Taste responsiveness to two steviol glycosides in three species of nonhuman primates

Taste responsiveness to two steviol glycosides in three species of nonhuman primates Primates have been found to differ widely in their taste perception and studies suggest that a co- evolution between plant species bearing a certain taste substance and primate species feeding on these plants may contribute to such between-species differences. Considering that only platyrrhine primates, but not catarrhine or prosimian primates, share an evolutionary history with the neotrop- ical plant Stevia rebaudiana, we assessed whether members of these three primate taxa differ in their ability to perceive and/or in their sensitivity to its two quantitatively predominant sweet- tasting substances. We found that not only neotropical black-handed spider monkeys, but also paleotropical black-and-white ruffed lemurs and Western chimpanzees are clearly able to perceive stevioside and rebaudioside A. Using a two-bottle preference test of short duration, we found that Ateles geoffroyi preferred concentrations as low as 0.05 mM stevioside and 0.01 mM rebaudioside A over tap water. Taste preference thresholds of Pan troglodytes were similar to those of the spider monkeys, with 0.05 mM for stevioside and 0.03 mM for rebaudioside A, whereas Varecia variegata was slightly less sensitive with a threshold value of 0.1 mM for both substances. Thus, all three primate species are, similar to human subjects, clearly more sensitive to both steviol glycosides compared to sucrose. Only the spider monkeys displayed concentration-response curves with both stevioside and rebaudioside A which can best be described as an inverted U-shaped function sug- gesting that Ateles geoffroyi, similar to human subjects, may perceive a bitter side taste at higher concentrations of these substances. Taken together, the results of the present study do not support the notion that a co-evolution between plant and primate species may account for between-species differences in taste perception of steviol glycosides. Key words: taste preference thresholds, stevioside, rebaudioside A, Western chimpanzees, spider monkeys, black-and-white ruffed lemurs Comparative studies on the sense of taste in primates found marked (Laska et al. 1996), body mass (Simmen and Hladik 1998), and differences between species in their ability to perceive a given taste phylogenetic relatedness (Glaser et al. 1995; Nofre et al. 1996). The substance and in their sensitivity to a given taste substance (Glaser latter explanation implies that species that are phylogenetically 1986; Laska and Hernandez Salazar 2004; Wielbass et al. 2015). closely related to each other should share a higher proportion of Possible explanations for the observed interspecific differences in the taste receptors specialized for the detection of certain taste sub- perception of substances described as sweet by humans, for ex- stances compared to species that are less closely related. Both psy- ample, include, but are not restricted to: the degree of frugivory chophysical tests of primate sweet-taste perception and genetic V C The Author (2017). Published by Oxford University Press. 63 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 64 Current Zoology, 2018, Vol. 64, No. 1 studies lend some support to this notion: only Old World primates station of the Universidad Veracruzana, near the town of Catemaco, (catarrhines) display taste responses to the sweet-tasting proteins in the province of Veracruz, Mexico. The animals were housed as thaumatin and monellin, whereas all prosimians and New World part of social groups in outdoor enclosures of 50–200 m that were primates (platyrrhines) tested so far are indifferent to these two sub- adjacent to indoor enclosures of 20 m . They were 7, 8, 9, 10, and stances (Glaser et al. 1978). Thaumatin is found in the west African 14 years old at the start of the study. The black-and-white ruffed katemfe fruit Thaumatococcus daniellii, and monellin in the fruit of lemurs were housed at Kolma ˚ rden Wildlife Park, Sweden, in a 117 3 2 the west African serendipity berry Dioscoreophyllum cumminsii, m indoor exhibit with access to a 100 m outdoor island with nat- suggesting that a co-evolution between these two plant species and ural vegetation. They were 11, 12, and 20 years old at the start of Old World primates feeding on their fruits might have occurred the study. which ultimately led to the expression of a taste receptor that is re- With all three species, we performed the tests in a smaller room sponsive to thaumatin and monellin (Hladik 1993). This notion is adjacent to the indoor exhibit which held several compartments in supported by genetic studies reporting that the ability of the mam- which the animals were tested separately to avoid competition and malian T1R2–T1R3 sweet-taste receptor to bind proteins of high distraction. All animals were trained to voluntarily enter the test molecular weight such as thaumatin and monelllin required consid- compartments and were completely accustomed to the procedure erable modifications of its recognition sites which are likely to have described below. The animals were fed fresh fruit and vegetables taken place during primate evolution (Temussi 2002; Li et al. 2011). (spider monkeys: one time per day; black-and-white ruffed lemurs: Stevia rebaudiana is a neotropical plant species belonging to the two times per day; chimpanzees: three times per day). The chimpan- family Asteraceae and native to Paraguay (Soejarto 2002). Its leaves zees and the black-and-white ruffed lemurs were additionally pro- contain intensely sweet-tasting ent-kaurene diterpene glycosides vided with commercial primate chow pellets and all three species among which two, stevioside and rebaudioside A, have been identi- had permanent access to water. Thus, no water deprivation schedule fied as quantitatively predominant and are commercially interesting was adopted. The amount of food offered daily to the animals was as low-calorie sweeteners (DuBois and Prakash 2012). Both sub- such that