Molecular Cloning and Characterization of npy Gene and Its Response to Starvation-Refeeding Strategy in Seriola aureovittata

Yellowtail kingfish (Seriola aureovittata), a pelagic marine finfish species with a worldwide distribution, is regarded as an emerging candidate for the aquaculture industry owing to its fast growth, superior flesh quality, and farming suitability in both sea cages and land-based facilities in China. The species has high economic value and is the second most produced Seriola species in the world following Japanese yellowtail Seriola quinqueradiata. Researchers worldwide have studied the role of regulatory factors neuropeptide Y (NPY) in fish feeding regulation. In recent years, there has been great progress in research on food intake in fish, however, very little attention has been paid to the endocrine regulation mechanism of food intake. Methods on strengthening the production performance of fish through appetite regulation is still a hot research topic.The control of food intake and energy metabolism in vertebrates are complex processes involving several neural pathways. Some hypothalamic signals are released by peripheral tissues that are associated with energy homeostasis or nutrient availability. Among the signaling molecules involved, NPY plays a key role. NPY is recognized as one of the most effective appetite regulators, which primarily function as a signaling factor to regulate a variety of biological processes such as food intake and glucose homeostasis. The orexigenic actions of NPY have been well investigated thoroughly over the past decades. Much evidence supports that NPY´s functional role as a regulator of energy homeostasis and appetite control is conserved across vertebrates, including teleosts. In several species, including rainbow trout, Nile tilapia, and grass carp, NPY injections increase food intake, supporting an orexigenic role. In line with this, food deprivation increased npy mRNA expression in the brain, such as seen for goldfish and Johnny carp. Moreover, refeeding normalized npy mRNA abundance following food deprivation. As S. aureovittata feeds heavily and fiercely, the breeding industries need to understand its feeding control mechanism. To make real-time adjustment to feeding strategy, it is necessary to obtain high-quality and high-yield aquatic products with the least input to maximize economic benefits. As a potent appetite stimulating factor, npy has been proven to promote feeding, but this gene has not been identified in S. aureovittata. Therefore, it is necessary to explore the variable rules of the npy gene in feeding and starvation compensation mechanism, to provide the special compound feed for breeding.To gain insight into the existence of npy in S. aureovittata, we used homologous cloning, RNA extraction and reverse-transcription to obtain the ORF sequence of npy. npy belongs to the pancreatic polypeptide (PP) family, which plays an important role in appetite regulation and energy expenditure in mammals and fish. The ORF of S. aureovittata npy is 300 nucleotides in size and encodes a 99-amino-acid precusor, with a calculated molecular mass and isoelectric point of 11.24 kDa and 5.02, respectively. The precursor protein is composed of a predicted signal peptide of 28-aa in size, 36-aa putative mature peptide, a GKR protein proteolytic site, and a 32-aa C terminus of unknown function. Bioinformatics analysis on the amino acid sequence identities and evolutionary relationships of the npy was performed. Comparison of homology of the precursor peptide sequences of npy analysis revealed that S. aureovittata npy displayed a high degree of identity with the counterparts of Seriola dumerili (99.0%), Morone saxatilis (98.0%), Micropterus salmoides (96.0%), and Scophthalmus maximus (94.9%), followed by Cynoglossus semilaevis (93.9%) and Oryzias latipes (92.9%). Phylogenetic analysis highly supported that the npy of S. aureovittata was closely related to that of S. dumerili. Furthermore, using real-time quantitative PCR, we found that the npy mRNA is widely expressed in 12 tissues, with abundant expression in the brain, followed by the pituitary and stomach. In addition, except for the intestine and gonad, npy was found to have no significant difference in all other detected tissues of both sexes. To establish the functional link between npy and feeding, the expression profiles of npy mRNA during food deprivation and refeeding were examined in S. aureovittata. We detected the 7 d, 14 d, and 21 d starvation and 7 d refeeding effect on npy mRNA levels. Results showed that fasting induced an increase of npy mRNA levels in brain, pituitary, and stomach when compared to the control groups. Interestingly, the pituitary npy transcripts significantly increased after 21 d of starvation compared with the control group. In addition, refeeding normalized npy mRNA abundance following food deprivation in the brain, pituitary, and stomach. These results indicated that npy is involved in the regulation of feeding and energy homeostasis in S. aureovittata. Collectively, we provided initial evidence for the existence of npy in S. aureovittata and suggested its involvement in the regulation of feeding, which plays an important role in the starvation compensation mechanism.In summary, we obtained the ORF sequence of npy and clarified its role as a potent orexigenic peptide in feeding regulation of S. aureovittata, which would be beneficial for specific feed for this species.