Behavioral study in the gray mouse lemur (Microcebus murinus) using compounds considered sweet by humans.код для вставкиСкачать
American Journal of Primatology 62:43–48 (2004) BRIEF REPORT Behavioral Study in the Gray Mouse Lemur (Microcebus murinus) Using Compounds Considered Sweet by Humans ALAIN SCHILLING1, VICKTORIA DANILOVA2, and GORAN HELLEKANT2n 1 Département d’Écologie et Gestion de la Biodiversite´, CNRS-MNHN UMR, Brunoy, France 2 Department of Animal Health and Biomedical Sciences, University of Wisconsin– Madison, Madison, Wisconsin This study presents the results from two-bottle preference (TBP) tests performed on the gray mouse lemur (Microcebus murinus), a small Malagasy primate. We found that of 18 compounds considered sweet by humans, M. murinus preferred only six: D-tryptophan, dulcin, fructose, sucrose, SC45647, and xylitol. The animals neither preferred nor rejected acesulfame-K, alitame, aspartame, N-4-cyanophenyl-N0 -cyanoguanidineacetate (CCGA), cyanosuosan, cyclamate, monellin, saccharin, suosan, super-aspartame, N-trifluoroacetyl-L-glutamyl-4-aminophenylcarbonitrile (TGC), and thaumatin. Together with previously recorded tastenerve responses in M. murinus to acesulfame-K, alitame, aspartame, cyclamate, monellin, saccharin, and suosan [Hellekant et al., Chem Senses 18:307–320, 1993b], the current results suggest that these compounds either do not taste sweet to M. murinus or they have an aversive taste component. In this work we also relate these findings to phylogeny. Am. J. Primatol. 62:43–48, 2004. r 2004 Wiley-Liss, Inc. Key words: sweet taste; mouse lemur; two-bottle preference; taste coding; taste qualities; receptors INTRODUCTION Although many studies have demonstrated anatomical or morphological differences among primates that can be related to phylogeny, few investigators have systematically explored the influence of phylogenetic factors in taste. One reason for this may be that species differences were not evident because ‘‘taste primaries,’’ such as NaCl, sucrose, and quinine, were used as the taste stimuli. However, with the introduction of new compounds, such as the potent sweetener aspartame and the extremely bitter compound denatonium benzoate, n Correspondence to: Goran Hellekant, Department of Animal Health and Biomedical Sciences, University of Wisconsin–Madison, 1656 Linden Dr., Madison, WI 53706. E-mail: email@example.com Received 7 August 2003; revision accepted 8 October 2003 DOI 10.1002/ajp.20004 Published online in Wiley InterScience (www.interscience.wiley.com). r 2004 Wiley-Liss, Inc. 44 / Schilling et al. phylogenetic factors became important. For example, of some 30 compounds distinctly sweet taste to humans, more than one-third do not taste sweet to the South American primate Callithrix jacchus jacchus, as judged by its behavior and recordings from its taste nerves [Danilova et al., 1998b, 2002]. The nocturnal Malagasy primate (Microcebus murinus) is phylogenetically more distant from humans than C. jacchus. Consequently, its sense of taste probably differs more from that of humans compared to C. jacchus. M. murinus belongs to the Lemuridae family, which is part of the prosimii infraorder and is one of the smallest extant primates [Nowak, 1991]. As a result of about 54 million years of isolation on Madagascar, it has developed some remarkable features. One is a seasonal change of body weight of up to 100% [Petter-Rousseaux, 1980]. These weight changes have been shown to correlate with variations of ‘‘preference thresholds’’ for sucrose [Simmen and Hladik, 1988]. This correlation led us to investigate the relationship between weight changes and taste in two groups of M. murinus (heavy and light). We found no relationship between seasonal weight and taste-nerve response to sucrose [Hellekant et al., 1993a]. However, in a second study we recorded the taste-nerve responses to a number of other compounds considered sweet by humans and found that some produced no taste response, while others, such as acesulfame-K, dulcin, saccharin, stevioside, suosan, and xylitol elicited strong responses [Hellekant et al., 1993b]. We sought to determine how the above sweeteners taste to M. murinus. One way to assess this is with