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Primates

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Functional decline of sweet taste sensitivity of colobine monkeys

  • Emiko Nishi
  • Nami Suzuki-Hashido
  • Takashi Hayakawa
  • Yamato Tsuji
  • Bambang Suryobroto
  • Hiroo Imai
Original Article

Abstract

For many primates, sweet taste is palatable and is an indicator that the food contains carbohydrates, such as sugars and starches, as energy sources. However, we have found that Asian colobine monkeys (lutungs and langurs) have low sensitivity to various natural sugars. Sweet tastes are recognized when compounds bind to the sweet taste receptor TAS1R2/TAS1R3 in the oral cavity; accordingly, we conducted a functional assay using a heterologous expression system to evaluate the responses of Javan lutung (Trachypithecus auratus) TAS1R2/TAS1R3 to various natural sugars. We found that Javan lutung TAS1R2/TAS1R3 did not respond to natural sugars such as sucrose and maltose. We also conducted a behavioral experiment using the silvery lutung (Trachypithecus cristatus) and Hanuman langur (Semnopithecus entellus) by measuring the consumption of sugar-flavored jellies. Consistent with the functional assay results for TAS1R2/TAS1R3, these Asian colobine monkeys showed no preference for sucrose or maltose jellies. These results demonstrate that sweet taste sensitivity to natural sugars is low in Asian colobine monkeys, and this may be related to the specific feeding habits of colobine monkeys.

Keywords

Colobine monkeys Taste sensitivity Natural sugars Sweet taste receptor TAS1R2/TAS1R3 

Introduction

For animals, the perception of sweet taste can provide important information regarding the quality and quantity of nutrients in foods. For frugivorous and omnivorous animals, including primates, sweet taste perception in response to sugars is particularly important for the detection of energy resources (Laska et al. 1999) because plants convert CO2 to starches by photosynthesis and store this energy as various types of sugars (Lemoine et al. 2013). In mammals, sweet taste compounds in the oral cavity are detected by the sweet taste receptor TAS1R2/TAS1R3, which is a heterotrimeric G-protein coupled receptor expressed on the surface of the tongue (Zhao et al. 2003). After stimuli, natural sugars and artificial sweeteners bind to TAS1R2/TAS1R3, the signal is transmitted to the brain, and animals recognize a sweet taste (Roper and Chaudhari 2017). The amino acid sequence of TAS1R2/TAS1R3 exhibits divergence among animal taxa; these replacements might cause functional and behavioral differences (Li et al. 2011).

Cercopithecidae are divided into two subfamilies, Cercopithecinae and Colobinae. Colobine monkeys are called leaf-eating monkeys; leaves represent half of their feeding repertoire (Tsuji et al. 2013). Previous studies have shown that Javan lutungs (Trachypithecus auratus), proboscis monkeys (Nasalis larvatus), and red leaf monkeys (Presbytis rubicunda) prefer plant species with a high protein-to-fiber ratio in leaves (Kool 1992; Matsuda et al. 2013). For Javan lutungs, when protein levels in the foliage are sufficiently high, digestibility may be an important factor in leaf selection and digestibility is determined by the amount of non-structural carbohydrates, such as starches (Kool 1992).

In a previous study of Japanese macaques (Macaca fuscata)—another member of Cercopithecidae (Old World monkeys)—we found that TAS1R2/TAS1R3 has high sensitivity to maltose and sucrose, while human TAS1R2/TAS1R3 reacts strongly to sucrose but weakly to maltose (Nishi et al. 2016). They also have high salivary alpha-amylase activity (Ohya et al. 1986; Janiak 2016), which hydrolyzes starch to maltose. Japanese macaques consume many starchy plant parts, such as leaves and nuts, including acorns (Hill 1997; Tsuji 2010), which contain many starches (Iwamoto 1982), suggesting that Japanese macaque feeding habits are related to high maltose sensitivity.