leftovers were still present on the floor the next morning. stances have been reported to be 100–300 times sweeter than su- Thus, it was unlikely that ravenous appetite affected the animals’ in- crose as perceived by humans although numbers may vary gestive behavior. depending on the psychophysical method and reference concentra- tion used (DuBois et al. 1991; Carakostas et al. 2012; Upreti et al. Taste stimuli 2012). However, there is consensus that both stevioside and rebau- We used the following two steviol glycosides: stevioside (CAS# dioside A have a bitter side taste for humans when presented at 57817-89-7), and rebaudioside A (CAS# 58543-16-1). The sub- higher concentrations (Schiffman et al. 1995) which may limit their stances were obtained from Shanghai Xunxin Chemical Co. use as sugar substitutes. (Shaoxing, China), and Xinghua Green Biological Preparation Co. Considering that only platyrrhine primates, but not catarrhine or (Jiangsu, China), respectively, and were of the highest available pur- prosimian primates, share an evolutionary history with Stevia ity ( 99.5%). rebaudiana, we decided to investigate whether members of these three primate taxa differ in their ability to detect and/or in their sen- Procedure sitivity to the two predominant sweet-tasting substances found in We used a two-bottle preference test of short duration (Richter and this neotropical plant. It was therefore the aim of the present study Campbell 1940). The animals were allowed to drink for 1 min from to assess the taste responsiveness of a prosimian primate species, the a pair of simultaneously presented graduated cylinders (of 120 ml black-and-white ruffed lemur Varecia variegata variegata, a platyr- volume for the spider monkeys and the black-and-white ruffed rhine primate species, the black-handed spider monkey Ateles geof- lemurs, and of 700 ml volume for the chimpanzees) with metal froyi, and a catarrhine primate species, the Western chimpanzee Pan drinking spouts. We performed four such 1-min trials per day and troglodytes verus to the two major steviol glycosides found in Stevia animal, two of them in the morning, and two in the afternoon. rebaudiana. More specifically, we determined taste preference To determine taste preference thresholds the animals were given thresholds for stevioside and rebaudioside A, and assessed whether the choice between tap water and defined concentrations of a steviol high concentrations of these tastants are rejected by the animals. To glycoside dissolved in tap water. With both substances, testing this end, we employed a two-bottle preference test of short duration started at a concentration of 1 mM and proceeded in the following (Richter and Campbell 1940). This method allows to directly meas- steps (0.1, 0.01 mM, etc.) until an animal failed to show a significant ure absolute preferences and largely rules out the influence of postin- preference. Subsequently, they were presented with intermediate gestive factors on the animals’ ingestive behavior. concentrations (between the lowest concentration that was preferred and the first concentration that was not) in order to determine the Materials and Methods preference threshold value more exactly. To maintain the animals’ motivation and willingness to cooperate, testing of the different con- Animals centrations did not follow a strict order but was pseudo- We assessed the taste responsiveness to two steviol glycosides in two randomized. This was true for both within a given session (morning adult female and one adult male Western chimpanzees Pan troglo- or afternoon) or between sessions. We presented each pair of stimuli dytes verus, four adult female and one adult male black-handed spi- 10 times per individual animal, and the position of the stimuli was der monkeys Ateles geoffroyi, and three adult male black-and-white pseudo-randomized in order to counterbalance possible position ruffed lemurs Varecia variegata variegata. The chimpanzees were preferences. Care was taken that an animal sampled both stimuli at housed, together with two other individuals, at Bora ˚ s Zoo, Sweden, 3 2 in a 261 m indoor exhibit with access to a 560 m outdoor island least once during each trial. The order in which the two steviol with natural vegetation. They were 27, 33, and 48 years old at the glycosides were tested was the same for all three species: (1) stevio- start of the study. The spider monkeys were housed at the field side, (2) rebaudioside A. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Nicklasson et al.  Steviol glycoside taste in nonhuman primates 65 Data analysis For each animal, we recorded the amount of liquid consumed from each bottle, summed it for the 10 trials with a given stimulus com- bination, converted it to percentages (relative to the total amount of liquid consumed from both bottles), and took 66.7% (i.e., 2/3 of the total amount of liquid consumed) as the criterion of preference. We chose this rather conservative criterion for reasons of comparability of data as the same criterion had been used in previous studies on sweet-taste responsiveness with other primate species (Larsson et al. 2014; Laska 1996, 1997, 2000; Laska et al. 1996, 1998, 1999, 2001; Wielbass et al. 2015), and in order to avoid misinterpretation due to a too liberal criterion. Additionally, we performed binomial tests, and regarded an animal as significantly preferring one of the two stimuli if it reached the criterion of 66.7% and consumed more from the bottle containing the preferred stimulus in at least 8 out of 10 trials (binomial test, P< 0.05). Thus, we defined taste preference threshold as the lowest concen- tration at which the animals met both criteria mentioned above. Preliminary analyses of the data indicated that there were no system- atic differences in choice behavior and liquid consumption between the first and the second presentation of a session, or between the morning and the afternoon session, respectively. Intraindividual variability of the amount of liquid consumed across the ten trials with a given stimulus combination was low and averaged <20%. Thus, a theoretically possible bias in the overall preference score due to excessive drinking in aberrant trials did not occur. Compliance with ethical standards The experiments reported here comply with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication no. 86-23, revised 1996) and also with current Swedish and Mexican laws. This study was approved by Gothenburg’s Animal Care and Use Committee (Go ¨ teborgs djurfo ¨ rso ¨ ksetiska n€ amnd, protocol #75-2016, for the chimpanzees), the Swedish Board of Agriculture (Jordbruksverket, protocol #5.2.19-5974/15, Figure 1. Mean taste responses (6 SD) of three black-and-white ruffed lemurs Varecia variegata, five black-handed spider monkeys Ateles geoffroyi, and for the black-and-white ruffed lemurs), and the Ethical Board of the three Western chimpanzees Pan troglodytes to aqueous solutions of stevio- Federal Government of Mexico’s Secretariat of Environment and side and rebaudioside A tested against tap water. Each data point represents Natural Resources (SEMARNAT; Official permits no. 09/GS-2132/ the mean value of 10 trials of 1 min per animal. The dotted horizontal lines at 05/10, for the spider monkeys). 66.7% and at 50% indicate the criterion of preference and the chance level, respectively. Results lower and detectable concentrations tested (Figure 1). Accordingly, Taste preference thresholds for stevioside were found to be 0.1 mM the concentration-response function with the best goodness-of-fit in the black-and-white ruffed lemurs, and 0.05 mM in the spider for the spider monkeys can be described as an inverted U-shaped monkeys and the chimpanzees, respectively (Figure 1, left panels). function (third-order polynomial regression: R ¼ 0.86 with stevio- Taste preference thresholds for rebaudioside A were 0.1 mM in the side, and R ¼ 0.90 with rebaudioside A), whereas the correspond- black-and-white ruffed lemurs, 0.01 mM in the spider monkeys, and ing functions for the black-and-white ruffed lemurs and the 0.03 mM in the chimpanzees (Figure 1, right panels). All animals chimpanzees can be described as a sigmoid function (symmetric sig- failed to show a significant preference for the lowest concentrations 2 2 moid regression: R ¼ 0.98 and 0.94 with stevioside, and R ¼ 0.96 presented, suggesting that the preference for higher concentrations and 0.92 with rebaudioside A). was indeed based on the chemical nature of the stimuli. In most cases, interindividual variability of scores was low for both sub- and suprathreshold concentrations tested (Figure 1) and with only few Discussion exceptions all animals of a given species either reached the criterion The results of the present study demonstrate that black-and-white of preference (> 66.7% of total consumption, plus binomial test, ruffed lemurs, black-handed spider monkeys, and Western chimpan- P< 0.05) with a given stimulus combination or all animals failed to zees are clearly able to perceive the two quantitatively predominant do so. sweet-tasting diterpene glycosides of the neotropical plant Stevia Only the spider monkeys, but not the black-and-white ruffed rebaudiana. Further, they show that all three primate species are lemurs and the chimpanzees, displayed a marked decrease of >10% able to detect concentrations as low as 0.1 mM or even lower with in their preference with the highest concentration of both stevioiside and rebaudioside A tested (1 mM) relative to at least one of the both substances and are thus, similar to human subjects, clearly Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 66 Current Zoology, 2018, Vol. 64, No. 1 more sensitive to both stevioside and rebaudioside A compared to Table 1. Comparison of taste thresholds (mM) for steviol glyco- sides and sucrose in primates sucrose. Our finding that not only spider monkeys, but also black-and- Species Stevioside Rebaudioside A Sucrose white ruffed lemurs and Western chimpanzees are able to perceive the two steviol glycosides tested here is not trivial considering that Prosimian primates a a b Varecia variegata variegata 0.1 0.1 25 other plant-derived high-potency sweeteners such as thaumatin and Platyrrhine primates monellin have been found to be perceptible only for catarrhine pri- a a c Ateles geoffroyi 0.05 0.01 3 mate species that co-evolved with the plant species in which these Catarrhine primates proteins are found, but not for prosimian or platyrrhine primates a a d Pan troglodytes verus 0.05 0.03 20 (Glaser et al. 1978). At least two possible explanations may account e e e Homo sapiens 0.0053 0.0046 10 for the finding that not only a representative of the New World pri- mates, but also representatives of the prosimian and Old World pri- Please note that taste preference thresholds in nonhuman primates are com- mates, respectively, are able to perceive steviol glycosides that have pared with taste detection thresholds in human subjects. Present study. so far exclusively been found in a neotropical plant species: first, it Wielbass et al. (2015). may simply be that between-species differences in the ability or in- Laska et al. (1996). ability to perceive sweet-tasting substances do not necessarily re- Sjo ¨ stro ¨ m (unpublished data). quire that a co-evolution between animal and plant species has van Gemert (2011). occurred. This notion is supported by the finding that only catar- rhine primates, but not prosimian and platyrrhine primates have been found able to detect the artificial sweeteners aspartame and glycosides compared to sucrose. However, the differences in thresh- neotame (Glaser et al. 1992). Similarly, only prosimian and catar- old values between the disaccharide and the two steviol glycosides is rhine primates, but not platyrrhine primates display taste responses generally smaller in the nonhuman primates (factor of 60–400 with to the artificial sweeteners AMPA (N-a-L-aspartyl-(R)-a-methylphe- stevioside, and 250–667 with rebaudioside A) compared to the nethylamine) and ASME (N-a-L-aspartyl-L-(O-tert-butyl)serine me- human subjects (factor of 1900 with stevioside, and 2200 with thyl ester) (Glaser et al. 