eyes is to be coded. Code: At onset of behavior. 8. Eyes undetermined The eyes of the monkey are not visible to the observer. Behavioral events Mouth and tongue 1. Tongue protrusion The tongue is visible and directed out of the mouth. The tongue is not in contact with any object. Code: When tongue is visible. Modifiers for tongue protrusion

Tongue protrusion flat The tongue is visible and directed out of the mouth on the horizontal plane, with no upward or downward direction. The tongue is directed forward. The tongue is not in contact with any object. Code: At peak of behavior. Tongue protrusion angled in downward The tongue is visible and directed out of the direction mouth angled in a downward direction. The tongue is not in contact with any object. Code: At peak of behavior. Tongue protrusion angled in upward The tongue is visible and directed out of the direction mouth angled in an upward direction. The tongue is not in contact with any object. Code: At peak of behavior.

Repetitive tongue protruding The tongue is visible and directed out of the mouth repeatedly and at a high frequency in a short period of time (i.e. 2 seconds). The tongue is not in contact with any object. Code: At peak of behavior. 2. Tongue protrusion gape The tongue is visible and directed forward out of the mouth whilst the monkey has its mouth wide open in a gape. Code: At peak of behavior. 3. Mouth gape The monkey has its mouth wide open. The tongue is not visibly directed forward out of the mouth. Code: At peak of behavior.

4. Lip stretching The monkey stretches its lips backwards, showing its teeth. The mouth can be slightly open, forming a narrow gape. Code: At peak of behavior.

5. Lip smacking The monkey brings together its lips repetitively in a savoring-like motion. There are very slight mouth apertures immediately before the lips come together. Code: At peak of behavior. Nose 6. Wrinkles nose The monkey wrinkles its nose, possibly elevating its upper lip and dilating its nostrils. Code: At peak of behavior. Others 7. Withdraw from dropper After having tried the stimulus, the monkey abruptly moves away from the dropper. Code: At peak of behavior.