The Bitter Taste Receptor Genes of the Raccoon Dog (Nyctereutes procyonoides)

Shuai Shang1,2, Honghai Zhang2,*, Xiaoyang Wu2, Qinguo Wei2, Jun Chen1, Huanxin Zhang1, Huaming Zhong2 and Xuexi Tang1,*

1College of Marine Life, Ocean University of China, Qingdao, Shandong, China

2College of Life Science, Qufu Normal University, Qufu, Shandong, China

ABSTRACT

Mammals have five basic tastes, including sweet, bitter, umami, sour and salty. Among these tastes, the bitter sense is believed to help animals identify poisonous compounds. Bitter taste receptors coded by bitter taste receptor (Tas2r) genes has been identified in many species, but the evolution and repertoire of Tas2r genes in raccoon dog (Nyctereutes procyonoides) was still unknown. As a true omnivorous mammal, the Tas2r genes may play a critical role in food selection of raccoon dogs. Thus, we explored the evolution of Tas2r genes in raccoon dogs. In this study, Tas2r genes including eleven intact genes and four pseudogenes from raccoon dogs were identified for the first time. We also obtained Tas2r genes from fourteen additional species based on their genome sequences and our previous study, including thirteen carnivorous species and one omnivorous species. Then, we constructed a phylogenetic tree using Tas2r genes sequences from these fifteen species. Phylogenetic analyses showed that most Tas2r genes of raccoon dogs were closed to other canid-species, indicating that the Tas2r genes were very conservative. Besides, we also detected positive selection in 11 intact genes in raccoon dogs. The results of the positive selection analyses indicated that Tas2r10 and Tas2r67 had positively selected sites and other intact genes in raccoon dogs were under purifying selection. Overall, the phylogenetic and evolutionary relationship of Tas2r genes in raccoon dogs was first studied in this study. Most of the raccoon dog’ Tas2r genes were under purifying selection, indicating that its true omnivorous diet was an important role in the Tas2r evolution. Our study provided valuable data of Tas2r genes in raccoon dogs, and provided novel insights into conservation concern of natural populations.

Article Information

Received 25 January 2017

Revised 13 June 2017

Accepted 21 November 2017

Available online 01 June 2018

Authors’ Contribution

HHZ and XXT designed the experiments. SS, XYW and JC performed the experiments. SS, HMZ and QGW analyzed the data. HXZ, HHZ and XXT contributed material. SS wrote the paper.

Key words

Raccoon dog, Tas2r genes, Phylogenetic analysis, Positive selection, Diet.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.4.1361.1366

* Corresponding authors: [email protected]; [email protected]

0030-9923/2018/0004-1361 $ 9.00/0

Introduction

Mammals have five basic tastes, including sweet, bitter, umami, sour and salty (Li and Zhang, 2014). Bitter taste receptors which were coded by bitter taste receptor (Tas2r) genes play an important role in identifying the toxins and choosing food in mammals (Chandrashekar et al., 2000). The Tas2r gene repertoires of vertebrates diverge tremendously in size, from 10 pseudogenes in the dolphin (Tursiops truncatus) (Lei et al., 2015) to 80 in the Latimeria chalumnae (Syed and Korsching, 2014). The relationship between the number of Tas2r genes and diets in 54 vertebrate species suggested that dietary toxins were major selective force for shaping the diversity of Tas2r repertoire (Li and Zhang, 2014). By analyzing the evolutionary trajectories of Tas2r genes in the 41 Laurasiatherian species, Liu found that insectivores in Laurasiatherian tended to have more functional Tas2r genes in comparison to carnivoresand omnivores (Liu et al., 2016). However, no positive correlation were detected between diet composition and Tas2r genes in carnivores (Shang et al., 2017).

Although Tas2r genes have been identified in many species (He et al., 2017), including seven canids, the evolution and repertoire of Tas2r genes in raccoon dog (Nyctereutes procyonoides) were still unknown. As a nocturnal canid, the raccoon dog originally came from East Asia (Ward and Wurster-Hill, 1990). Raccoon dogs have spread over eastern, central and north Europe since its introduction from Asia (Kowalczyk and Bunevich, 2009). Previous studies mainly focused on the diet, habitat use, seasonal physiology, hair and reproduction of the raccoon dog (Kauhala et al., 1993; Hirasawa et al., 2006; Matsuyama et al., 2006; Kowalczyk and Bunevich, 2009). As a true omnivore (Hirasawa et al., 2006), the raccoon dog may face poisonous compounds in food selection, but the research of Tas2r genes in the raccoon dog was obscure. In the present study, we first identified and explored the evolution of Tas2r genes in raccoon dogs.

