Xu-Fang Liang

The draft genome of the grass carp (Ctenopharyngodon idellus) provides insights into its evolution and vegetarian adaptation

The grass carp is an important farmed fish, accounting for ∼16% of global freshwater aquaculture, and has a vegetarian diet. Here we report a 0.9-Gb draft genome of a gynogenetic female adult and a 1.07-Gb genome of a wild male adult. Genome annotation identified 27,263 protein-coding gene models in the female genome. A total of 114 scaffolds consisting of 573 Mb are anchored on 24 linkage groups. Divergence between grass carp and zebrafish is estimated to have occurred 49–54 million years ago. We identify a chromosome fusion in grass carp relative to zebrafish and report frequent crossovers between the grass carp X and Y chromosomes. We find that transcriptional activation of the mevalonate pathway and steroid biosynthesis in liver is associated with the grass carps adaptation from a carnivorous to an herbivorous diet. We believe that the grass carp genome could serve as an initial platform for breeding better-quality fish using a genomic approach.

Programming effects of high-carbohydrate feeding of larvae on adult glucose metabolism in zebrafish, Danio rerio

The aim of the present study was to determine the potential long-term metabolic effects of early nutritional programming on carbohydrate utilisation in adult zebrafish (Danio rerio). High-carbohydrate diets were fed to fish during four ontogenetic stages: from the first-feeding stage to the end of the yolk-sac larval stage; from the first-feeding stage to 2 d after yolk-sac exhaustion; after yolk-sac exhaustion for 3 or 5 d. The carbohydrate stimuli significantly increased the body weight of the first-feeding groups in the short term. The expression of genes was differentially regulated by the early dietary intervention. The high-carbohydrate diets resulted in decreased plasma glucose levels in the adult fish. The mRNA levels and enzyme activities of glucokinase, pyruvate kinase, α-amylase and sodium-dependent glucose co-transporter 1 were up-regulated in the first-feeding groups. There was no significant change in the mRNA levels of glucose-6-phosphatase (G6Pase) in any experimental group, and the activity of G6Pase enzyme in the FF-5 (first feeding to 2 d after yolk-sac exhaustion) group was significantly different from that of the other groups. The expression of phosphoenolpyruvate carboxykinase gene in all the groups was significantly decreased. In the examined early programming range, growth performance was not affected. Taken together, data reported herein indicate that the period ranging from the polyculture to the external feeding stage is an important window for potential modification of the long-term physiological functions. In conclusion, the present study demonstrates that it is possible to permanently modify carbohydrate digestion, transport and metabolism of adult zebrafish through early nutritional programming.

Transcriptome analysis of food habit transition from carnivory to herbivory in a typical vertebrate herbivore, grass carp Ctenopharyngodon idella.

BackgroundAlthough feeding behavior and food habit are ecologically and economically important properties, little is known about formation and evolution of herbivory. Grass carp (Ctenopharyngodon idella) is an ecologically appealing model of vertebrate herbivore, widely cultivated in the world as edible fish or as biological control agents for aquatic weeds. Grass carp exhibits food habit transition from carnivory to herbivory during development. However, currently little is known about the genes regulating the unique food habit transition and the formation of herbivory, and how they could achieve higher growth rates on plant materials, which have a relatively poor nutritional quality.ResultsWe showed that grass carp fed with duckweed (modeling fish after food habit transition) had significantly higher relative length of gut than fish before food habit transition or those fed with chironomid larvae (fish without transition). Using transcriptome sequencing, we identified 10,184 differentially expressed genes between grass carp before and after transition in brain, liver and gut. By eliminating genes potentially involved in development (via comparing fish with or without food habit transition), we identified changes in expression of genes involved in cell proliferation and differentiation, appetite control, circadian rhythm, and digestion and metabolism between fish before and after food habit transition. Up-regulation of GHRb, Egfr, Fgf, Fgfbp1, Insra, Irs2, Jak, STAT, PKC, PI3K expression in fish fed with duckweed, consistent with faster gut growth, could promote the food habit transition. Grass carp after food habit transition had increased appetite signal in brain. Altered expressions of Per, Cry, Clock, Bmal2, Pdp, Dec and Fbxl3 might reset circadian phase of fish after food habit transition. Expression of genes involved in digestion and metabolism were significantly different between fish before and after the transition.ConclusionsWe suggest that the food habit transition from carnivory to herbivory in grass carp might be due to enhanced gut growth, increased appetite, resetting of circadian phase and enhanced digestion and metabolism. We also found extensive alternative splicing and novel transcript accompanying food habit transition. These differences together might account for the food habit transition and the formation of herbivory in grass carp.