behavioral observations. In the current study we used another approachFthe two-bottle preference (TBP) test. MATERIALS AND METHODS Five female gray mouse lemurs (M. murinus) were used in this study. The animals were born in the laboratory breeding colony of Brunoy (MNHN, France; European Institutions Agreement 962773) and raised from birth under controlled conditions of temperature (24–261), relative humidity (55%), and an artificial photoperiodic regimen of 5 months of long days/short nights and 3 months of short days/long nights [Perret, 1992]. The experiments were carried out during the long-days/short-nights phase. Under such a regimen, increased physiological and behavioral activity is associated with a seasonal activation of reproductive function in this highly photoperiod-dependent species. The animals were housed individually and fed ad libitum. The animals had uninterrupted access to water before and after the tests. During the TBP tests, each animal was offered 10 ml of water and 10 ml of one of the compounds listed in Table I. Their concentrations (see Table I) were chosen to be equisweet to 0.3 M sucrose in terms of human taste. The intake of water and the compound in question was recorded during two consecutive TBP test sessions of 10-hr night periods, and the position of the bottles was switched between measurements. The preference ratio was calculated for each animal and session as the amount of sweetener consumed, divided by the total amount of liquid consumed. The mean preference ratios were then calculated for each animal and the results were averaged for five animals. Thus equal consumption of both water and sweetener yields a preference ratio of 0.5; complete preference yields a preference ratio of 1. The results were first assessed by analysis of variance (ANOVA). Then a twotailed t-test was used to compare mean preference ratios to 0.5. A P-value o0.05 was considered significant. Sweet Taste in Mouse Lemur / 45 TABLE I. List of Compounds Used in TBP Experiments With M. murinus Compounds Acesulfame-K Alitame Aspartame CCGA (N-4-cyanophrnyl-N’-cyanoguanidineacetate) Cyanosuosan Cyclamate D-tryptophan Dulcin Fructose Monellin Saccharin SC-45647 Sucrose Suosan Super-aspartame TGC (N-trifluoroacetyl-L-glutamyl-4-aminophenylcarbonitrile) Thaumatin Xylitol Concentration 7.3 mM 0.21mM 3.6 mM 0.21 mM 2.5 mM 33.5 mM 19.5 mM 1.67 mM 0.45 M 3 uM 2 mM 0.04 mM 0.3 M 2.17 mM 0.11 mM 0.17 mM 1.5 uM 820 mM Potencies of sweeteners in human compared with sucrose were presented in Danilova et al. . RESULTS Figure 1 presents the average intake of water and each compound, and Fig. 2 shows the average preference ratios for each sweetener. In the figures, the compounds are arranged in order of decreasing preference ratio, and fall into two groups. The first group of sweeteners consisted of D-tryptophan, dulcin, fructose, sucrose, SC45647, and xylitol. The consumption of these sweeteners was significantly higher than the consumption of water. The second group (acesulfame-K, alitame, aspartame, N-4-cyanophenyl-N0 cyanoguanidineacetate (CCGA), cyanosuosan, cyclamate, monellin, saccharin, suosan, super-aspartame, N-trifluoroacetyl-L-glutamyl-4-aminophenylcarbonitrile (TGC), and thaumatin) was neither rejected nor preferred over water. The preference ratios for these compounds did not significantly differ from 0.5. DISCUSSION Although M. murinus has a ‘‘sweet tooth’’ and likes many solutions considered sweet by humans [Hellekant et al., 1993a; Simmen and Hladik, 1988], the results described above show that only one-third of 18 compounds that taste distinctly sweet to humans elicited a significant preference in the TBP tests. As mentioned above, we used concentrations equisweet to humans. It is possible, and perhaps likely, that some of the compounds in the second group might have induced significant behavioral effects at higher concentrations. However, because the major emphasis of this study was to compare the sense of taste in M. murinus with that in humans, we used only concentrations that clearly tasted sweet to humans. Furthermore, when these concentrations were applied to Old World monkeys, they elicited taste responses. This indicates that the results from the compounds in the second group reflected phylogenetic differences, and were unrelated to the concentrations of the compounds. 