In rearing conditions, mandarin fish accept only live prey fish, refusing dead prey fish or artificial diets . Although pervious research showed the methods of mandarin fish domestication , little is known about the mechanism of food habits domestication. In the present study, we domesticated the mandarin fish to feed on artificial diets, and found a part of mandarin fish could accept artificial diets easily (Group X), but another part could not accept completely (Group W). To uncover the molecular mechanism why mandarin fish refuses artificial diets, we conducted the transcriptome sequencing and metabolome analysis. The results showed that the differentially expressed gene between the two groups were enriched in metabolism, in which the global and overview maps and lipid metabolism were the most enriched. And the rich factors of steroid biosynthesis and glycerolipid metabolism were the highest. The metabolome results showed that the pathways with different metabolites were mostly enriched in the metabolic pathways, which were consistent with transcriptome sequencing results. Previous research has shown that the most important pathways related to the domestication of dead prey fish in mandarin fish included the retinal photosensitivity, circadian rhythm, appetite control, learning and memory pathway . Our results showed that metabolism, especially lipid metabolism, might contribute to the domestication of artificial diets, which was different from the domestication of dead prey fish, as the different constituents between dead prey fish and artificial diets.
We then analyzed the pathways in which the differential genes or metabolites were involved, the common pathways which showed the most enriched differential genes and metabolites, were retinol metabolism, glycerolipid metabolism and biosynthesis of unsaturated fatty acids. For retinol metabolism, retinol, 9-cis-retinol and 11-cis-retinol metabolites were higher in the Group X, consistently the RDH (retinoldehydrogenase) gene expression was higher in the Group X, suggesting a better visual acuity in the mandarin fish which could be easy to accept artificial diets. Animals make food choices based on the physiological, nutritional, environmental, and sociocultural factors , sensory system is of significance to food choices. It is critical for mandarin fish to catch prey fish though the perception of shape and motion with well-developed scotopic vision . Salmo spp. shows the same motion and shape of food and the offered food pellet can be captured immediately before it falls down to the bottom of the tank because they have high visual acuity, thus can feed swiftly by darting . But mandarin fish has low visual acuity and feed only by stalking, it can not recognize the prey before the time when food pellet fall to the bottom of tank, thus makes it difficult to feed mandarin fish with artificial diets . The retinol metabolism dysfunction might be contributed to the lower visual ability in the mandarin fish which refused artificial diets.
In glycerolipid metabolism pathway, the gene expression of triacylglycerol lipase was higher in the mandarin fish of Group X, and the glycerophosphoric acid metabolite was also higher in the Group X. In the biosynthesis of unsaturated fatty acids pathway, stearoyl-CoA gene expression and docosapentaenoic acid (DPA) metabolite were higher in the Group X. These results suggested that mandarin fish which could accept artificial diets well, might be attributed to the better capacity of glycerolipid metabolism and unsaturated fatty acids biosynthesis. Live food diets (such as zooplankton) and dry formulated diets have different fat levels and influences in European grayling . Artificial diets might have more fat and energy than live prey fish, suggesting that mandarin fish which accept artificial diets could make good use of fat, while the fish which refuse artificial diets could not.
To elucidate the regulatory mechanism of up-regulated gene expression in the mandarin fish of Group X, we analyzed the differentially expressed genes based on transcriptome sequencing. The results showed TFIIF gene expression was significantly increased in the Group X. TFIIF communicates with a number of factors to regulate gene transcription. It has been reported that TFIIF directly binds to basal factors of TFIID, TFIIE and TFIIB . TFIIF has been shown to be necessary for most, if not all, preinitiation complex formation and gene transcription . It suggested that in the mandarin fish which accepted artificial diets, the up-regulated transcription of genes, involved in retinol metabolism, glycerolipid metabolism and biosynthesis of unsaturated fatty acids, might be contributed to the increased TFIIF expression.
To uncover why TFIIF was upregulated in the mandarin fish of Group X, the expressions of histone methyltransferases were analyzed based on transcriptome. The expression of histone-lysine N-methyltransferase ezh1 was significantly decreased in the Group X. Histone methyltransferases EZH1 and EZH2 catalyze the tri-methylation of H3K27, which serves as an epigenetic signal for chromatin condensation and transcriptional repression . In mice, Ezh1 was required for neonatal heart regeneration after myocardial infarction and overexpression of Ezh1 promoted heart regeneration by upregulating cardiac muscle growth genes . Furthermore, we observed the protein level of tri-methylation of histone H3 at lysine 27 was lower in the Group X, suggesting an active function of gene expression. The decreased abundance of histone H3K27me3 was also found in FOXO1 (forkhead box protein O1) in HFD (high fat diets) fed rats, which persisted even after 8 weeks of diet reversal . In addition, the total DNA methylation level of TFIIF was significantly higher in the mandarin fish of Group X than those of Group W. The mRNA level of TFIIF was higher in fish of Group X, our results showed a positive effect of DNA methylation on gene expression. In soybean, the genome-wide methylation profiles showed that hyper-methylated genes had higher gene expression, in ventricular septal defect patients, genome-wide DNA methylation data showed 12 hypermethylated genes had a higher gene expression. In Arabidopsis, upon loss of CpG methylation, there was target-speciﬁc enrichment of H3K27me3 in heterochromatin that correlated with transcriptional reactivation, it is suggested that there was an antagonistic effect between CpG methylation and H3K27me3 . It is hypothesized that the lower ezh1 expression in the mandarin fish of Group X, could be contributed to the decreased methylation at 'Lys-27' of histone H3, and then up-regulating the expression and methylation of TFIIF gene.