Because Japanese macaques consume many starches and have high maltose sensitivity, it is plausible that colobine monkeys also have high maltose sensitivity to detect starches, thereby facilitating the selection of more digestible leaves. Alternatively, the colobines might have low sensitivity to natural sugars, such as maltose, since other mammals with specialized feeding habits have lost receptor sensitivity within this sensory realm, as evidenced by the pseudogenization of TAS1R1 in the panda (Jiang et al. 2014) and TAS1R2 in felids (Li et al. 2005). However, owing to a lack of behavioral or molecular experiments, the sweet taste sensitivity of colobine monkeys is not yet clear. To clarify the relationship between feeding habits and sweet taste sensitivity in colobine monkeys, we conducted assays of receptor responses to sugars (sucrose, maltose, glucose, fructose, lactose, and sorbitol) and behavioral preference tests using sucrose and maltose.

Methods

Sweet taste compounds

The natural sugars maltose, sucrose, glucose, fructose, lactose, and sorbitol (all purchased from Wako, Osaka, Japan) were used to prepare sweet taste solutions. The compounds were dissolved in an assay buffer (10 mM HEPES, 130 mM NaCl, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, pH 7.4) for in vitro functional assays. To prevent potential osmotic effects on cells, which affect intracellular calcium, a maximum concentration of 100 mM was used.

Cell culture

HEK293T cells were provided by Dr. Matsunami (Duke University) via Dr. Misaka (Tokyo University) for the functional assays. Cells were cultivated in a 5% CO2 incubator at 37 °C with low-glucose Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Japan, Tokyo, Japan) containing 10% fetal bovine serum.

Transfection of wild-type and chimeric sequences

Genomic DNA of a silvery lutung and a Hanuman langur was obtained from samples provided by the Japan Monkey Centre (Aichi, Japan). Samples consisted of a blood draw obtained during a veterinary examination and a liver sample collected during a necropsy. Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (QIAGEN GmbH, Hilden, Germany) (Table S1). In addition, genomic DNA of a Javan lutung was extracted from fecal samples collected at Pangandaran Nature Reserve (Java, Indonesia) using a QIAamp DNA Stool Mini Kit (QIAGEN GmbH). All DNA extracts were stored frozen and used as template for PCR experiments.

To identify the coding sequences of the Javan lutung (Trachypithecus auratus: Ta), silvery lutung (Trachypithecus cristatus), and Hanuman langur (Semnopithecus entellus) TAS1R2 and TAS1R3 genes, PCR was performed to amplify six exons each of TAS1R2 and TAS1R3 using Tks Gflex DNA Polymerase (Takara Bio Inc., Shiga, Japan) and specific primers designed based on the in-house next-generation shotgun sequences of Javan lutung genomic DNA (Table S1).

Amplified exons of TAS1R2 and TAS1R3 were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 3130xl (Applied Biosystems, Inc., Foster City, CA, USA). The sequences were deposited in the DNA Data-Base of Japan (DDBJ) under accession numbers LC370003 (Ta_TAS1R2) and LC370004 (Ta_TAS1R3). Multiple alignments of nucleotide and amino acid sequences were constructed using ClustalW (version 2.1) (Larkin et al. 2007). All exons of Ta_TAS1R2 and Ta_TAS1R3 were inserted into the mammalian expression vector pEAK10 (Edge BioSystems, Inc., Gaithersburg, MD, USA) using the In-Fusion HD Cloning Kit (Takara Bio Inc.). Japanese macaque (Mf) TAS1R2 and TAS1R3 were prepared as described previously (Nishi et al. 2016). TAS1R2 and TAS1R3 expression vectors were transfected into HEK293T cells with Gα 16-gust44 (Ueda et al. 2003) using Lipofectamine 3000 (Life Technologies, Inc., Carlsbad, CA, USA). To construct chimeric receptors, the Venus flytrap (VFD) domain (TAS1R2: 1–1482 bp, TAS1R3: 1–1479 bp) and cysteine-rich (CRD) + transmembrane (TMD) domain (Ta_TAS1R2: 1483–2520 bp, Ta_TAS1R3: 1480–2553 bp, Mf_TAS1R3: 1480–2559 bp) were amplified by PCR and inserted head-to-tail into the pEAK10 vector using the In-fusion HD Cloning Kit.

