Is the Nutritional Value of Fish Fillet Related to Fish Maturation or Fish Age? Integrated Analysis of Transcriptomics and Metabolomics in Blunt Snout Bream (Megalobrama amblycephala).

BACKGROUND/AIMS: Fish is a protein-rich food and is increasingly favored by consumers. It has been well recognized that the flesh composition of fish is closely related to its maturation and growth stage, but few studies have explored these differences. Additionally, hormone residues in fish after artificial induction of reproduction also attract consumer concern. In this study, we attempt to address these concerns by using a combination of transcriptomics and metabolomics analyses to identify key regulated pathways, genes, and metabolites that may affect the flesh nutrition of one typical aquaculture species in China, blunt snout bream (Megalobrama amblycephala). METHODS: The four groups of fish were used for transcriptomics and metabolomics analyses, including one-year-old immature (group I), two-year-old immature (group II), two-year-old mature (group III) and successfully spawned (group IV) female M. amblycephala after artificial induction of reproduction. RESULTS: We identified a total of 1460 differential compounds and 1107 differentially expressed unigenes in muscle among the different groups. Differential metabolites related to fish age (group II vs group I, group III vs group I) were largely enriched in "Glycerophospholipid metabolism", "Linoleic acid metabolism", "α-Linolenic acid metabolism", and "Biosynthesis of unsaturated fatty acids". Between these two pairwise comparisons, metabolites that are beneficial to human health, such as docosapentaenoic acid, α-Linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were found to be significantly decreased in two-year-old (group II, group III) compared with one-year-old (group I) M. amblycephala. Only one differential metabolite related to fish maturation, a triglyceride, was detected between groups III and II. Transcriptomics data showed that differently expressed genes (between group III vs group II, group III vs group I) related to maturation were highly enriched in "Cell adhesion molecules (CAMs)", "Sphingolipid metabolism" and "Phagosome". DEGs (between group II vs group I, group III vs group I) relating to fish age were enriched in the "cGMP-PKG signaling pathway", "FoxO signaling pathway", and "AMPK signaling pathway". The gene-metabolite interaction network showed pivotal genes, including fumarate hydratase and GNPAT, which played a major role in the regulation of glycerphospholipid metabolism. The nutritional components were also measured, which verified the metabolomics results. Moreover, the metabolomics results showed that after 24 hours of artificial hormone injection, the drug was completely metabolized. CONCLUSION: Integrated analysis demonstrated that the nutrition value of fish fillet was much more related to fish age compared with maturation status in M. amblycephala females.

Evolution of Fish Let-7 MicroRNAs and Their Expression Correlated to Growth Development in Blunt Snout Bream.

The lethal-7 (let-7) miRNA, known as one of the first founding miRNAs, is present in multiple copies in a genome and has diverse functions in animals. In this study, comparative genomic analysis of let-7 miRNAs members in fish species indicated that let-7 miRNA is a sequence conserved family in fish, while different species have the variable gene copy numbers. Among the ten members including let-7a/b/c/d/e/f/g/h/i/j, the let-7a precursor sequence was more similar to ancestral sequences, whereas other let-7 miRNA members were separate from the late differentiation of let-7a. The mostly predicted target genes of let-7 miRNAs are involved in biological process, especially developmental process and growth through Gene Ontology (GO) enrichment analysis. In order to identify the possible different functions of these ten miRNAs in fish growth development, their expression levels were quantified in adult males and females of Megalobrama amblycephala, as well as in 3-, 6-, and 12-months-old individuals with relatively slow- and fast-growth rates. These ten miRNAs had similar tissue expression patterns between males and females, with higher expression levels in the brain and pituitary than that in other tissues (p < 0.05). Among these miRNAs, the relative expression level of let-7a was the highest among almost all the tested tissues, followed by let-7b, let-7d and let-7c/e/f/g/h/i/j. As to the groups with different growth rates, the expression levels of let-7 miRNAs in pituitary and brain from the slow-growth group were always significantly higher than that in the fast-growth group (p < 0.05). These results suggest that let-7 miRNA members could play an important role in the regulation of growth development in M. amblycephala through negatively regulating expression of their target genes.

Screening of Biomarkers Related to Ovarian Maturation and Spawning in Blunt Snout Bream (Megalobrama amblycephala) Based on Metabolomics and Transcriptomics.

In fish breeding practices, gamete maturity of females is vital to reproductive success. For some species, it is possible to estimate the female maturation status based on abdomen observation, but quite difficult for some species which mature at big size. To screen out the potential biomarker in fish blood relating to female maturation, we employed the approach integrating the UPLC-MS/MS and RNA-seq techniques to investigate the metabolites and genes reflecting the sexual maturation and spawning of female blunt snout bream Megalobrama amblycephala. The study included four groups, 1-year-old immature female individuals, 2-year-old immature female individuals, 2-year-old sexually mature female individuals, and 2-year-old sexually mature female individuals after 24 h of successful spawning. The upregulated metabolites in mature females were involved in "steroid hormone biosynthesis," "metabolic pathways," "glycerophospholipid metabolism," etc. compared with those of immature individuals. As the key intermediate of steroid hormone biosynthesis, 17α-hydroxypregnenolone exhibited the highest level in 2-year-old mature females than in the immature females. Meanwhile, the metabolites (i.e., dodecanoic acid and myristic acid) participating in fatty acid synthesis exhibited much lower levels in the females after spawning than those before spawning. In addition to the metabolites, the genes involved in ovarian steroidogenesis were significantly upregulated in the 2-year-old immature females compared to the 1-year-old immature females, indicating that the ovarian steroidogenesis plays important roles in ovarian development of M. amblycephala at the early stages. The significant upregulation of genes (i.e., itpr1, camk2, and mekk2) involved in the "GnRH signaling pathway" was observed in the mature females compared with the immature females, which indicated that the estrogen levels increased after female maturation in M. amblycephala. Moreover, many genes (e.g., gck, creb1, tf2-9, ryr2, asgr1, and creb1) regulating insulin secretion and thyroid hormone synthesis were significantly downregulated after female spawning. The dynamics of gene expression and metabolites observed in this study provide novel cues for guiding fish practical artificial reproduction.