Sour, but acceptable: Taste responsiveness to five food-associated acids in zoo-housed white-faced sakis, Pithecia pithecia

White-faced sakis ( Pithecia pithecia ) are commonly considered as frugivores but are unusual among primates as they do not specialize on ripe fruits but rather include a high proportion of unripe fruits into their diet, even during seasons when ripe fruits are available. Using a two-bottle preference test of short duration we therefore assessed whether this dietary specialization affects the taste responsiveness and sour-taste tolerance of four adult white-faced sakis for five food-associated acids. We found taste preference thresholds of the sakis to be 1-10 mM for citric acid, 0.5-20 mM for ascorbic acid, 2-10 mM for malic acid, 0.1-1 mM for tannic acid, and 2-20 mM for acetic acid, respectively. When given the choice between a reference solution of 50 mM sucrose and mixtures containing varying concentrations of sucrose plus citric acid, the sakis displayed a high sour-taste tolerance and required only 100 mM of sucrose (when mixed with 10 mM citric acid) or 200 mM of sucrose (when mixed with 30 or 50 mM citric acid), respectively, to prefer the sweet-sour mixture over the purely sweet 50 mM sucrose reference solution. These results demonstrate that white-faced sakis have a well-developed taste sensitivity for food-associated acids which is not inferior to that of primates specializing on ripe fruits. Compared to other platyrrhine primates, the sakis displayed a markedly higher sour-taste tolerance. These results may therefore reflect an evolutionary adaptation to the dietary specialization of the white-faced sakis to sour-tasting unripe fruits.


Introduction
Comparative studies on taste perception have demonstrated marked differences between species in the ability to perceive a given taste quality and in their sensitivity for a given taste substance.Both of these phenomena are thought to reflect evolutionary adaptations to dietary specializations [3,62].Cats, for example, lost the ability to perceive sweet-tasting substances, presumably because of a relaxed purifying selection pressure on the gene coding for the mammalian sweet-taste receptor driven by their strictly carnivorous diet [24].Dolphins and whales even lost the ability to perceive all taste qualities except for salty taste, possibly due to the fact that they swallow their prey whole and therefore without evaluating its taste [63].
Similarly, dietary specializations may also explain the marked between-species differences in sensitivity for a given taste substance that have been reported.Taste thresholds for bitter-tasting substances in vertebrates, for example, have been found to correlate positively with their degree of herbivory (Li and Zhang, 2014), and sensitivity for sweet-tasting carbohydrates has been reported to be higher in nectar-feeding hummingbirds compared to birds feeding on seeds or insects [2].
Primates are known to comprise a wide variety of dietary specializations which include, but are not restricted to, frugivores, folivores, herbivores, insectivores, graminivores, gummivores, and omnivores (Fleagle, 2013).Accordingly, they should be a particularly interesting taxon to study between-species differences in taste perception.Indeed, the proportion of animal matter in the diet of nonhuman primates has been found to correlate negatively with taste sensitivity to the prototypical umami taste substance monosodium glutamate [31].Similarly, taste sensitivity of nonhuman primates for food-associated carbohydrates has been reported to correlate positively with their degree of frugivory [43].
The taste of fruits is usually composed of a mixture of taste qualities, with sweet and sour being the predominant taste sensations.In general, young fruits contain more acids than ripe ones due to their conversion to sugars, that is, gluconeogenesis during maturation [41].In turn, when fruits turn overripe, that is, during fermentation and putrefaction, sour-tasting acids are produced by acidogenic microbes such as Lactobacillus or Acetobacter by reducing carbohydrates to acids [52].Accordingly, the ratio between sweetness and sourness is thought to be a reliable indicator of the degree of ripeness of fruits and thus of their palatability and nutritional value [12].
Fruits contain different acidic tastants [41].Citric acid and malic acid are the most common acids found in fruits and both are used by plants to store respiratory energy [51].Ascorbic acid, also known as vitamin C, is vital for several physiological processes in mammals.Ascorbic acid is particularly important for primates as they are unable to synthesize their own requirements of this vitamin and must therefore acquire it through their diet [7,15].Acetic acid is a byproduct of the fermentation processes of plant material [52] and may therefore indicate the overripeness or putrefaction of a given fruit and thus its edibility.Finally, tannic acid is often found in the skin and husks of fruit and serves as a defense mechanism which deters animals from consuming the fruit as tannic acid binds proteins and amino acids, thus preventing their absorption [9,45].
White-faced sakis are platyrrhine (=New World) primates that are known to include a high proportion of fruits into their diet [39].However, in contrast to most other frugivorous primate species, they do not serve as seed dispersers but rather feed on the seeds of the fruits they consume and are thought to exploit the lipids and proteins that these seeds contain [33].Further, unlike most other frugivorous primates, white-faced sakis do not specialize on ripe fruits but rather include a high proportion of unripe fruits into their diet, even during seasons when ripe fruits are available [36].This is remarkable taking into account that the pulp of ripe fruits has a markedly higher nutritional value than the pulp of unripe fruits [44].The sakis' predilection for unripe fruits has been suggested to be a behavioral and ecological adaptation to avoid food competition with sympatric ripe-fruit specialists [38].
Considering the common unattractiveness of strong sour taste sensations among animals [15], there are two plausible mechanisms how white-faced sakis, for example, may cope with feeding on a diet which includes a high proportion of unripe and thus sour-tasting fruits: their sense of taste may be less sensitive to food-associated sour-tasting acids compared to that of ripe-fruit specialists and/or they may be more tolerant towards acidic tastants than primates feeding on ripe fruits.Similar mechanisms have been reported to underlie between-species differences in bitter-taste sensitivity and tolerance: mammalian herbivores feeding on plants containing bitter and potentially toxic compounds have been found to display either a lower bitter-taste sensitivity [17] or a higher bitter-taste tolerance [22] compared to e.g.carnivorous mammals.
Behavioral studies on sour-taste perception in nonhuman primates are scarce and only rarely assessed a species' responsiveness to more than one food-associated acid [30,54].Nevertheless, marked between-species differences in both sensitivity for and tolerance towards sour tastants have been reported [16,32] and suggest a possible link with dietary specialization.
It was therefore the aim of the present study: 1. to determine the taste responsiveness of four white-faced sakis to the five food-associated acids citric acid, ascorbic acid, malic acid, acetic acid, and tannic acid, 2. to determine the sour-taste tolerance of the four white-faced sakis to mixtures containing varying concentrations of citric acid and sucrose compared to a reference solution of 50 mM sucrose, and 3. to compare the obtained results on taste preference thresholds and sour-taste tolerance with existing data from other primate and nonprimate species, to further assess the importance of sour-taste perception and the behavioral relevance of sour-taste for food selection.
We hypothesized that white-faced sakis, due to the high proportion of unripe fruit in their diet would be 1.less sensitive to food-associated acids compared to primates specializing on ripe fruits, and 2. more tolerant to sour taste compared to ripe-fruit specialists and nonfrugivorous primates.

