Genetic Technologies for Growth Enhancement in Catfish

Abstract

The effects of CRISPR/Cas9 knockout of the myostatin (MSTN) gene in channel catfish, Ictalurus punctatus, was investigated. A total of 209 fish survived microinjection over 3 years with an average mutation rate of 58%. Successful generation of MSTN F1 mutants was achieved in 2019 by individually mating two pairs of control females with MSTN mutant males. The offspring of both families inherited the mutation at a high rate of 88%. Growth was generally higher in MSTN mutants when compared to controls at all life stages and in both pond and tank environments. Heterozygous F1 mutants were 218% larger than controls at the stocker stage in ponds. Mean expression level of MSTN was 2.90 times higher in controls than in MSTN F1 progeny (p=0.009). When challenged with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), MSTN mutants performed equally or better than controls.

The effects of CRISPR/Cas9 knockout of the melanocortin-4 recepter (MC4R) gene in channel catfish was investigated. Growth was generally higher in MC4R mutants when compared to controls at all life stages and in pond and tank environments. There were no significant differences in body weight between MC4R F1 progeny and controls generated in 2018, although there was a positive relationship between zygosity and growth. F1 homozygous/bi-allelic mutants were 30% larger than F1 heterozygotes at market size grown in earthen ponds (p=0.022).

Channel catfish have limited ability to synthesize n-3 fatty acids, due to a lack of elongases and desaturases. The ccβA-msEvol2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, beta-actin promoter was inserted into the channel catfish melanocortin-4 receptor (MC4R) gene using the 2-Hit 2-Oligo with Plasmid (2H2OP) method. SgRNA one (MC4R-A) targeting exon one of the MC4R gene resulted in a knockout mutation rate of 92% with 69% homozygosity/bi-allelism, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Evol2/MC4R fish were 41.81% larger than controls at 6-months post hatch (p=0.005). There was no significant increase in overall omega three content, however observed mean eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) content were 92.16% (p=0.203) and 32.82% (p=0.380) higher, respectively, in Evol2/MC4R fish than controls.

Combining ability for body weight of fingerlings grown in aquaria and food size fish grown in earthen ponds generated from channel catfish females and blue catfish, I. furcatus, males was calculated using a 6 x 6 diallel design. At 14-months post hatch, and a mean weight of 9.69g±8.78g, the GCAD, GCAS and SCA were 4.5%, 8.4% 20.8% of total variance, respectively. At 40-months, and a mean weight of 836g±250.71, the GCAD, GCAS and SCA for the interspecific matings were 1.1%, 0.0 and 4.6% of total variance, respectively. The heaviest and lightest families at 40-months post hatch were not the same as those at 14-months post hatch, indicating genotype-environment interactions.

Keywords: Channel catfish, Ictalurus punctatus, blue catfish, Ictalurus furcatus, myostatin, melanocortin-4 receptor, elongase, omega-3 fatty acid, growth, gene editing, transgenesis, CRISPR/Cas9, heritability, combining ability, growth, breeding, factorial design