| Blunt snout bream(Megalobrama amblycephala), commonly known as Wuchang fish, belongs to the Megalobrama, Cyprinidae, Cypriniformes, Actinopterygii. It is the economically most important herbivorous fish species in China. So far, little is known about the potential mechnism of food digestion and absorption in fish species, especially the herbivorous fish. Why these herbivorous fish eat kinds of grass but could grow fast and how these fish degrade cellulose and obtain enough energy to maintain life activities? To explore these questions, a typical herbivorous endemic fish species, M. amblycephala, was selected. In this study, we sequenced the whole genome of M. amblycephala, obtained protein coding genes, SNP, non-coding RNA and provided novel insights into the adaptation to herbivorous diet. In addition, we investigated endogenous digestive genes(cellulase, amylases and proteases) and olfactory and taste receptor genes that are associated with food digestion and food choice in three different feeding habits fish species. To better understand the role of ribonuclease(RNase1) in herbivorous M. amblycephala, we characterized its expression patterns and biological functions. Moreover, the composition and diversity of the gut microbial communities in herbivorous M. amblycephala and other feeding habits fish were determined by using bacterial 16 S rRNA sequencing. Collectively, we integrate the new genome draft with meta-genome data and with genomic data from other fish not only shedding light on the evolution and adaptation of M. amblycephala to herbivorous diet, but also providing valuable resources for the genetic improvement and nutrition research of this and other species. 1. Study on the whole genome sequencing of M. amblycephala and adaption to herbivorous dietWe assembled a 1.116 Gb reference genome sequence of M. amblycephala from 142.55 Gb(approximately 130-fold coverage) of clean data. The M. amblycephala genome has an average GC content of 37.3%, similar to cyprinid Cyprinus carpio(37.0%) and Danio rerio(36.7%). The contig and scaffold N50 lengths reached 49 Kb and 839 Kb, respectively. We annotated a total of 23,696 protein-coding genes. Of the predicted genes, 99% are supported by transcript data and/or by the existence of homologs in other species. In addition, we identified 1,408 non-coding RNAs including 474 miRNAs, 110 rRNA, 530 tRNAs, and 294 sn RNAs. Transposable elements(TE) comprise approximately 34%(381.3 Mb) of the M. amblycephala genome. DNA transposons(23.80%) and long terminal repeat retrotransposons(LTRs)(9.89%) are the most abundant TEs in M. amblycephala and the proportion of LTRs in M. amblycephala is highest in comparison with other teleosts. A phylogenetic analysis showed that M. amblycephala is closest with Ctenopharyngodon idellus, approximately 13.1 million years ago. To illuminate the evolutionary process resulting in the adaptation to grass diet, we analyzed the functional properties of expanded gene families in the M. amblycephala and C. idellus lineage. We find that over-represented KEGG pathways are olfactory transduction, immune-related pathways, lipid metabolic related process, as well as xenobiotics biodegradation and metabolism. Moreover, 20 genes encoding enzymes involved in lipid and carbohydrate metabolism were found to be under positive selection in both fish species. 2. Comparative studies of endogenous digestive system in different feeding habits fish speciesTo explore the genetic adaptation to herbivorous diet, we investigated genes that might be associated with digestion. No genes encoding potentially cellulose-degrading enzymes including endoglucanase, exoglucanase and beta-glucosidase were identified in the genome of M. amblycephala, suggesting that cellulose-degrading may largely depend on their gut microbiome. Genes that encode proteases(including pepsin, trypsin, cathepsin and chymotrypsin) and amylases(including alpha-amylase and glucoamylase) were identified in the genomes of M. amblycephala, carnivorous and omnivorous fish, indicating that herbivorous M. amblycephala has a protease repertoire that is not substantially different from those of the carnivorous and omnivorous. In the M. amblycephala genome, we identified 179 functional olfactory receptor(OR) genes, which is larger than those of other teleosts. Intriguingly, we found a massive expansion of beta-type OR genes in the genomes of the herbivorous M. amblycephala and C. idellus, while very few exist in other teleosts. The gene copy number and the evolution of taste receptor genes such as umami, sweetness and bitterness are dramatically different in fish with differernt feeding habits. The umami T1R1 gene has been lost in herbivorous M. amblycephala, suggesting loss of expressing a functional umami taste receptor, but the sweetness T1R2 gene and bitterness T2 Rs were duplicated. On the contrary, in carnivorous Gadus morhua and Cynoglossus semilaevis, the umami T1R1 gene is expanded but the sweetness T1R2 and bitterness T2 Rs genes have been lost. 3. Functional novelty of ribonuclease 1 in herbivorous M. amblycephalaRibonuclease(RNase1) is an important digestive enzyme that has been used to study the physiological functions and adaptive evolution to the herbivorous diet in some herbivores. Here, we identified the RNase1 genes from the genomes of M. amblycephala, C. idellus, C. carpio, D. rerio and Oryzias latipes. All of them have only one copy in their genome. The five identified fish RNase1 genes all have the signature motifs of the RNase A superfamily: CKXXNTF, the catalytic histidine–lysine–histidine triad and six conserved cysteines. Gene expression analysis showed that no expression of Ma-RNase1 was detected in early developmental stages but a weak expression was detected at 120 and 144 hours post-fertilization(hpf). Ma-RNase1 was only expressed in the liver and heart of one year old fish but strongly expressed in the liver, spleen, gut and kidney of two years old fish, suggesting the functional differentiation during fish growing. A biological functional analysis of the recombinant protein demonstrated that M. amblycephala RNase1 had a relatively strong ribonuclease activity at its optimal pH 6.1, which is consistent with the pH of its intestinal microenvironment. In addition, RNase1 protein also exhibited potent antimicrobial activity against the Gram-negative and Gram-positive bacteria. 4. Comparative studies of exogenous digestive system in different feeding habits fish speciesTo explore the diversities and complexities of gut communities in herbivorous M amblycephala and other feeding habits fish species and assess the potential roles of the gut microbiota in food digestion, we utilized a meta-analysis of 16 S rRNA gene sequence for comparison. A total of 985,356 quality-filtered sequences obtained from the 24 samples, resulted in identification of a total of 7,349 OTUs with ≥ 97% sequence similarity of 53 bacterial phyla. PCoA test showed that gut bacterial communities of carnivorous and herbivorous fishes formed distinctly different clusters in PCoA space. Although fish in different feeding habits fish species shared a large size of OTUs comprising a core microbiota community and dominanted by Proteobacteria, Firmicutes, Fusobacteria, Acidobacteria with some difference in the abundance, at the genus level a strong distinction existed. Cellulose-degrading bacteria Clostridium, Citrobacter and Leptotrichia were dominant in the herbivorous M. amblycephala and C. idellus(more than 7%), while only 2% was found in the carnivorous and dominanted by Cetobacterium and protease-producing bacteria Halomonas. The omnivorous and filter-feeding fishes were enriched with Clostridium, Cetobacterium and Halomonas. Moreover, cellulase and amylase activities in herbivorous fishes were significantly higher than in the carnivorous, while trypsin activity in the carnivorous was much higher than in the herbivorous. These results indicate a strong influence of the fish feeding habits on the structure and diversity of gut microbiota and most importantly the relative high abundance of cellulose-degrading bacteria might play a significant role in food degesion in the herbivorous fish. |
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