46 / Schilling et al. 10 * * 8 water * sweetener * * 6 * 4 2 0 Fig. 1. Consumption of sweeteners and water in TBP tests. Data were averaged for five animals. Stimuli concentrations are listed in Table I. Error bars are SE. Asterisks indicate significant difference between sweetener and water consumption (Po0.05). 1 0.9 0.8 0.7 * * * * * * 0.6 0.5 0.4 0.3 0.2 0.1 0 Fig. 2. Preference ratios from TBP tests. Data were averaged for five animals. Stimuli concentrations are listed in Table I. Error bars are SE. Asterisks indicate significant difference from preference ratio 0.5 (P o0.05). Sweet Taste in Mouse Lemur / 47 In the following, we relate these results with those from earlier studies in M. murinus and other mammals. We then discuss the results in the context of the recent identification of a sweet taste receptor family (T1Rs). With regard to the preferred compounds, we know from our earlier electrophysiological work [Hellekant et al., 1993a, b] that xylitol, sucrose, and dulcin elicit CT responses in M. murinus. Consequently, they have a taste to M. murinus. Although we have no electrophysiological data for M. murinus regarding fructose, D-tryptophan, or SC45647, it is likely that these compounds also would have evoked a taste-nerve response, because they elicit taste-nerve responses in all other species studied, from rodents to primates. Of the 12 nonpreferred compounds, acesulfame-K, alitame, aspartame, cyclamate, monellin, saccharin, and suosan elicited a CT response in our earlier study of M. murinus. Since they produced a taste-nerve response at the same concentrations as used here, lack of taste cannot explain the behavioral results. Therefore, the taste sensation they evoked was either not attractive or was a combination of an aversive taste with an attractive one. Of the five remaining nonpreferred compounds, super-aspartame and thaumatin elicited no CT response. Although it is possible that they could stimulate some other taste nerves, this indicates that they do not taste to M. murinus. Since we lacked electrophysiological data for CCGA, cyanosuosan, and TGC, we were unable to draw any conclusions regarding those compounds. Of the preferred compounds, the mouse lemur shares a preference for xylitol, fructose, sucrose, dulcin, D-tryptophan, and SC45647 with nonprimates, such as hamsters [Danilova, 1998a] and mice [Bachmanov et al., 2001; Inoue et al., 2001; Ninomiya et al., 1992], and higher primates, such as chimpanzees, rhesus monkeys, and marmosets [Danilova et al., 1998a; Hellekant et al., 1997a,b; Hobi and Glaser, 1983; Nofre et al., 1996; Plata-Salaman et al., 1992, 1993]. Furthermore, some of the compounds that were not preferred by M. murinus are preferred by higher primates. For example, marmosets, rhesus monkeys, and chimpanzees, all of which are more closely related to humans than M. murinus, prefer alitame, super-aspartame, acesulfame-K, and cyanosuosan–compounds that taste pleasantly sweet to humans. This points to phylogenetic differences in the sense of taste, not only between primates and nonprimates, but also among primates. This conclusion is supported by a recent finding that the members of a recently identified sweet receptor family, T1R, vary among primates. Li et al.  found eight sequence variant sites in T1R3, and 19 in T1R2 that were different between primates tasting and not tasting aspartame. This may also explain observed differences in the ability of New and Old World primates to taste other compounds, such as thaumatin or monellin [Brouwer et al., 1973; Hellekant, 1976; Glaser et al., 1978]. The genetical differences in T1Rs between species show that the presence or absence of a taste-nerve response or behavioral response is not determined by dietary factors. It is true that in the case of a compound that has a taste, dietary factors may lead to shifts in intake or preference, but diet does not abolish or produce taste-nerve response to a compound. The only exceptions to this are the compounds known as taste modifiers; however, their effects do not last long. 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