Functional assays were conducted as described previously (Nishi et al. 2016). Calcium 4 (Molecular Devices, Inc., Eugene, OR, USA) was used as an intracellular Ca2+ indicator. Fluorescence was measured at 525 nm following excitation at 485 nm using the FlexStation 3 Microplate Reader (Molecular Devices Japan, Inc., Tokyo, Japan). The calcium response amplitudes are expressed as ΔF/F, which is defined as the ratio of the ligand-dependent increase in fluorescence to the fluorescence before ligand addition. The response of cells that were transfected with the empty pEAK10 vector and Gα16gust44 was defined as the mock response (TAS1R-independent response). ΔF/F values were fitted to the Hill equation (y = ([max] − [min])/(1 + (x/EC50) × [rate])). After the Shapiro–Wilk test was used to confirm whether the ΔF/F values follow a normal distribution, comparisons across concentrations were evaluated by one-way analysis of variance (p < 0.05) or non-parametric Kruskal–Wallis tests. Dunnett’s tests or Steel’s test were used to determine the concentration at which significantly greater responses were observed compared to that at 0 mM (p < 0.05) (Table S2). Student’s t tests were used to compare the responses of chimeric receptors to 0 and 30 mM sucrose (p < 0.05) (Table S3). Fitting and statistical analyses, except Steel’s tests, were implemented in Igor Pro v7.06 (Wave Metrics, Inc., Lake Oswego, OR, USA); Steel’s tests were performed using R version 3.4.3 (R Development Core Team, Vienna, Austria).

Immunocytochemistry

TAS1R2/TAS1R3 post-transfected cells were cultivated on a 48-well plate coated with Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated for 24 h after transfection and washed with phosphate-buffered saline (Wako). Cells were washed with phosphate-buffered saline after every subsequent step. We fixed cells with 4% paraformaldehyde (Wako) for 15 min. After they were permeabilized by 0.5% Triton X-100 for 5 min, cells were incubated with 5% skim milk (Wako) for 30 min. The anti-human TAS1R2 antibody and anti-human TAS1R3 antibody (SC-50305 and SC-22458; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), diluted in skim milk (1:100), were added to cells as primary antibodies. Cells were incubated for 24 h at 4 °C. Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Thermo Fisher Scientific) and Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific), diluted with skim milk (1:500), were added as secondary antibodies. Cells were incubated for 1 h at room temperature in the dark to detect primary antibodies. Finally, cells were washed and observed by fluorescence microscopy (BZ-X710; Keyence Corporation, Osaka, Japan).

Behavioral experiment

Sugar-flavored and non-sugar-flavored jellies were prepared as follows. Non-sugar-flavored jellies were made of deionized water with the addition of 2.0% (w/w) agar (Wako). Sugar-flavored jellies were also made using 300 mM sucrose or maltose solution instead of water. The behavioral experiment was performed in two silvery lutungs (15-and 16-year-old male, born in the Japan Monkey Centre) and one Hanuman langur (19-year-old male, reared in the Japan Monkey Centre since 2015) at the Japan Monkey Centre and two Japanese macaques (7- and 9-year-old males, born at the Primate Research Institute, Kyoto University) at the Primate Research Institute, Kyoto University. Colobine monkeys may freely move between the indoor and outdoor enclosures and had social contact with other individuals. Japanese macaques were kept in individual cages in the same room. Access to food and water was not restricted.

To measure the sweet taste preference of monkeys, a food-choice test was conducted. In this test, one of the baskets contained non-sugar-flavored jellies and another contained sugar-flavored jellies (15 pieces each). In a trial, monkeys were exposed to two baskets at the same time and jelly intake was recorded using a video camera. This experiment was conducted twice a day, before feeding. The position of baskets was randomly changed in each experiment. Ten trials each were performed for sucrose-flavored and maltose-flavored jellies. The intake index of sugar-flavored jellies was calculated as follows: [intake index of sugar-flavored jellies] = [intake of sugar-flavored jellies]/15. Because the monkeys almost always consumed all of the jellies, the first 15 intakes were calculated and preference was evaluated. The experiment was completed when the monkeys consumed all of the jellies or stopped eating for 10 min. The average intake index of each sugar-flavored jelly was calculated for each monkey (n = 10). The average intake index of each sugar-flavored jelly was compared with 50%, the level expected by chance, using Student’s t tests (p < 0.05) (Table S3).

