Expansion of sweet taste receptor genes in grass carp (Ctenopharyngodon idellus) coincided with vegetarian adaptation

Abstract

Background: Taste is fundamental to diet selection in vertebrates. Genetic basis of sweet taste receptor in the shaping of food habits has been extensively studied in mammals and birds, but scarcely studied in fishes. Grass carp is an excellent model for studying vegetarian adaptation, as it exhibits food habit transition from carnivory to herbivory.

Results: We identified six sweet taste receptors (gcT1R2A-F) in grass carp. The four gcT1R2s (gcT1R2C-F) have been suggested to be evolved from and paralogous to the two original gcT1R2s (gcT1R2A and gcT1R2B). All gcT1R2s were expressed in taste organs and mediated glucose-, fructose- or arginine-induced intracellular calcium signaling, revealing they were functional. In addition, grass carp was performed to prefer fructose to glucose under a behavioral experiment. Parallelly, compared with gcT1R2A-F/gcT1R3 co-transfected cells, gcT1R2C-F/gcT1R3 co-transfected cells showed a higher response to plant-specific fructose. Moreover, food habit transition from carnivory to herbivory in grass carp was accompanied by increased gene expression of certain gcT1R2s.

Conclusions: We suggested that the gene expansion of T1R2s in grass carp was an adaptive strategy to accommodate the change in food environment. Moreover, the selected gene expression of gcT1R2s might drive the food habit transition from carnivory to herbivory in grass carp. This study provided some evolutional and physiological clues for the formation of herbivory in grass carp.

Keywords: Adaptive evolution; Food habit transition; Gene expansion; Herbivory; Taste receptor.

Fig. 1

The gene structures of T1R2 genes in grass carp (a) and zebrafish (b). The black lines indicate introns, and the black boxes indicate exons. The six gcT1R2 genes we obtained contained 6 exons and 5 introns as well as the genomic structure of two zebrafish T1R2 genes

Fig. 2

Synteny analysis of T1R2 genes. The synteny analysis performed by searching gene(s) flanking T1R2 in genomes of zebrafish, medaka and fugu using map viewer of NCBI. GenBank accession numbers of the adjacent genes of zebrafish T1R2.1 (NM_001039831.1) and T1R2.2 (NM_001083856.1) in the figure were as follows: sult1st3, NM_183348.2; gpr153, XM_009304261.1; acot7, NM001004617.1; hes2, NM_001045353.1; espn, NM_001123282.1; arhgef16, NM_001123283.1; ybx1, NM_001126457.1; ppih, NM_001009902.2; prdm16; XM_005167301.2; tprg1l, XM_001922766.5; wrap73, NM_199893.1; tp73, NM_183340.1; rer1, XM_005167299.2; aak1a, XM_005167316.2; ddx51, NM_001003864.1; rad 21 L1, NM_001080050.1; fkbp1aa, NM199945.1. GenBank accession numbers of the adjacent genes of medaka tas1r2a (NM_001104858.1), tas1r2b (NM_001104723.1) and tas1r2c (NM_001104724.1) in the figure were as follows: prdm16, XM_011476903.1; tprg1l, XM_004070640.2; wrap73, XM_004070359.2; tp73, XM_004070358.2; rer1, XM_011476898.1; gpr153, XM_011476896.1; acot7, XM_011476894.1; hes2, XM_004070354.2; espn, XM_011476893.1; arhgef16, XM_011476892.1; ybx1, NM_001104673.1; ppih, XM_004070353.2. GenBank accession numbers of the adjacent genes of fugu tas1r2a (NM_001105217.1) and tas1r2b (NM_001105218.1) in the figure were as follows: prdm16, XM_003963257.1; tprg1l, XM_003963136.1; wrap73, XM_003963258.1; tp73, XM_003963138.1; rer1, XM_003963139.1; gpr153, XM_003963260.1; acot7, XM_003963140.1; hes2, XM_003963261.1; espn, XM_003963263.1; arhgef16, XM_003963264.1; ybx1, XM_003963141.1; ppih, XM_003963144.1

Fig. 3

Molecular phylogenetic analysis by maximum likelihood method of T1R2. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. The tree with the highest log likelihood (− 39,600.8248) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 2.8034)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 1.3917% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 73 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 503 positions in the final dataset. Evolutionary analysis was conducted in MEGA7. The T1R2s amino acid sequences of fishes and mammals used are given in the electronic Additional file 3: Table S2

Fig. 4

A timetree inferred using the reltime method and the general time reversible model of fish T1R2 genes. The timetree was computed using 4 calibration constraints. The estimated log likelihood value is − 21,587.6247. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.6461)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 9.5713% sites). The analysis involved 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1353 positions in the final dataset. Evolutionary analysis was conducted in MEGA7. The T1R2s nucleotide sequences of fishes used are given in the electronic Additional file 4: Table S3

Fig. 5

Tissue distributions of gcT1R2s. Relative mRNA expression was quantified using real-time PCR and normalized against EF1 as a housekeeping gene. All values represent the mean ± S.E.M. (n = 6). Values marked with different lowercase letters are significantly different (one-way ANOVA, P < 0.05)

Fig. 6

Ca²⁺ changes in fluorescence intensity of 20 single HEK293T cells upon taste substances. HEK293T cells were stimulated with 200 mM glucose (a), 200 mM fructose (b), and 100 mM arginine (c). Images were recorded at 6.54 s intervals up to 183.16 s using 488 nm excitation filter and 516 nm emission filter and analyzed using FV10-ASW 3.1 Viewer software. The backgrounds of the emission intensities were subtracted. Data are expressed as the ratio of the fluorescence intensities of 20 single HEK293T cells per dish and initial intensity (F/F0)

Fig. 7

The behavioral experiment of perceiving the sugar in grass carp. a Schematic drawing of experimental setup; b The ratio of fish chose to different experimental feed placement areas at 0.5 h after the transparent net opening (%). The behavioral experiment was repeated for five times. All values represent the mean ± S.E.M. (n = 5). Values marked with different lowercase letters are significantly different (one-way ANOVA, P < 0.05)

Fig. 8

The gene expressions of gcT1R2s in the tongue of grass carp transition from carnivory to herbivory. Relative mRNA expression was quantified using real-time PCR and normalized against EF1 as a housekeeping gene. All values represent the mean ± S.E.M. (n = 6). Values marked with different lowercase letters are significantly different (one-way ANOVA, P < 0.05)

Fig. 9

The gene expressions of gcT1R2s in the gut of grass carp transition from carnivory to herbivory. Relative mRNA expression was quantified using real-time PCR and normalized against EF1 as a housekeeping gene. All values represent the mean ± S.E.M. (n = 6). Values marked with different lowercase letters are significantly different (one-way ANOVA, P < 0.05)