1996). In all these cases, any co-evolution rebaudioside A). Small between-species variations in the binding do- between primate and plant species as a driving force for between- mains of the T1R2-T1R3 sweet-taste receptor are thought to ac- species differences in sweet-taste perception can be excluded as the count for such differences in the receptor’s affinity to certain ligands taste substances in question do not occur in nature. and thus in an organism’s sensitivity to different sweet-tasting sub- Second, it might be that prosimian and catarrhine primates have stances (Liu et al. 2011). co-evolved with plant species bearing taste substances that are struc- A comparison of the taste threshold values between human and turally similar to the steviol glycosides tested here which might have nonhuman primates suggests the former to be more sensitive for the led to modifications at the sweet-taste receptor allowing for the two steviol glycosides tested here than the latter. However, it should binding and recognition of stevioside and rebaudioside A. This no- be considered that the sophisticated psychophysical signal detection tion is supported by the finding that two intensely sweet-tasting di- procedures employed with human subjects are commonly regarded terpene glycosides named rubusoside and suavioside A which are to yield lower threshold values compared to the simple two-bottle structurally closely related to stevioside and rebaudioside A have preference test used with the nonhuman primates which provides been identified in the leaves of a paleotropical plant, the Chinese only a conservative approximation of an animal’s taste sensitivity blackberry Rubus suavissimus (Tanaka et al 1981; Hirono et al. (Spector 2003). As the differences between the human taste detec- 1990; Ohtani et al. 1991). Thus, the possibility that prosimian and tion threshold values and the taste preference threshold values in the catarrhine primates may have been exposed to these sweet-tasting spider monkeys and chimpanzees were only a factor of 10 or even substances over an evolutionarily relevant period of time and thus less than that, one can assume the two nonhuman primate species to may have evolved a correspondingly modified sweet-taste receptor have a similar sensitivity for the taste of stevioside and rebaudioside that is also responsive to the sweet-tasting compounds of Stevia A as human subjects. With both steviol glycosides, Varecia variegata rebaudiana cannot be excluded. had slightly higher threshold values compared to Ateles geoffroyi In this context, it is also interesting to note that a recent genetic and Pan troglodytes which is in line with the finding that the black- study assessing variation in the sweet-taste receptor protein T1R3 and-white ruffed lemur is also slightly less sensitive for sucrose com- reported that the sweet-tasting protein brazzein which is found in pared to the spider monkey and the chimpanzee (Table 1). the fruits of the West African plant Pentadiplandra brazzeana is Nevertheless, all three species of nonhuman primates tested here are likely to be perceptible for most catarrhine primates, but not for gor- clearly sensitive enough to detect the average concentrations of ste- illas (Guevara et al. 2016). The authors speculate that gorilla- vioside (21.5 mM) and rebaudioside A (11.2 mM) found in fresh specific mutations at this receptor might be a counter-adaptation to leaves of Stevia rebaudiana (Woelwer-Rieck et al. 2010). To the best the deceptive, that is: non-caloric, sweet signal of brazzein. Thus, of our knowledge, no study so far reported on behavioral observa- the possibility that co-evolution between animals and plants might tions of nonhuman primates ingesting Stevia leaves in the wild. affect even phylogenetically closely related species in a different Similarly, no study so far assessed behavioral responses of captive manner should also be considered. primates to the presentation of Stevia leaves. Such studies would Table 1 compares the taste preference threshold values for ste- allow for a first, tentative conclusion as to whether nonhuman pri- vioside and rebaudioside A obtained in the present study with those mates may reject Stevia leaves due to the possible bitter side taste of obtained in the same primate species for sucrose and with taste de- stevioside and rebaudioside A at high concentrations. tection threshold values for the same three substances obtained in Finally, our finding that the spider monkeys displayed human subjects. Similar to humans, all three primate species tested concentration-response curves with both stevioside and rebaudioside here were found to be considerably more sensitive to the two steviol A which can best be described as an inverted U-shaped function Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Nicklasson et al.  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Synthetic study of the relationship be- Woelwer-Rieck U, Lankes C, Wawrzum A, Wu ¨ st M, 2010. Improved HPLC tween structure and sweet taste properties of steviol glycosides. Molecules method for the evaluation of the major steviol glycosides in leaves of Stevia 17:4186–4196. rebaudiana. Eur Food Res Technol 231:581–588. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Zoology Oxford University Press

Taste responsiveness to two steviol glycosides in three species of nonhuman primates

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Abstract