Results

First, we sequenced the coding region of the Javan lutung TAS1R2 and TAS1R3 genes. Amino acid differences between the TAS1R2 and TAS1R3 sequences of the Javan lutung and Japanese macaque are summarized in Tables 1 and 2. Compared with Mf_TAS1R3, we identified a two-nucleotide deletion at the C-terminus of the TAS1R3 gene (nucleotide positions 2543–2544), which was previously reported in Francois’s lutung (Trachypithecus francoisi) and the purple-faced langur (Semnopithecus vetulus) (Liu et al. 2014). This deletion leads to a TAS1R3 sequence that is two amino acid residues shorter in lutungs and langurs than in other primates (Table 2). However, sequencing data did not indicate any premature stop codons, and there was no evidence of gene pseudogenization.
Table 1

Amino acid differences between Japanese macaque and Javan lutung TAS1R2

Position

VFD

Number

7

16

52

53

67

104

118

119

127

132

173

184

212

225

341

342

348

365

369

375

407

Trachypithecus auratus

A

R

N

F

M

I

D

N

D

M

N

T

S

N

Q

G

S

N

D

T

Y

Macaca fuscata

T

W

D

Y

I

V

E

D

N

V

D

A

G

D

R

D

P

S

G

N

H

Position

VFD

CRD

TMD

Number

422

423

435

436

437

456

490

501

533

547

555

616

631

635

649

686

693

697

729

733

741

Trachypithecus auratus

E

K

Q

L

F

D

V

L

Y

R

Q

L

L

V

T

M

M

I

L

S

L

Macaca fuscata

K

E

E

I

S

G

I

S

F

S

R

F

F

A

A

T

V

M

F

G

V

Table 2

Amino add differences between Japanese macaque and Javan lutung TAS1R3

Position

VFD

Number

3

44

47

52

97

133

170

195

198

224

228

232

233

234

247

249

250

258

294

341

349

Trachypithecus auratus

G

S

G

S

Y

R

S

T

V

G

T

A

H

S

H

D

G

D

S

H

V

Macaca fuscata

C

G

E

G

H

Q

G

V

A

S

A

S

R

G

R

N

S

E

R

R

A

Position

VFD

CRD

TMD

Number

383

407

411

417

443

470

480

483

530

534

539

542

543

544

553

576

577

619

632

651

787

Trachypithecus auratus

T

T

N

M

H

N

T

L

Q

D

S

N

Q

D

S

L

S

L

L

L

A

Macaca fuscata

A

A

S

V

R

D

I

P

R

G

T

S

R

E

R

F

G

I

R

F

V

Position

TMD

Number

796

846

848

849

850

851

852

Trachypithecus auratus

A

D

R

E

T

Macaca fuscata

G

G

Q

G

K

H

E

TAS1R sequences include three domains: the Venus flytrap domain VFD (TAS1R2: 1–1482 bp, TAS1R3: 1–1494 bp), CRD (TAS1R2: 1483–1694 bp, TAS1R3: 1495–1701 bp), and TMD (TAS1R2: 1695–2520 bp, Mf_TAS1R3: 1702–2559 bp, Ta_TAS1R3: 1702–2557 bp). In total, 66.7% of amino acid differences between Javan lutung and Japanese macaque TAS1R2 were located in the VFD, a large extracellular domain, 23.8% were located in the TMD, and 9.5% were located in the CRD, which is a short chain between the VFD and TMD. In TAS1R3, 59.2% of amino acid differences were located in the VFD, 26.5% were in the TMD, and 14.3% were located in the CRD.

Next, to characterize the functions of Javan lutung TAS1R2/TAS1R3, we performed an in vitro functional assay and examined the response to various natural sugars. Ta_TAS1R2/TAS1R3 did not show responses to any of the sugars (Fig. 1a–f). Mf_TAS1R2/TAS1R3 showed a dose-dependent response to sucrose (Fig. 1a), maltose (Fig. 1b), and glucose (Fig. 1c), as reported previously.
Fig. 1

Responses of Javan lutung (Ta) and Japanese macaque (Mf) TAS1R2/TAS1R3 to various natural sugars. Sucrose (a), maltose (b), glucose (c), fructose (d), sorbitol (e), and lactose (f) were added to Ta_TAS1R2/TAS1R3 (filled circle) and Mf_TAS1R2/TAS1R3 (empty circle). Values represent the mean ± SD of three independent measurements. The blank arrow indicates the Mf_TAS1R2/TAS1R3 concentration for which ΔF/F is significantly greater than that for 0 mM