Single Nucleotide Polymorphisms in Growth Hormone Gene and Their Association with Growth Traits in Siniperca chuatsi (Basilewsky)

Growth hormone (GH) has been considered as a candidate gene for growth traits in fish. In this study, polymorphisms of the GH gene were evaluated for associations with growth traits in 282 Siniperca chuatsi individuals. Using directly sequencing, four single nucleotide polymorphisms (SNPs) were identified in GH gene, with two mutations in intron 4 (g.4940A>C, g.4948A>T), one mutation in exon 5 (g.5045T>C) and one in intron 5 (g.5234T>G). Notably, three of them were significantly associated with growth performance, particularly for g.4940A>C which was highly correlated with all the four growth traits. In conclusion, our results demonstrated that these SNPs in GH gene could influence growth performance of S.chuatsi and could be used for marker-assisted selection (MAS) in this species.

Gene structure and expression of leptin in Chinese perch

Leptin is an important hormone involved in regulation of food intake, energy expenditure and reproduction in mammals, but its role in acanthomorph fishes remains scant. In the present study, we characterized leptin gene structure and its tissue expression in Chinese perch (Siniperca chuatsi). In contrast to typical leptin gene organization of 3 exons and 2 introns in other vertebrates, Chinese perch leptin gene consisted of 2 exons and 1 intron. This is the first leptin gene characterized in Perciformes, and is also the first leptin gene lacking an intron reported in Perciformes. The unique gene structure, the conservation of both cysteines that form the single disulfide bridge in leptin, and stable clustering in phylogenetic analyses substantiate the unambiguous orthology of mammalian and fish leptins, despite low amino acid identity. Polymorphism of leptin gene was examined in wild and cultivated populations of Chinese perch by direct sequencing of 120 fish. No SNP was found in leptin gene. Leptin mRNA of Chinese perch was highly expressed in liver, and expressed at low levels in brain, visceral adipose tissue, intestine, spleen and muscle.

Leptin expression in mandarin fish Siniperca chuatsi (Basilewsky): Regulation by postprandial and short-term fasting treatment.

Most fish species possess duplicate leptin genes (LEP). Mandarin fish (Siniperca chuatsi) leptin A gene (sLEP-A) have been cloned in the previous study. In the present study, we cloned and characterized leptin B gene (sLEP-B) in mandarin fish, including a 471bp open reading frame (ORF) encoding a 158-amino acid protein. The three-dimensional (3D) structural model of sLEP-B protein showed a highly conserved of tertiary structure similar to that of other vertebrates. Genomic sequencing results indicated that sLEP-B possessed only one intron. This is the first report of the loss of an intron in LEP-B in Perciformes. The different distribution patterns of sLEPs suggest different physiological roles of these two genes. The presence of HNF3β, a liver-enriched transcription factor, only in sLEP-A indicated abundant expression and metabolic function of sLEP-A in the liver. In an in vivo experiment, the expressions of brain sLEP-A and sLEP-B were observed to increase after a meal. During the short-term fasting, the expressions of sLEPs in mandarin fish brain were decreased significantly. A persistent and significant increase in hepatic sLEP-A expression supported a relationship between leptin and food intake in mandarin fish. These results suggest that sLEP-A plays an important role in the regulation of energy homeostasis in this carnivorous fish, and sLEP-B is probably a specialized gene responsible for the central nervous system (CNS) control of energy regulation.