Primates have been found to differ widely in their taste perception and studies suggest that a co- evolution between plant species bearing a certain taste substance and primate species feeding on these plants may contribute to such between-species differences. Considering that only platyrrhine primates, but not catarrhine or prosimian primates, share an evolutionary history with the neotrop- ical plant Stevia rebaudiana, we assessed whether members of these three primate taxa differ in their ability to perceive and/or in their sensitivity to its two quantitatively predominant sweet- tasting substances. We found that not only neotropical black-handed spider monkeys, but also paleotropical black-and-white ruffed lemurs and Western chimpanzees are clearly able to perceive stevioside and rebaudioside A. Using a two-bottle preference test of short duration, we found that Ateles geoffroyi preferred concentrations as low as 0.05 mM stevioside and 0.01 mM rebaudioside A over tap water. Taste preference thresholds of Pan troglodytes were similar to those of the spider monkeys, with 0.05 mM for stevioside and 0.03 mM for rebaudioside A, whereas Varecia variegata was slightly less sensitive with a threshold value of 0.1 mM for both substances. Thus, all three primate species are, similar to human subjects, clearly more sensitive to both steviol glycosides compared to sucrose. Only the spider monkeys displayed concentration-response curves with both stevioside and rebaudioside A which can best be described as an inverted U-shaped function sug- gesting that Ateles geoffroyi, similar to human subjects, may perceive a bitter side taste at higher concentrations of these substances. Taken together, the results of the present study do not support the notion that a co-evolution between plant and primate species may account for between-species differences in taste perception of steviol glycosides. Key words: taste preference thresholds, stevioside, rebaudioside A, Western chimpanzees, spider monkeys, black-and-white ruffed lemurs Comparative studies on the sense of taste in primates found marked (Laska et al. 1996), body mass (Simmen and Hladik 1998), and differences between species in their ability to perceive a given taste phylogenetic relatedness (Glaser et al. 1995; Nofre et al. 1996). The substance and in their sensitivity to a given taste substance (Glaser latter explanation implies that species that are phylogenetically 1986; Laska and Hernandez Salazar 2004; Wielbass et al. 2015). closely related to each other should share a higher proportion of Possible explanations for the observed interspecific differences in the taste receptors specialized for the detection of certain taste sub- perception of substances described as sweet by humans, for ex- stances compared to species that are less closely related. Both psy- ample, include, but are not restricted to: the degree of frugivory chophysical tests of primate sweet-taste perception and genetic V C The Author (2017). Published by Oxford University Press. 63 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 64 Current Zoology, 2018, Vol. 64, No. 1 studies lend some support to this notion: only Old World primates station of the Universidad Veracruzana, near the town of Catemaco, (catarrhines) display taste responses to the sweet-tasting proteins in the province of Veracruz, Mexico. The animals were housed as thaumatin and monellin, whereas all prosimians and New World part of social groups in outdoor enclosures of 50–200 m that were primates (platyrrhines) tested so far are indifferent to these two sub- adjacent to indoor enclosures of 20 m . They were 7, 8, 9, 10, and stances (Glaser et al. 1978). Thaumatin is found in the west African 14 years old at the start of the study. The black-and-white ruffed katemfe fruit Thaumatococcus daniellii, and monellin in the fruit of lemurs were housed at Kolma ˚ rden Wildlife Park, Sweden, in a 117 3 2 the west African serendipity berry Dioscoreophyllum cumminsii, m indoor exhibit with access to a 100 m outdoor island with nat- suggesting that a co-evolution between these two plant species and ural vegetation. They were 11, 12, and 20 years old at the start of Old World primates feeding on their fruits might have occurred the study. which ultimately led to the expression of a taste receptor that is re- With all three species, we performed the tests in a smaller room sponsive to thaumatin and monellin (Hladik 1993). This notion is adjacent to the indoor exhibit which held several compartments in supported by genetic studies reporting that the ability of the mam- which the animals were tested separately to avoid competition and malian T1R2–T1R3 sweet-taste receptor to bind proteins of high distraction. All animals were trained to voluntarily enter the test molecular weight such as thaumatin and monelllin required consid- compartments and were completely accustomed to the procedure erable modifications of its recognition sites which are likely to have described below. The animals were fed fresh fruit and vegetables taken place during primate evolution (Temussi 2002; Li et al. 2011). (spider monkeys: one time per day; black-and-white ruffed lemurs: Stevia rebaudiana is a neotropical plant species belonging to the two times per day; chimpanzees: three times per day). The chimpan- family Asteraceae and native to Paraguay (Soejarto 2002). Its leaves zees and the black-and-white ruffed lemurs were additionally pro- contain intensely sweet-tasting ent-kaurene diterpene glycosides vided with commercial primate chow pellets and all three species among which two, stevioside and rebaudioside A, have been identi- had permanent access to water. Thus, no water deprivation schedule fied as quantitatively predominant and are commercially interesting was adopted. The amount of food offered daily to the animals was as low-calorie sweeteners (DuBois and Prakash 2012). Both sub- such that leftovers were still present on the floor the next morning. stances have been reported to be 100–300 times sweeter than su- Thus, it was unlikely that ravenous appetite affected the animals’ in- crose as perceived by humans although numbers may vary gestive behavior. depending on the psychophysical method and reference concentra- tion used (DuBois et al. 1991; Carakostas et al. 2012; Upreti et al. Taste stimuli 2012). However, there is consensus that both stevioside and rebau- We used the following two steviol glycosides: stevioside (CAS# dioside A have a bitter side taste for humans when presented at 57817-89-7), and rebaudioside A (CAS# 58543-16-1). The sub- higher concentrations (Schiffman et al. 1995) which may limit their stances were obtained from Shanghai Xunxin Chemical Co. use as sugar