It is possible that the lack of a response of Ta_TAS1R2/TAS1R3 to sugars can be explained by a lack of Ta_TAS1R2 and Ta_TAS1R3 co-expression on the cell surface. It has been reported that a lack of heterodimer formation prevents TAS1R2 and TAS1R3 from responding to sweet taste compounds (Shimizu et al. 2014). Additionally, sugars are aqueous molecules, and the binding sites for sugars in TAS1R2/TAS1R3 have to be exposed on the surface of the cell membrane. To confirm the co-expression of Ta_TAS1R2 and Ta_TAS1R3 on the cell membrane of HEK293T cells, the post-transfected cells were stained with anti-TAS1R2 or anti-TAS1R3 antibodies (Fig. 2). Under a fluorescence microscope, we detected TAS1R2 (red) and TAS1R3 (green) in cells transfected with TAS1R2/TAS1R3 of the Javan lutung and Japanese macaque, respectively. We detected the colocalization of TAS1R2-specific and TAS1R3-specific fluorescence in several cells. These results showed that Ta_TAS1R2 and Ta_TAS1R3 are co-expressed on the surface of HEK293T cells, similar to Mf_TAS1R2/TAS1R3.
Fig. 2

Immunocytochemistry using HEK293T cells transfected with TAS1R2 and TAS1R3 expression vectors. TAS1R2 and TAS1R3 expression were validated for Mf_TAS1R2/TAS1R3, Ta_TAS1R2/TAS1R3, and pEAK10 vector post-transfected cells by immunocytochemistry. Red fluorescence indicates TAS1R2; green fluorescence indicates TAS1R3

We then conducted a functional assay using chimeric TAS1R2/TAS1R3 of the Japanese macaque and Javan lutung to identify the site related to the sucrose response (Fig. 3). The putative sucrose binding site is located in the VFD of TAS1R2 (Masuda et al. 2012). We did not observe significant responses for Mf_TAS1R2/Ta_TAS1R3. We then measured the responses of various types of chimeric TAS1R2/TAS1R3. We found that MfTa_TAS1R2/Mf_TAS1R3 (p = 0.0179), MfTa_TAS1R2/Ta_TAS1R3 (p = 0.00511), and Mf_TAS1R2/MfTa_TAS1R3 (p = 0.00924) showed significant responses to 30 mM sucrose. Additionally, the TAS1R2 VFD of Mf_TAS1R2/Mf_TAS1R3 replaced with that of Javan lutungs exhibited a reduced response to sucrose. Although not all of the cells expressing the VFD of Mf_TAS1R2 responded to sucrose, these results suggest that the VFD of Ta_TAS1R2 is involved in low sensitivity to sucrose.
Fig. 3

a Comparisons of the responses of chimeric TAS1R2/TAS1R3. Responses to 0 mM (blank bar) and 30 mM (filled bar) sucrose are shown. Bottom figures are schematic diagrams of TAS1R2 and TAS1R3; circles indicate the VFD and squares indicate the CRD (small) and TMD (large). Filled circles and squares show Ta sequences and open circles and squares show Mf sequences. Values represent the mean ± SD of five independent measurements. Asterisks indicate that the response to 30 mM sucrose was significantly greater than that to 0 mM sucrose. b Protein structures for TAS1R2 and TAS1R3, including the deletion in TAS1R3

The functional assay and immunocytochemistry results showed that Ta_TAS1R2/TAS1R3 has a low sensitivity to natural sugars. This suggests that the Javan lutung has a low sensitivity to sugars. This phenotype may involve the TAS1R2 VFD. We compared the Javan lutung TAS1R2 VFD sequence to the corresponding human and Japanese macaque sequences, both of which respond to sucrose. Nine amino acid sites in the Ta_TAS1R2 VFD differed from the residues in humans and Japanese macaques. Then, we compared the sequences of the Javan lutung with those of other colobine monkeys (Table 3). Because the silvery lutung (Trachypithecus cristatus) and Hanuman langur (Semnopithecus entellus) have identical amino acids at these nine residues, we conducted behavioral experiments in these two colobine species as well as Japanese macaques to verify their sweet taste sensitivity.
Table 3

Colobine monkey-specific amino acids in the TAS1R2 VFD

 