Animals
Four captive white-faced sakis (Pithecia pithecia), housed at Furuviksparken (Furuvik, Sweden), participated in the study.The group comprised two adult males (Kariakov and Engelbrekt, 18 and 12 years of age, respectively) and two adult females (Lisha and Anita, 15 and 8 years of age, respectively).All individuals were born in captivity.The sakis were housed in an indoor enclosure of 633 m 3 which was connected to a 127 m 3 outdoor enclosure.The indoor enclosure contained a 15 m 3 test cage with a sliding door allowing for the temporary separation of the animals for individual testing.Both the indoor and the outdoor enclosure as well as the test cage were equipped with natural vegetation and with a variety of structures to climb and perch.Environmental enrichment included structural (e.g.ladders, ropes, swings, boxes), social (the indoor enclosure was designed as a walk-through exhibit for visitors, and shared with turtles and iguanas), and food enrichment (e.g.food items were hidden at various places to elicit foraging behavior).
The animals were fed fresh fruits and vegetables, seeds, nuts, insects, and a tamarin cake (Mazuri Zoo Foods, Witham, Essex, Great Britain).Occasionally, the sakis were also given access to other food items such as edible branches, leaves and flowers placed into the enclosure.Fruits and vegetables were fed three times each day, around 08:00, 12:00, and 15:00, respectively, while water was available ad libitum.Food leftovers were still present the following morning, suggesting that it was unlikely that ravenous appetite affected the outcome of the taste tests performed in the present study.

Ethical note
The experiments reported here comply with the American Society of Primatologists' Principles for the Ethical Treatment of Primates, with the European Union Directive on the Protection of Animals Used for Scientific Purposes (EU Directive 2010/63/EU), and with current Swedish animal welfare laws.The ethical board of Furuviksparken AB approved the study prior to its commencement (Protocol number: 2023-3).