Materials and Methods

Samples, primers and DNA extraction

The sample was obtained from Dalai Lake. Genomic DNA of the raccoon dog was isolated from the muscle tissue using QIAGEN DNeasy kits. The primers were designed by Primer premier 5.0 (Lalitha, 2000), which can be found in Supplementary Material S1. PCR condition was as follow: 95°C 30s, 52-58°C 40s, 72°C 45s, and 72°C elongation for 10 min. Then polymerase chain reaction (PCR) productions were sequenced in both directions. The sequences of the newly generated were submitted in GenBank under the accession number (KT426765-KT426779), and additional Tas2r gene sequences used in this study was present in Supplementary Material S2.

Ethical approvals

This research was approved by the Qufu Normal University Institutional Animal Care and Use Committee with the permit number of QFNU2015-004. This study has no harm for the canid species. Meanwhile, it may help us protect these animals better.

Sequencing data and phylogenetic analysis

The DNA sequences of intact genes were translated into protein sequences by the MEGA 6 (Tamura et al., 2013). Then the protein sequences (Supplementary Material S2) were used for multiple sequence alignment by Multiple Sequence Comparison by Log-Expectation (MUSCLE), free available online at http://www.ebi.ac.uk/Tools/msa/muscle (version 3.8.31) (Edgar, 2004). The software was also used for analyzing the percent identity matrix. The aligned protein data were used for phylogenetic analysis using the software MEGA 6. The neighbor-joining statistical method of analysis and the bootstrap consensus tree was inferred from 1000 replicates (Felsenstein, 1985; Saitou and Nei, 1987). And the zebra fish V1R gene was used as outgroup. The best fitting model was calculated by ProtTest 3.4 (Abascal, 2005).

Selective pressure on Tas2r genes

In order to detect the selective pressure on the intact bitter taste receptor genes in raccoon dogs, we used the site model in the codeml program in PAML (Yang, 2007). The ω ratios of nonsynonymous to synonymous substitution (dN/dS) being less than 1, equal to 1 and more than 1 indicate purifying selection, neutral evolution and positive selection, respectively. Twice the log likelihood differences (2Δl) were detected in the Chi-square test. The LRT suggest positive selection if there is a significant difference between the two models. Then we use three distinct different models, including random effect likelihood (REL), fixed-effect likelihood (FEL) and single likelihood ancestor counting (SLAC) to analyze the potential selective pressure of 11 intact genes (Kosakovsky-Pond and Frost, 2005).

Results

Tas2r gene amplification and sequencing

The dog Tas2r genes were used to design the primers of the raccoon dogs. The PCR products of Tas2r genes were obtained from the raccoon dogs, including 11 genes (at least 270 codons, start codon, stop codon and seven transmembrane domains) and 4 pseudogenes (Fig. 1). The similarity of the Tas2r protein sequence between dog and raccoon dog ranged from 97.6% to 99.25%, thus the newly Tas2r genes of the raccoon dog were reliable (Supplementary Material S3). Meanwhile, we also used Tas2r genes from 14 additional species (Fig. 1). The results showed that the intact and pseudogenes were different among 15 species. For example, several Tas2r intact genes in raccoon dog were pseudogenized in other species.

Phylogenetic analysis

In the present study, we carried out a phylogenetic analysis based on the intact Tas2r genes from 15 mammals. Intact Tas2r genes which were homologous to 11 intact Tas2r genes from the raccoon dog were used in the analysis. The pseudogenes and other intact genes were not used in the phylogenetic analysis. Total 145 Tas2r genes and one zebra fish V1r gene were used in the phylogenetic analysis. The result showed that most Tas2r genes of raccoon dog were closed to other canid species (except for Tas2r40 and Tas2r39) (Fig. 2). And Tas2r genes from the Feliodea species tended to cluster together. Tas2r genes of canid species were near to the carnivorous species and far from the omnivorous species.

Table I.- Positive selection of 11 intact Tas2r genes.

Gene	Species	Lnl M7	Lnl M8	P-values	M8	SLAC	FEL	REL
Tas2r2	8	-1402.88	-1401.38	0.22	31	0	0	31
Tas2r5	8	-1459.87	-1457.99	0.15	26 73 76	0	0	0
Tas2r7	9	-1425.02	-1423.17	0.15	15 26 30 31 180 213 308	0	0	0
Tas2r10	8	-1494.8	-1490.98	0.02	6 7 9 40 164 200 318			7 9
Tas2r12	9	-1523.52	-1523.19	0.72	171 262	0	0	0
Tas2r38	9	-1486.22	-1483.36	0.057	46 65 105 299 300 312	0	0	0
Tas2r39	9	-1535.46	-1534.66	0.45	149 205	0	0	0
Tas2r40	9	-1524.8	-1523.44	0.26	84 112 229	0	0	0
Tas2r41	9	-1508.97	-1508.97	0.99	0	0	0	0
Tas2r42	8	-1548.74	-1546.37	0.093	39 63 82 216 279 291 310 315 319 320 321 322	0	0	315 321
Tas2r67	9	-1573.53	-1565.84	0.00045	86 308 310 312	0	0	86 308 312

Positively selected sites with posterior probability >0.95 are in bold and < 0.95 in plain text.