Subfamily

Species

Amino acid position

7

67

118

119

173

348

423

436

490

Cercopithecidae

Colobinae

Javan lutung

Trachypithecus auratus

A

M

D

N

N

S

K

L

V

Silvery lutung

Trachypithecus cristatus

Francois’s lutung

Trachypithecus francoisi a

Hanuman langur

Semnopithecus entellus

Purple-faced langur

Semnopithacus vetulus a

Black snub-nosed monkey

Rhinopithecus bieti a

T

D

Gray snub-nosed monkey

Rhinopithecus brelichi a

T

D

Golden snub-nosed monkey

Rhinopithecus roxellana a

T

D

Black-shanked douc

Pygathrix nigripes a

T

D

Red-shanked douc

Pygathrix nemaeus a

T

D

Proboscis monkey

Nasalis larvatusa

T

D

Sumatran Surili

Presbytis melalophos a

T

D

King colobus

Colobus polykomos a

T

D

I

Cercopithecinae

Japanese macaque

Macaca fuscata

T

I

E

D

D

P

E

I

I

Apes

 

Human

Homo sapiens

T

I

E

D

D

P

E

I

I

aLiu et al. (2014)

We exposed animals to sugar-flavored and non-flavored jelly simultaneously and measured their intake index. As expected, Japanese macaques showed significantly higher intake indexes (86 ± 26, p = 0.0117 in Macaca fuscata 1 and 73 ± 28, p = 0.0103 in Macaca fuscata 2) of sucrose-flavored than non-flavored jelly (Fig. 4a). Japanese macaques also tended to consume more maltose-flavored (62 ± 35, p = 0.442 in Macaca fuscata 1 and 71 ± 34, p = 0.112 in Macaca fuscata 2) than non-flavored jelly (Fig. 4b). On the other hand, colobine monkeys did not show significant differences (indexes = 45–59, p > 0.280 for sucrose, 42–50, p > 0.290 for maltose) between the intake of sugar-flavored and non-flavored jelly (Fig. 4a, b). These results are consistent with the molecular results for TAS1R2/TAS1R3 in colobines.
Fig. 4

Intake indexes of sugar-flavored jellies for each monkey. Bars represent the mean intake indexes of sucrose-flavored (a) and maltose-flavored (b) jellies ± SE of ten independent trials. The dotted line indicates a 50% intake index. Asterisks indicate that the sugar-flavored jelly intake rete is significantly greater than 50%

Discussion

In this study, Javan lutung TAS1R2/TAS1R3 showed no response to natural sugars, while some chimeric TAS1R2/TAS1R3 with the VFD of Mf_TAS1R2 showed a response to 30 mM sucrose. These results suggest that the ability of the VFD of Ta_TAS1R2 to bind to sucrose is low, and therefore Ta_TAS1R2/TAS1R3 cannot respond to the sucrose solution. According to a previous study, S40, Y103, D142, P277, D278, E302, and R383 located in the VFD of TAS1R2 are related to sucrose binding (Zhang et al. 2010). Because all of these amino acids in Javan lutung TAS1R2 are identical to those of Japanese macaque TAS1R2, it is possible that other amino acids are important for sucrose binding. Relaxed selection on the ancestral TAS1R2 in colobine monkeys was reported in CODEML analysis of ten colobine species (Liu et al. 2014); evidence for positive selection at particular sites in TAS1R2 was also detected. In the present study, we obtained new TAS1R2 amino acid sequences for three colobine species. Based on an alignment of colobine TAS1R2 sequences with human and Japanese macaque TAS1R2, both of which respond to sucrose, I67M, E118D, D119N, P348S, E423K, and I436L were identified as colobine-specific substitution in the VFD (Table 3). These amino acids are different from the previously suggested positively selected sites (Liu et al. 2014). While site-specific mutations are required to elucidate whether these amino acids are involved in sucrose binding, these amino acid residues may explain the lack of a response for Ta_TAS1R2 as well as other colobine TAS1R2 loci.

