Generation of fast growth Nile tilapia (Oreochromis niloticus) by myostatin gene mutation

Tilapia is the second most prolific species grown in aquaculture after carp, and is widely grown in >100 countries. Myostatin (MSTN) has been proved to be a negative regulator of skeletal muscle growth. Mutation of MSTN gene resulted in significant increase in both body size and muscle mass in vertebrates, mostly with a species-specific effect. To generate a new strain in Nile tilapia (Oreochromis niloticus) with fast growth traits, the molecular characterization, gene expression and functional studies of mstn gene were extensively investigated. Phylogenetic and synteny analyses indicated that mstnb, but not mstna, might be the orthologous gene of mammalian MSTN gene. Expression pattern analyzed by qRT-PCR demonstrated that mstnb is abundant in muscle, while mstna is dominantly expressed in the brain. Fluorescence in situ hybridization (FISH) analysis also demonstrated that mstnb is exclusively expressed in the basement membrane of white skeletal muscle fibers, further suggesting the possible role of mstnb gene in tilapia muscle growth. To verify the roles of mstnb in skeletal muscle growth, we obtain the mstnb(-/-) mutant using CRISPR/Cas9 gene editing technology. Compared with the wild type (WT) fish, the mstnb(-/-) mutants showed a typical double-muscle phenotype with increased muscle mass, sticking out between head and dorsal fin, from 5 months after hatching (mah). Morphological observation revealed an excessive proliferation of white muscle fibers at 7 mah. The average body weights (21.67 +/- 8.26 g), body heights (4.46 +/- 0.65 cm) and body widths (2.36 +/- 0.53 cm) of the mstnb(-/-) fish were 49.45%, 32.74% and 37.21% higher than those of the mstnb(+/+) fish (body weight, 14.50 +/- 8.60 g; body height, 3.36 +/- 0.45 cm; body width, 1.72 +/- 0.18 cm) at 5 mah. The results showed that the growth performance parameters of the mstnb(-/-) fish, including weight gain rate (1.99 times), condition factor (1.77 times) and specific growth rate (1.23 times), were significantly higher than the mstnb(+/+) fish under laboratory conditions at 5 mah. In addition, the parameters of WGR, CF, SGR and FE were significantly increased in mstnb(-/-) Nile tilapia than those of mstnb(+/+) Nile tilapia and GIFT tilapia for a 90-day feeding trial under the wild natural environment. Interestingly, sexually dimorphic effects on muscle growth have been found with an increase of growth performance of skeletal muscle in mstn(-/-) XY fish, but not mstn(-/-) XX fish at 7 mah. Taken together, this study, for the first time, proved that mstn gene mutation might promote the generation of new fast-growing male strain of Nile tilapia.