Procedures
We employed a two-bottle preference test of short duration [50].The animals were allowed for 2 minutes to drink from a pair of simultaneously presented 150 ml cylinders with metal drinking spouts.We performed up to six trials per day and animal, spread throughout the day, taking care to keep intertrial intervals of at least 30 minutes between consecutive trials and after each meal.We conducted tests on 5-6 days per week.

Determination of taste preference thresholds
Here, the animals were given the choice between a 50 mM sucrose solution and a food-associated acid of a defined concentration dissolved in a 50 mM sucrose solution.With all substances except tannic acid, testing started at a concentration of 50 mM, followed by lower concentrations of 20, 10, 5 mM, etc. until an animal failed to show a significant preference for the 50 mM sucrose solution over the acid plus sucrose mixture.With tannic acid, testing starting at a concentration of 1 mM followed by 0.5, 0.2, 0.1 mM, etc. Subsequently, solutions with intermediate concentrations (between the lowest acid concentration that was rejected and the first concentration that was not) were used in order to determine preference threshold values more precisely.
Testing did not follow a strict descending staircase approach, but instead followed a pseudo-randomized order of presumably more or less attractive concentrations.This was done to maintain the animals' willingness to cooperate.With all four animals, the food-associated acids were tested in the following order: 1. citric acid, 2. ascorbic acid, 3. malic acid, 4. tannic acid, 5. acetic acid.

Assessment of sour-taste tolerance
Here, the animals were given the choice between a 50 mM sucrose solution and a mixture composed of varying defined concentrations of sucrose and 10, 30 or 50 mM of citric acid.The concentration of sucrose in the citric acid solution was 10, 20, 50, 100, 200, and 400 mM, respectively, until an animal failed to display a preference for the sucrose solution without citric acid.Accordingly, this experiment addressed the question how much sweetness must be added to a sourtasting solution to make it as attractive as a relatively weak but purely sweet-tasting reference solution?The experiment followed the same pseudo-randomized procedure for presenting the solutions to the animals as the taste preference threshold testing.
The order in which the citric acid concentrations were tested were the same for all animals and followed this order: 1. 10 mM citric acid, 2. 30 mM citric acid, 3. 50 mM citric acid.

Data analysis
In both experiments the total amount of the liquid consumed from each bottle was recorded for each individual, summed for the 10 trials with a given stimulus combination and converted into percentages.66.7 % of the total amount of liquid consumed was taken as the criterion of preference.Accordingly, the taste avoidance criterion is defined as 33.3 % of the total liquid consumed.This rather conservative criterion was used to easily compare the present data with data from previous studies which used the same criterion with other primate species or taste substances [11,[27][28][29][30]32,35,40,43,47,48,61] and in order to minimize the risk of misinterpretation of data due to a too liberal criterion.
Additionally, two-tailed binomial tests were performed to determine if an animal showed a significant preference for one of the two stimuli.An individual was only considered as displaying a statistically significant preference if the criterion of 66.7 % was reached and if the individual consumed more from the preferred solution in ≥ 8 out of the 10 trials (binomial test, p < 0.05).Taste preference thresholds were therefore defined as the lowest concentration at which an animal met both criteria mentioned above.

Taste preference thresholds
The taste preference threshold of the four white-faced sakis was 1-10 mM for citric acid, 0.5-20 mM for ascorbic acid, 2-10 mM for malic acid, 0.1-1 mM for tannic acid and 2-20 mM for acetic acid (Fig. 1).Malic acid yielded the lowest interindividual variability with a dilution factor of 5 between the most-and least-responsive animal.With citric acid, tannic acid and acetic acid the sakis' responsiveness varied by a dilution factor of 10 between the most-and least-responsive animal, whereas ascorbic acid yielded the highest interindividual variability with a dilution factor of 40 between the most-and least-responsive animal.

Sour-taste tolerance
When given the choice between a reference solution of 50 mM sucrose and mixtures containing varying concentrations of sucrose plus citric acid, all four sakis required only 100 mM of sucrose when mixed with 10 mM citric acid to significantly prefer the sweet-sour mixture over the purely sweet 50 mM sucrose reference solution (Fig. 2).Similarly, the sakis required only 200 mM of sucrose when mixed with 30 or 50 mM citric acid, respectively, to significantly prefer the sweet-sour mixture over the purely sweet 50 mM sucrose reference solution (Fig. 2).