substitutes. (Shaoxing, China), and Xinghua Green Biological Preparation Co. Considering that only platyrrhine primates, but not catarrhine or (Jiangsu, China), respectively, and were of the highest available pur- prosimian primates, share an evolutionary history with Stevia ity ( 99.5%). rebaudiana, we decided to investigate whether members of these three primate taxa differ in their ability to detect and/or in their sen- Procedure sitivity to the two predominant sweet-tasting substances found in We used a two-bottle preference test of short duration (Richter and this neotropical plant. It was therefore the aim of the present study Campbell 1940). The animals were allowed to drink for 1 min from to assess the taste responsiveness of a prosimian primate species, the a pair of simultaneously presented graduated cylinders (of 120 ml black-and-white ruffed lemur Varecia variegata variegata, a platyr- volume for the spider monkeys and the black-and-white ruffed rhine primate species, the black-handed spider monkey Ateles geof- lemurs, and of 700 ml volume for the chimpanzees) with metal froyi, and a catarrhine primate species, the Western chimpanzee Pan drinking spouts. We performed four such 1-min trials per day and troglodytes verus to the two major steviol glycosides found in Stevia animal, two of them in the morning, and two in the afternoon. rebaudiana. More specifically, we determined taste preference To determine taste preference thresholds the animals were given thresholds for stevioside and rebaudioside A, and assessed whether the choice between tap water and defined concentrations of a steviol high concentrations of these tastants are rejected by the animals. To glycoside dissolved in tap water. With both substances, testing this end, we employed a two-bottle preference test of short duration started at a concentration of 1 mM and proceeded in the following (Richter and Campbell 1940). This method allows to directly meas- steps (0.1, 0.01 mM, etc.) until an animal failed to show a significant ure absolute preferences and largely rules out the influence of postin- preference. Subsequently, they were presented with intermediate gestive factors on the animals’ ingestive behavior. concentrations (between the lowest concentration that was preferred and the first concentration that was not) in order to determine the Materials and Methods preference threshold value more exactly. To maintain the animals’ motivation and willingness to cooperate, testing of the different con- Animals centrations did not follow a strict order but was pseudo- We assessed the taste responsiveness to two steviol glycosides in two randomized. This was true for both within a given session (morning adult female and one adult male Western chimpanzees Pan troglo- or afternoon) or between sessions. We presented each pair of stimuli dytes verus, four adult female and one adult male black-handed spi- 10 times per individual animal, and the position of the stimuli was der monkeys Ateles geoffroyi, and three adult male black-and-white pseudo-randomized in order to counterbalance possible position ruffed lemurs Varecia variegata variegata. The chimpanzees were preferences. Care was taken that an animal sampled both stimuli at housed, together with two other individuals, at Bora ˚ s Zoo, Sweden, 3 2 in a 261 m indoor exhibit with access to a 560 m outdoor island least once during each trial. The order in which the two steviol with natural vegetation. They were 27, 33, and 48 years old at the glycosides were tested was the same for all three species: (1) stevio- start of the study. The spider monkeys were housed at the field side, (2) rebaudioside A. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Nicklasson et al.  Steviol glycoside taste in nonhuman primates 65 Data analysis For each animal, we recorded the amount of liquid consumed from each bottle, summed it for the 10 trials with a given stimulus com- bination, converted it to percentages (relative to the total amount of liquid consumed from both bottles), and took 66.7% (i.e., 2/3 of the total amount of liquid consumed) as the criterion of preference. We chose this rather conservative criterion for reasons of comparability of data as the same criterion had been used in previous studies on sweet-taste responsiveness with other primate species (Larsson et al. 2014; Laska 1996, 1997, 2000; Laska et al. 1996, 1998, 1999, 2001; Wielbass et al. 2015), and in order to avoid misinterpretation due to a too liberal criterion. Additionally, we performed binomial tests, and regarded an animal as significantly preferring one of the two stimuli if it reached the criterion of 66.7% and consumed more from the bottle containing the preferred stimulus in at least 8 out of 10 trials (binomial test, P< 0.05). Thus, we defined taste preference threshold as the lowest concen- tration at which the animals met both criteria mentioned above. Preliminary analyses of the data indicated that there were no system- atic differences in choice behavior and liquid consumption between the first and the second presentation of a session, or between the morning and the afternoon session, respectively. Intraindividual variability of the amount of liquid consumed across the ten trials with a given stimulus combination was low and averaged <20%. Thus, a theoretically possible bias in the overall preference score due to excessive drinking in aberrant trials did not occur. Compliance with ethical standards The experiments reported here comply with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication no. 86-23, revised 1996) and also with current Swedish and Mexican laws. This study was approved by Gothenburg’s Animal Care and Use Committee (Go ¨ teborgs djurfo ¨ rso ¨ ksetiska n€ amnd, protocol #75-2016, for the chimpanzees), the Swedish Board of Agriculture (Jordbruksverket, protocol #5.2.19-5974/15, Figure 1. Mean taste responses (6 SD) of three black-and-white ruffed lemurs Varecia variegata, five black-handed spider monkeys Ateles geoffroyi, and for the black-and-white ruffed lemurs), and the Ethical Board of the three Western chimpanzees Pan troglodytes to aqueous solutions of stevio- Federal Government of Mexico’s Secretariat of Environment and side and rebaudioside A tested against tap water. Each data point represents Natural Resources (SEMARNAT; Official permits no. 09/GS-2132/ the mean value of 10 trials of 1 min per animal. The dotted horizontal lines at 05/10, for the spider monkeys). 