The food safety assessment of all-female common carp (Cyprinus carpio) (cyp17a1+/-;XX genotype) generated using genome editing technology.

There are several technical challenges and public issues concerning genome editing applications before they become viable in commercial aquaculture. Recently, we developed a novel strategy to generate all-female (AF) common carp, which exhibited a growth advantage over the control carp, using genetic editing through single gene-targeting manipulation. Here, we found that the body weight of the AF common carp was higher by 22.58% than that of the control common carp. Because the genotype of the AF common carp was cyp17a1+/-;XX, the contents of sex steroids were normally synthesized, as they were comparable to that of the control female carp. To evaluate the food safety of the AF carp, Wistar rats were fed a diet containing control female carp (control, C) or all-female (AF) carp at an incorporation rate of 5, 10 and 20% (w/w) for 90 days. Compared with those fed control carp, the rats fed AF common carp exhibited no significant difference in body weight, food intake, feed conversion ratio, hematology, serum biochemistry, urine test, relative organ weight, gross necropsy, and histopathological examination. This is the first food safety assessment of the farmed fish strain cultured using CRISPR/Cas9, which will further advance the fishery development of genome-edited animals.

Transcriptome analysis of grass carp (Ctenopharyngodon idella) between fast- and slow-growing fish.

Grass carp is one of the most important freshwater aquaculture species in China. However, the mechanisms underlying the growth of muscle tissue in the fish are unclear. High-throughput RNA-Seq was used to analyze the transcriptome of grass carp muscle tissue between fast- and slow-growing fish family groups. Twenty-four individuals each from 4 fast-growing families and 4 slow-growing families were used to reduce background noise. 71 up-regulated and 35 down-regulated genes were identified in the differentially expressed genes (DEGs). GO and KEGG enrichment analyses revealed the DEGs were involved in the GH/IGF axis, calcium metabolism, protein and glycogen synthesis, oxygen transport, cytoskeletal and myofibrillar components. IGFBP1 was up-regulated in big fish while GHR2 was down-regulated. Glutamic pyruvate transaminase 2, an indicator of liver tissue damage, was down-regulated in big grass carp, which indicates that the fish was better adapted to an artificially formulated diet. GAPDH, the rate-limiting enzyme in glycolytic flux was highly expressed in fast-growing grass carp, reflecting enhanced carbohydrate metabolism. Higher expression of ALAS2 and myoglobin 1 in big grass carp, related to oxygen transport might promote aerobic exercise along with food intake and muscle growth. Genes for cytoskeletal and myofibrillar components such as tropomyosin, meromyosin, and troponin I were also up-regulated in big grass carp. These results provide valuable information about the key genes for use as biomarkers of growth in selective breeding programs for grass carp and contribute to our understanding of the molecular mechanisms and regulative pathways regulating growth in fish.

The draft genome of blunt snout bream (Megalobrama amblycephala) reveals the development of intermuscular bone and adaptation to herbivorous diet.

The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.

PPARγ, an important gene related to lipid metabolism and immunity in Megalobrama amblycephala: cloning, characterization and transcription analysis by GeNorm.

In order to be able to modulate and improve the function of PPARγ and decrease further some metabolic diseases of M. amblycephala, we have cloned and identified the full-length cDNA of PPARγ in M. amblycephala and examined its transcription patterns at different embryo developmental stages and in different tissues of adult and immature fish. We also accurately normalized seven reference genes by GeNorm and calculated their gene transcription normalization factors. The full-length of PPARγ was 1968 bp, consisting of 218 bp 5'-untranslated region, 1,533 bp open reading frame encoding 510 amino acids residues and 217 bp 3'-untranslated region. M. amblycephala PPARγ peptide was predicted to consist of 4 conserved domains, i.e. N-terminal domain, DNA-binding domain, ligand binding domain and flexible hinge region. PPARγ mRNAs were detected in all studied tissues of adult and immature fish including adipose tissue, gill, heart, liver, spleen, kidney, white muscle, intestine, brain and gonad. In adult fish, PPARγ transcription in liver was highest, followed by gills and it was lowest in female gonads. Moreover, the differences among liver, gill, intestine/brain, spleen/white muscle, kidney and female gonads were greatly significant (p<0.01). The transcription of PPARγ in male gonads was significantly higher than in female gonads (p<0.01). In immature fish, the transcription of PPARγ was highest in intestines followed by adipose tissue, and it was lowest in hearts and white muscles. A great difference was observed (p<0.01) in the transcription of PPARγ among adipose tissue, intestines, liver and heart/white muscles. At different embryo developmental stages, PPARγ transcription in unfertilized spermatozoa was greatly higher than in unfertilized ovum (p<0.01) and it was highest among different embryo developmental stages. The transcription of PPARγ increased gradually during 2 cells stage and 32 cells stage and then decreased until gastrula stage at which it was lowest. The transcription of PPARγ increased again on first day after hatching. There was a significant difference (p<0.01) in the transcription of PPARγ between 2 cells stage and 32 cells stage and it was same between 32 cells stage and gastrula stage. These results revealed that transcription of PPARγ showed a tissue-dependent regulation and a developmental-stage-dependent regulation that are valuable and helpful to improve the function of PPARγ and to decrease some metabolic diseases in the culture of M. amblycephala.