Induction of liver GST transcriptions by tert-butylhydroquinone reduced microcystin-LR accumulation in Nile tilapia (Oreochromis niloticus)

The cyanobacterial toxin, MC-LR, is predominantly presented during toxic cyanobacterial blooms and is consumed by phytoplanktivorous fish and zooplanktivorous fish directly. Detoxification of MC-LR in liver was believed to begin with conjugate formation with GSH, catalyzed by GSTs. MC-LR GSH conjugates display increased solubility and are subjected to accelerated biliary excretion. In this study, we showed that the mRNA transcriptions of GSTA, GPX and UCP2 were increased within 8h following MC-LR exposure in isolated hepatocytes of Nile tilapia, confirming the roles of phase II enzymes, especially GSTs, in MC-LR detoxification in tilapia. The widely used food-additive, synthetic antioxidant, tert-butylhydroquinone (tBHQ) has been shown to induce phase II enzymes including GSTs, via the antioxidant responsive elements (ARE) locate in the regulatory regions of these genes. Our results also showed that the transcription of various GSTs, including GSTA, GSTR2 and GSTT were significantly induced by tBHQ in Nile tilapia. In consistence, fish fed on tBHQ-containing diet (0.01 percent tBHQ) showed significantly reduced MC-LR accumulation in liver tissues 48 h after an oral administration of a single dose of 250 μg MC-LR/kg body weight (bwt). The findings in this research suggested that tBHQ could reduce MC-LR accumulations in liver, likely through the induction of phase II metabolizing enzymes such as GSTs. Subacute effects of tBHQ and its potential applications in fishery need to be further investigated.

Molecular cloning and tissue expression of uncoupling protein 1, 2 and 3 genes in Chinese perch (Siniperca chuatsi)

Uncoupling proteins (UCPs) are mitochondrial anion carrier proteins, which play important roles in several physiological processes, including thermogenesis, reactive oxygen species generation, growth, lipid metabolism and insulin secretion. Although the roles of UCPs are well understood in mammals, little is known in fish. To investigate the thermogenesis roles in Chinese perch (Siniperca chuatsi), we cloned the UCP1, 2 and 3. The UCP1 consisted of six exons and five introns, and the UCP2 consisted of eight exons and seven introns. The UCP1 was primarily expressed in liver, UCP2 was ubiquitously expressed, and UCP3 was primarily expressed in muscle. The mRNA levels of UCP1 and UCP2 in liver, and UCP3 in muscle were significantly increased after prolonged cold exposure, but did not change after prolonged heat exposure, suggesting that Chinese perch might have a mechanism of response to cold environment, but not to hot environment. The intestinal UCP1 mRNA level was significantly up-regulated after prolonged heat exposure, while the UCP2 mRNA level was significantly up-regulated after prolonged cold exposure, suggesting that the two paralogs might play different roles in intestine of Chinese perch. In addition, the phylogenetic analysis could shed new light on the evolutionary diversification of UCP gene family.

Population genetic structure of Siniperca chuatsi in the middle reach of the Yangtze River inferred from mitochondrial DNA and microsatellite loci

Abstract The Chinese mandarin fish (Siniperca chuatsi) is currently one of the most important economic freshwater fish in China, whereas the wild resource has declined dramatically in recent years. In this study, we examined the genetic structure and diversity of five populations from the middle reach of the Yangtze River using mitochondrial cytochrome b sequences and microsatellite markers. This research revealed high genetic diversity and low genetic differentiation of S. chuatsi from these regions. The pairwise Fst values of the two markers showed low and no-significant differentiation among populations. AMOVA analysis of two markers and the haplotype genealogy of the Cytb gene confirmed these results. The STRUCTURE analysis of the microsatellite marker implied that the dam upon the tributary of the Yangtze River blocked the gene flow among those regions. This research will be useful in breeding programs and conservation management of this species.

Transcriptome analysis of grass carp (Ctenopharyngodon idella) fed with animal and plant diets

Numerous studies have been focused on the replacement of fish meal by other alternative protein sources. However, little is currently known about the molecular mechanism of utilization of diets with different protein sources in fish. Grass carp is a typical herbivorous fish. To elucidate the relationship between gene expression and utilization of animal and plant diets, transcriptome sequencing was performed in grass carp fed with chironomid larvae and duckweed. Grass carp fed with duckweed had significantly higher relative length of gut than those fed with chironomid larvae. 4435 differentially expressed genes were identified between grass carp fed with chironomid larvae and duckweed in brain, liver and gut, involved in cell proliferation and differentiation, appetite control, circadian rhythm, digestion and metabolism pathways. These pathways might play important roles in utilization of diets with different protein sources in grass carp. And the findings could provide a new insight into the replacement of fish meal in artificial diets.