66.7% and at 50% indicate the criterion of preference and the chance level, respectively. Results lower and detectable concentrations tested (Figure 1). Accordingly, Taste preference thresholds for stevioside were found to be 0.1 mM the concentration-response function with the best goodness-of-fit in the black-and-white ruffed lemurs, and 0.05 mM in the spider for the spider monkeys can be described as an inverted U-shaped monkeys and the chimpanzees, respectively (Figure 1, left panels). function (third-order polynomial regression: R ¼ 0.86 with stevio- Taste preference thresholds for rebaudioside A were 0.1 mM in the side, and R ¼ 0.90 with rebaudioside A), whereas the correspond- black-and-white ruffed lemurs, 0.01 mM in the spider monkeys, and ing functions for the black-and-white ruffed lemurs and the 0.03 mM in the chimpanzees (Figure 1, right panels). All animals chimpanzees can be described as a sigmoid function (symmetric sig- failed to show a significant preference for the lowest concentrations 2 2 moid regression: R ¼ 0.98 and 0.94 with stevioside, and R ¼ 0.96 presented, suggesting that the preference for higher concentrations and 0.92 with rebaudioside A). was indeed based on the chemical nature of the stimuli. In most cases, interindividual variability of scores was low for both sub- and suprathreshold concentrations tested (Figure 1) and with only few Discussion exceptions all animals of a given species either reached the criterion The results of the present study demonstrate that black-and-white of preference (> 66.7% of total consumption, plus binomial test, ruffed lemurs, black-handed spider monkeys, and Western chimpan- P< 0.05) with a given stimulus combination or all animals failed to zees are clearly able to perceive the two quantitatively predominant do so. sweet-tasting diterpene glycosides of the neotropical plant Stevia Only the spider monkeys, but not the black-and-white ruffed rebaudiana. Further, they show that all three primate species are lemurs and the chimpanzees, displayed a marked decrease of >10% able to detect concentrations as low as 0.1 mM or even lower with in their preference with the highest concentration of both stevioiside and rebaudioside A tested (1 mM) relative to at least one of the both substances and are thus, similar to human subjects, clearly Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 66 Current Zoology, 2018, Vol. 64, No. 1 more sensitive to both stevioside and rebaudioside A compared to Table 1. Comparison of taste thresholds (mM) for steviol glyco- sides and sucrose in primates sucrose. Our finding that not only spider monkeys, but also black-and- Species Stevioside Rebaudioside A Sucrose white ruffed lemurs and Western chimpanzees are able to perceive the two steviol glycosides tested here is not trivial considering that Prosimian primates a a b Varecia variegata variegata 0.1 0.1 25 other plant-derived high-potency sweeteners such as thaumatin and Platyrrhine primates monellin have been found to be perceptible only for catarrhine pri- a a c Ateles geoffroyi 0.05 0.01 3 mate species that co-evolved with the plant species in which these Catarrhine primates proteins are found, but not for prosimian or platyrrhine primates a a d Pan troglodytes verus 0.05 0.03 20 (Glaser et al. 1978). At least two possible explanations may account e e e Homo sapiens 0.0053 0.0046 10 for the finding that not only a representative of the New World pri- mates, but also representatives of the prosimian and Old World pri- Please note that taste preference thresholds in nonhuman primates are com- mates, respectively, are able to perceive steviol glycosides that have pared with taste detection thresholds in human subjects. Present study. so far exclusively been found in a neotropical plant species: first, it Wielbass et al. (2015). may simply be that between-species differences in the ability or in- Laska et al. (1996). ability to perceive sweet-tasting substances do not necessarily re- Sjo ¨ stro ¨ m (unpublished data). quire that a co-evolution between animal and plant species has van Gemert (2011). occurred. This notion is supported by the finding that only catar- rhine primates, but not prosimian and platyrrhine primates have been found able to detect the artificial sweeteners aspartame and glycosides compared to sucrose. However, the differences in thresh- neotame (Glaser et al. 1992). Similarly, only prosimian and catar- old values between the disaccharide and the two steviol glycosides is rhine primates, but not platyrrhine primates display taste responses generally smaller in the nonhuman primates (factor of 60–400 with to the artificial sweeteners AMPA (N-a-L-aspartyl-(R)-a-methylphe- stevioside, and 250–667 with rebaudioside A) compared to the nethylamine) and ASME (N-a-L-aspartyl-L-(O-tert-butyl)serine me- human subjects (factor of 1900 with stevioside, and 2200 with thyl ester) (Glaser et al. 1996). In all these cases, any co-evolution rebaudioside A). Small between-species variations in the binding do- between primate and plant species as a driving force for between- mains of the T1R2-T1R3 sweet-taste receptor are thought to ac- species differences in sweet-taste perception can be excluded as the count for such differences in the receptor’s affinity to certain ligands taste substances in question do not occur in nature. and thus in an organism’s sensitivity to different sweet-tasting sub- Second, it might be that prosimian and catarrhine primates have stances (Liu et al. 2011). co-evolved with plant species bearing taste substances that are struc- A comparison of the taste threshold values between human and turally similar to the steviol glycosides tested here which might have nonhuman primates