Discussion
The results of the present study demonstrate that white-faced sakis have a well-developed taste sensitivity for food-associated acids which is not inferior to that of primates specializing on ripe fruits and which is clearly sufficient to perceive the acid concentrations present in the fruits they feed on in the wild.Further, the results show that the sakis displayed a higher sour-taste tolerance compared to other platyrrhine primates.

Taste preference thresholds
The two-bottle preference test of short duration employed here provides a conservative approximation of a species' taste sensitivity [57].It also allows for a direct measurement of relative preferences for or aversions to taste substances.The taste preference threshold values of the white-faced sakis for the five food-associated acids tested here fall into the range of those reported in other primates, suggesting that their gustatory system is as sensitive to sour tastants as that of primates specializing on ripe fruits or on other diets (Table 1).This is in contrast to our first hypothesis that white-faced sakis, due to the high proportion of unripe fruit in their diet, should be less sensitive to food-associated acids compared to ripe-fruit specialists such as spider monkeys (Ateles geoffroyi) and chimpanzees (Pan troglodytes), for example, or compared to non-frugivorous primates such as insectivorous squirrel monkeys (Saimiri sciureus), or gummivorous pygmy marmosets (Cebuella pygmaea), or omnivorous grey mouse lemurs (Microebus murinus).
Three hypothetical explanations, which are not mutually exclusive, may account for this finding: Specializing on ripe fruits is considered to be the evolutionarily ancestral state among frugivorous primates whereas specializing on unripe fruits is thought to be the derived state [1].This would imply that white-faced sakis may have kept the sour-taste sensitivity of their ancestors who probably specialized on ripe fruits.This, in turn, may explain why the white-faced sakis displayed a similar taste sensitivity to food-associated acids as ripe-fruit specialists, despite the high proportion of unripe and presumably sour-tasting fruit in their diet.
Other authors suggest that sour-taste perception in primates might be linked to the detection of vitamin C (= ascorbic acid) in food items [15].Unlike most other mammals, primates are unable to synthesize vitamin C [7], an ability that was lost in the least common ancestor of monkeys and apes after the divergence from strepsirrhines about 61-74 million years ago, as a result of pseudogenization of the GLO gene (L-gulono-γ-lactone oxidase).Relaxed purifying selection pressure on this gene, possibly due to a fruit-rich and thus vitamin C-rich diet, has been proposed as the underlying mechanism for this event [26].In this context it is interesting to note that herbivorous non-primate mammals such as guinea pigs (Cavia porcellus), goats (Capra hircus), horses (Equus caballus), sheep (Ovis aries), mule deer (Odocoileus hemionus), bank voles (Clethrionomys glareolus), and the common degu (Octodon degus) which are all able to synthesize their requirements of vitamin C tend to have higher taste preference thresholds and thus a lower sensitivity to food-associated acids compared to primates (Table 1).
Yet other authors proposed that the ability to detect acidic tastants at sufficiently low concentrations should be fundamental to all primates, irrespective of their dietary specialization as consumption of either too high concentrations or of too large amounts of food-associated acids may be detrimental to their health and thus to their inclusive fitness by causing damage to e.g.their teeth or their gut microbiome [15].
The taste preference threshold values obtained in the present study show that white-faced sakis are able to detect food-associated acids at concentrations that are well below those commonly present in wild fruits consumed by neotropical primates [5,53].Citric acid concentrations in wild fruits, for example, have been reported to range between 0.4 mM and 140 mM, with the majority of fruits containing more than 10 mM of citric acid which equals the taste threshold value of the least-responsive white-faced saki of the present study [14,34].Considering that fruits usually contain more than one type of organic acid which all contribute to their overall acidity it is more than likely that the taste sensation "sour" plays a prominent role in the gustatory evaluation of fruits by white-faced sakis.
Tannic acid is special among the food-associated acids tested here as, in addition to evoking the taste sensation "sour", it also evokes an astringent mouthfeel which has been described as a dry, puckering sensation on all mucosae of the oral cavity and tongue caused by its property of precipitating salivary proteins [10].Tannic acid serves as a defense mechanism which deters animals from consuming a fruit as it binds proteins and amino acids, thus preventing their absorption [9,45].Although tannic acid concentrations are usually higher in leaves than in fruits [13], they have also been reported to be higher in unripe fruits compared to ripe ones (Del Bubba et al., 2019).The tannic acid concentrations in unripe fruits that have been reported so far are generally higher than the taste preference thresholds for this polyphenol found in the present study (Del Bubba et al., 2019).Therefore, it is also more than likely that white-faced sakis experience the astringent mouthfeel elicited by tannic acid when evaluating fruits.This notion is supported by our observation that the animals of the present study puckered their mouth and smacked their lips when sampling the highest concentrations of tannic acid.