Selective pressure of Tas2r genes

To explore the evolutionary forces on the Tas2r genes, we conducted an analysis of site models by using the Tas2r2, Tas2r5, Tas2r7, Tas2r10, Tas2r12, Tas2r38, Tas2r39, Tas2r40, Tas2r41, Tas2r42 and Tas2r67 sequences as input data. The likelihood values, estimated parameters, and predicted positively selected sites were listed in Table I. The LRT was significantly different in Tas2r10 and Tas2r67, indicating that the positive selection was reliable in these genes. The results showed that two and three common positively selected sites were identified in Tas2r10 and Tas2r67, respectively (Table I).

The two common positively selected sites (codon 7and 9) in Tas2r10 were located on the transmembrane region (Fig. 3). And the common positively selected sites in Tas2r67 were located on the transmembrane region (codon 86) and an intracellular domain (codon 308 and 312) (Fig. 3).

Discussion

The Tas2r genes sequences were identified for the first time in the raccoon dog. The protein sequence identity between dog and raccoon dog ranged from 97.6% to 99.25% (Supplementary Material S3), suggesting that the dog gene-based primer sets can be used for the raccoon dog. Previous researches showed that the carnivorous species obtained 22 or more Tas2r genes (Hu and Shi, 2013; Li and Zhang, 2014; Liu et al., 2016; Shang et al., 2017), while in our research, 11 intact genes and 4 pseudogenes were obtained in the raccoon dog. The incomplete repertoire of Tas2r genes in the raccoon dog may be due to the high similarity but containing gaps in the template sequence at the annealing point of the primer (Monteiro-Ferreira et al., 2015). Several Tas2r intact genes in raccoon dog were pseudogenized in other species, for example, the Tas2r67 and Tas2r2 were not existed in human, but intact in raccoon dogs and other canid species; the Tas2r39, Tas2r40 and Tas2r41 was pseudogenized in ferret, but was intact in canid species. The pseudogenes and intact genes were different species, indicating that the Tas2r genes were species-specific.

The phylogenetic tree showed that most Tas2r genes of the raccoon dog were clustered together with canid species. We speculated that Tas2r genes were very conservative among species. Thus they could be used as a molecular marker to study the evolutionary relationships among animals (Shi and Zhang, 2006; Hu and Shi, 2013). The phylogenetic results also showed that 11 intact genes from raccoon dogs were intermingled across the tree which indicated that gene duplication events were not existent, which was similar to other carnivorous species (Hu and Shi, 2013). Due to lack of genome sequences in raccoon dog, the incomplete repertoire of Tas2r genes may not reflect the real events. More comparative data are needed to critically test it.

Previous study showed that the repertoire and evolution of Tas2r genes were shaped by many factors, including diet and feeding behaviors (Hong and Zhao, 2014; Li and Zhang, 2014; Liu et al., 2016; Shang et al., 2017; Zhong et al., 2017). Our results showed that the raccoon dog had a small number of Tas2r genes, which might suggest Tas2r genes playing a less important role. But minimal number of functional Tas2r genes did not imply a reduced importance of bitter taste, as it could be compensated by large tuning width (Behrens et al., 2014).

In human, the Tas2r10 was reported to identify 20 natural bitter compounds (Meyerhof et al., 2010). The results of positive selection of Tas2r genes in the raccoon dogs showed that two intact genes had positively selected sites, the Tas2r10 and Tas2r67. Meanwhile, except the Tas2r10 and Tas2r67, the remaining intact genes in raccoon dogs had no positively selected sites, suggesting that most genes were under purifying selection. Raccoon dogs are omnivores that feed on meat (insects, rodents birds, fish, carrion and reptiles) and plants (fruits, nuts and berries) (Sasaki and Kawabata, 1994; Sutor et al., 2010). They faced the potential poisonous compounds, including the bile acids, venom, and skin secretion from carrion, reptiles, insects and toxic plants. We speculated that Tas2r genes might be functionally important during their evolution due to its true omnivorous diet (Ward and Wurster-Hill, 1990). These taste capabilities could protect raccoon dogs from harmful substances and thus be helpful for their survival.

Conclusions

Overall, the phylogenetic and evolutionary relationship of Tas2r genes in raccoon dogs was first studied in this study. Most of the raccoon dog’s Tas2r genes were under purifying selection, indicating that its true omnivorous diet was an important role in the Tas2r evolution. Our study provided valuable data of Tas2r genes in raccoon dogs, and provided novel insights into conservation concern of natural populations.

Acknowledgments

This work was supported by the Special Fund for Forest Scientific Research in the Public Welfare (No. 201404420) and the National Natural Science Fund of China (No. 31372220, 31672313).

Supplementary material

There is supplementary material associated with this article. Access the material online at: http://dx.doi.org/10.17582/journal.pjz/2018.50.4.1361.1366

Statement of conflict of interest

Authors have declared no conflict of interest.

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