suggests the former to be more sensitive for the led to modifications at the sweet-taste receptor allowing for the two steviol glycosides tested here than the latter. However, it should binding and recognition of stevioside and rebaudioside A. This no- be considered that the sophisticated psychophysical signal detection tion is supported by the finding that two intensely sweet-tasting di- procedures employed with human subjects are commonly regarded terpene glycosides named rubusoside and suavioside A which are to yield lower threshold values compared to the simple two-bottle structurally closely related to stevioside and rebaudioside A have preference test used with the nonhuman primates which provides been identified in the leaves of a paleotropical plant, the Chinese only a conservative approximation of an animal’s taste sensitivity blackberry Rubus suavissimus (Tanaka et al 1981; Hirono et al. (Spector 2003). As the differences between the human taste detec- 1990; Ohtani et al. 1991). Thus, the possibility that prosimian and tion threshold values and the taste preference threshold values in the catarrhine primates may have been exposed to these sweet-tasting spider monkeys and chimpanzees were only a factor of 10 or even substances over an evolutionarily relevant period of time and thus less than that, one can assume the two nonhuman primate species to may have evolved a correspondingly modified sweet-taste receptor have a similar sensitivity for the taste of stevioside and rebaudioside that is also responsive to the sweet-tasting compounds of Stevia A as human subjects. With both steviol glycosides, Varecia variegata rebaudiana cannot be excluded. had slightly higher threshold values compared to Ateles geoffroyi In this context, it is also interesting to note that a recent genetic and Pan troglodytes which is in line with the finding that the black- study assessing variation in the sweet-taste receptor protein T1R3 and-white ruffed lemur is also slightly less sensitive for sucrose com- reported that the sweet-tasting protein brazzein which is found in pared to the spider monkey and the chimpanzee (Table 1). the fruits of the West African plant Pentadiplandra brazzeana is Nevertheless, all three species of nonhuman primates tested here are likely to be perceptible for most catarrhine primates, but not for gor- clearly sensitive enough to detect the average concentrations of ste- illas (Guevara et al. 2016). The authors speculate that gorilla- vioside (21.5 mM) and rebaudioside A (11.2 mM) found in fresh specific mutations at this receptor might be a counter-adaptation to leaves of Stevia rebaudiana (Woelwer-Rieck et al. 2010). To the best the deceptive, that is: non-caloric, sweet signal of brazzein. Thus, of our knowledge, no study so far reported on behavioral observa- the possibility that co-evolution between animals and plants might tions of nonhuman primates ingesting Stevia leaves in the wild. affect even phylogenetically closely related species in a different Similarly, no study so far assessed behavioral responses of captive manner should also be considered. primates to the presentation of Stevia leaves. Such studies would Table 1 compares the taste preference threshold values for ste- allow for a first, tentative conclusion as to whether nonhuman pri- vioside and rebaudioside A obtained in the present study with those mates may reject Stevia leaves due to the possible bitter side taste of obtained in the same primate species for sucrose and with taste de- stevioside and rebaudioside A at high concentrations. tection threshold values for the same three substances obtained in Finally, our finding that the spider monkeys displayed human subjects. Similar to humans, all three primate species tested concentration-response curves with both stevioside and rebaudioside here were found to be considerably more sensitive to the two steviol A which can best be described as an inverted U-shaped function Downloaded from https://academic.oup.com/cz/article-abstract/64/1/63/3056229 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Nicklasson et al.  Steviol glycoside taste in nonhuman primates 67 Glaser D, van der Wel H, Brouwer JN, DuBois GE, Hellekant G, 1992. (Figure 1, middle panels) suggests that Ateles geoffroyi, similar to Gustatory responses to the sweetener aspartame and their phylogenetic im- human subjects (Schiffman et al. 1995), may perceive a bitter side plications. Chem Senses 17:325–335. taste at higher concentrations of these substances. This notion is Guevara EE, Veilleux CC, Saltonstall K, Caccone A, Mundy NI et al., 2016. supported by electrophysiological findings in another New World Potential arms race in the coevolution of primates and angiosperms: primate, the common marmoset Callithrix jacchus: in this species, brazzein sweet proteins and gorilla taste receptors. Am J Phys Anthropol not only sweet-best fibers, but also bitter-best fibers of both the 161:181–185. chorda tympani and the glossopharyngeal nerve responded to stimu- Hellekant G, Danilova V, Ninomiya Y, 1997. Primate sense of taste: behav- lation with stevioside (Danilova et al. 2002). In a two-bottle prefer- ioral and single chorda tympani and glossopharyngeal nerve fiber recordings ence test, stevioside presented at a concentration of 0.62 mM was in the rhesus monkey Macaca mulatta. J Neurophysiol 77:978–993. clearly rejected by the marmosets, suggesting the bitter side taste Hirono S, Chou WH, Kasai R, Tanaka O, Tada T, 1990. Sweet and bitter even to prevail at this concentration (Danilova and Hellekant 2004). diterpene-glucosides from leaves of Rubus suavissimus. Chem Pharm Bull 38:1743–1744. Based on the data of the present study, it is not possible to decide Hladik CM, 1993. Fruits of the rain forest and taste perception as a result of evolu- whether the other two primate species tested here, black-and-white tionary interactions. In: Hladik CM, Hladik A, Linares OF, Pagezy H, Semple A ruffed lemurs and Western chimpanzees, may also perceive a bitter et al., editors. 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