The results of the behavioral experiment corresponded with the results of the functional analysis. Fossil records and dental morphology suggest that the last common ancestor of Cercopithecidae was a frugivore (Benefit 2000); therefore, cercopithecines maintain the feeding habit of a frugivore, while colobines tend to be folivores. Some mammals with specialized feeding habits and less exposure to specific tastes lose sensitivity to the particular taste, as evidenced by the pseudogenization of TAS1R1 in panda (Jiang et al. 2014) and TAS1R2 in felids (Li et al. 2005). The low preference for natural sugars and the functional decline of TAS1R2/TAS1R3 in colobine monkeys might be related to their feeding habits, in which they have minimal opportunity to consume fruits that contain many simple sugars (Kool 1993). Furthermore, Ta_TAS1R/TAS1R3 show low sensitivity to maltose, suggesting that the amount of starches is not an important factor in leaf selection by colobine monkeys. Colobine monkeys that feed on excessive carbohydrates or sugars, and not enough fiber, suffer from diarrhea and digestive disorders (Nijboer et al. 2006). Colobine monkeys have a peculiar ruminant-like stomach to digest cellulose and hemicellulose by bacterial fermentation (Lambert 1998). The consumption of too many ripe fruits might contribute to rapid over-fermentation and the overproduction of volatile fatty acids, leading to acidosis (Lambert 1998). The low sweet sensitivity in colobine monkeys might also be related to their digestive organs.

Notes

Acknowledgements

The authors would like to thank Drs. T. Ueda, T. Misaka, and H. Matsunami for providing cells and vectors, and Dr. K. A. Widayati, L. H. Purba, and S. Nila for supporting the collection of fecal samples from Javan lutungs. Our thanks are also due to T. Hoshino, T. Funahashi, and other keepers and veterinarians of the Japan Monkey Centre for taking care of the colobine monkeys, supporting behavioral experiments, and collecting genetic samples. We also thank A. Yamanaka and CHEMR of KUPRI for taking care of the Japanese macaques. This study was financed by JSPS KAKENHI (#16H01338, #15H02421, #15H05242, and #25257409 to HI; #12J04270 and #16K18630 to TH; and #12J04300 and #17K15203 to NSH), research grants from Kobayashi International Scholarship Foundation, and the JSPS bilateral research program between Japan and Indonesia.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

Colobine monkey behavioral experiments were conducted at the Japan Monkey Centre, Aichi, Japan. The study was approved by the Research Ethics Committee of the Japan Monkey Centre (#2017-014) based on the Ethical Guidelines for Research in Japan Monkey Centre (April 1st, 2016) as Collaborative Research of the Japan Monkey Centre (#2017013) The Japanese macaque experiment was conducted at the Primate Research Institute, Kyoto University. Both experiments were approved by the Animal Welfare and Animal Care Committee of Primate Research Institute, Kyoto University (#2017-178) and were in compliance with the Guidelines for Care and Use of Nonhuman Primates of the Primates Research Institute, Kyoto University (Version 3, issued in 2010). These guidelines were prepared based on the provisions of the Guidelines for Proper Conduct of Animal Experiments (June 1, 2006; Science Council of Japan), Basic Policies for the Conduct of Animal Experiments in Research Institutions under the Jurisdiction of the Ministry of Health, Labour and Welfare (effective on June 1, 2006; Ministry of Health, Labour and Welfare), Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions (Notice No. 71 of the Ministry of Education, Culture, Sports, Science and Technology dated June 1, 2006), and Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Notice No. 88 of the Ministry of the Environment dated April 28, 2006).

Supplementary material

10329_2018_679_MOESM1_ESM.pdf (90 kb)
Supplementary material 1 (PDF 89 kb)
10329_2018_679_MOESM2_ESM.pdf (80 kb)
Supplementary material 2 (PDF 79 kb)
10329_2018_679_MOESM3_ESM.pdf (44 kb)
Supplementary material 3 (PDF 43 kb)

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Copyright information

© Japan Monkey Centre and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Molecular Biology Section, Department of Cellular and Molecular Biology, Primates Research InstituteKyoto UniversityAichiJapan
  2. 2.Department of Wildlife Science (Nagoya Railroad Co., Ltd.), Primates Research InstituteKyoto UniversityAichiJapan
  3. 3.Japan Monkey CentreAichiJapan
  4. 4.Social Systems Evolution Section, Primates Research InstituteKyoto UniversityAichiJapan
  5. 5.Department of BiologyBogor Agricultural UniversityBogorIndonesia

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