Sour-taste tolerance
Our finding that the white-faced sakis required only 100 mM of sucrose when mixed with 10 mM citric acid, and only 200 mM of sucrose when mixed with 30 or 50 mM citric acid, respectively, to significantly prefer these sweet-sour mixtures over a purely sweet 50 mM sucrose reference solution demonstrates a high degree of sour-taste tolerance.This is in line with our second hypothesis that, due to the high proportion of unripe fruit in their diet, they should be more tolerant to sour Octodon degus 80 [8] Threshold values of the present study are given in bold typeface.Human data represent taste detection threshold values rather than taste preference threshold values and are given in italics.
taste compared to ripe-fruit specialists or non-frugivorous primates (Table 2).Indeed, spider monkeys which are known to specialize on ripe fruits [19] needed a four times higher concentration of sucrose mixed with 50 mM of citric acid to prefer this sweet-sour mixture over a purely sweet 50 mM sucrose reference solution than the white-faced sakis of the present study.Pigtail macaques (Macaca nemestrina) and olive baboons (Papio anubis), similar to white-faced sakis, have also been reported to include a considerable proportion of unripe fruits into their diet [6,25] which fits to the finding that both of these catarrhine primates display a high sour-taste tolerance which either matches or even surpasses that of the white-faced sakis (Table 2).Also in line with the notion that dietary specialization may affect sour-taste tolerance is our finding that squirrel monkeys which are known to include only a relatively low proportion of fruit in their diet [20] are markedly less sour-taste tolerant compared to more frugivorous primates (Table 2).
Having a high tolerance for acidic tastants and thus for sour and unripe fruits may provide an advantage over sympatric frugivores by reducing competition for ripe fruits [42].Further, it allows for consumption of fruits across longer periods of time as the temporal availability of unripe fruits has been reported to be longer than that of ripe fruits of tropical plants [37].This, in turn, enables species who accept the levels of acidic tastants in unripe fruits to gain the nutritional value of these fruits as well as to avoid seasonal bottlenecks in food availability that affect ripe-fruit specialists [21].However, while having an extended period of temporal availability, the pulp of unripe fruits has a markedly lower nutritional value compared to that of ripe fruits [44].Therefore, unripe fruit specialists must accept the trade-off between such fruit being less nutritionally valuable, but available for a longer period of time [58].White-faced sakis may overcome this trade-off by exploiting the seeds present in unripe fruits which are often higher in nutritional value than the fruit itself, mainly due to their high content of lipids and proteins [33].Furthermore, because of the relative rarity of species specializing on unripe rather than on ripe fruit, white-faced sakis may benefit from reduced foraging costs as they are able to consume fruits and their seeds at stages of maturity that are not palatable for other primates [42].
In conclusion, the results of the present study suggest that whitefaced sakis display a high tolerance rather than a low sensitivity towards food-associated acids which may reflect an evolutionary adaptation to their dietary specialization on sour-stasting unripe fruits.

Table 2
Concentrations of sucrose (mM) needed in mixtures with citric acid to induce a significant preference over a sweet-tasting reference solution of 50 mM sucrose.

Fig. 1 .
Fig. 1.Taste responses of four white-faced sakis to various concentrations of citric acid, ascorbic acid, malic acid, tannic acid, and acetic acid, respectively, dissolved in a 50 mM sucrose solution.Each data point corresponds to the mean value calculated from the 10 trials performed per individual and stimulus combination.The preference criterion (66.7 %), avoidance criterion (33.3 %), and chance criterion (50 %) are represented by the line with longer dashes, the line with shorter dashes, and the dotted line, respectively.Circle: Engelbrekt; Square: Kariakou; Triangle: Lisha; Diamond: Anita.

Fig. 2 .
Fig. 2. Taste responses of four white-faced sakis given a choice between a 50 mM sucrose solution and mixtures of 10 mM, 30 mM, and 50 mM citric acid, respectively, plus various concentrations (10-400 mM) of sucrose.Each data point corresponds to the mean value calculated from the 10 trials performed per individual and stimulus combination.The preference criterion (66.7 %) and chance criterion (50 %) are represented by the dashed line and the dotted line, respectively.Circle: Engelbrekt; Square: Kariakou; Triangle: Lisha; Diamond: Anita.

Table 1
Taste preference thresholds for food-associated acids in primates and nonprimate mammals (mM).