Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0−50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3−74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Response of cecropin transgenesis to challenge with Edwardsiella ictaluri in channel catfish Ictalurus punctatus.

Constructs bearing the cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (ß-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F3 channel catfish family transgenic for cecropin transgene driven by the CMV promoter, and one F1 channel catfish family transgenic for cecropin transgene driven by the common carp ß-actin promoter were produced. F3 and F1 individuals exhibited enhanced disease resistance when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). Inheritance of the transgene by the F1 and F3 generation was 15% and 60%, respectively. Growth rates of the cecropin transgenic and non-transgenic full siblings (controls) channel catfish were not different (P > 0.05). All transgenic fish showed significant resistance to infection by ESC at day 3 and day 4 post exposure (P = 0.005). No correlation was detected between body weight and time to death for all genetic groups (P = 0.34). Results of our study confirmed that genetic enhancement of E. ictaluri resistance can be achieved by cecropin transgenesis in channel catfish. In addition to survival rate, improving survival time is essential because the extension of survival time gives a better chance to apply treatments to stop the bacterial infection.

Comparison of growth performance among channel-blue hybrid catfish, ccGH transgenic channel catfish, and channel catfish in a tank culture system.

Fish is an essential source of high-quality protein for people worldwide. The present study was designed to compare the growth performance among the channel-blue hybrid catfish, channel catfish transgenic for the channel catfish growth hormone (ccGH) cDNA driven by the antifreeze protein promoter from an ocean pout Zoarces americanus (opAFP-ccGH), and non-transgenic channel catfish control. Mean body weight of channel-blue hybrid catfish was 15.80 and 24.06% larger than non-transgenic channel catfish control at 4 and 18 months of age, respectively. However, transgenic opAFP-ccGH channel catfish were 5.52 and 43.41% larger than channel-blue hybrid catfish and 22.19 and 77.91% larger than their controls at 4 and 18 months of age, respectively. Significant differences in mean body weight between the sexes within all genetic types were found. Males were larger than females (P < 0.001). However, mean body weight of non-transgenic males was not larger than transgenic opAFP-ccGH females or male and female hybrid catfish. Condition factor of transgenic opAFP-ccGH channel catfish was higher (P < 0.05) than that of full-sibling, non-transgenic channel catfish and hybrid catfish. The mean percentage body weight gain of GH transgenic channel catfish was 559%, the channel-blue hybrid catfish was 384.9% and their non-transgenic controls channel catfish was 352.6%.

Growth Differences of Growth Hormone Transgenic Female and Male Channel Catfish, Ictalurus punctatus, Grown in Earthen Ponds to Sexual Maturation.

This study compared growth performance between female and male transgenic channel catfish, Ictalurus punctatus, containing channel catfish growth hormone full-length cDNA driven by the ocean pout antifreeze protein promoter, opAFP-ccGH, the rainbow trout metallothionein promoter, rtMT-ccGH, or both constructs, and their non-transgenic siblings in earthen ponds at 16 and 48 months of age. Body weight between the transgenic and their non-transgenic siblings differed (P < 0.001) at all ages. Transgenic F2 opAFP-ccGH grew 1.51- to 2.58-, F2 rtMT-ccGH grew 1.44- to 2.99- and F1fish transgenic for both constructs grew 1.36- to 2.92- fold larger than their non-transgenic sibling controls, depending upon age and sex. Body weight of the transgenic GH males was significantly higher than those of the transgenic GH females at 16 months of age (P < 0.001). However, body weight of the transgenic GH females was significantly higher (P < 0.001) compared with those of the transgenic GH males at 48 months of age, but not for the double transgenics (P > 0.05). In the case of non-transgenic GH siblings, males were larger than females at both 16 and 48 months of age (P < 0.001). Sexually dimorphic responses to GH transgenes were the opposite after sexual maturation. When critically low dissolved oxygen levels were encountered, survival of transgenic male and female opAFP-ccGH channel catfish was lower than that of controls (P = 0.004), as well as rtMT-ccGH females (P = 0.11), which is not surprising since the largest fish are most likely to succumb during an oxygen depletion.

Cathelicidins enhance protection of channel catfish, Ictalurus punctatus, and channel catfish ♀ × blue catfish, Ictalurus furcatus ♂ hybrid catfish against Edwarsiella ictaluri infection.

Cathelicidins are a class of antimicrobial peptides (AMPs) known to possess rapid and direct antimicrobial activities against a variety of microorganisms. Recently identified cathelicidins derived from alligator and sea snake were found to be more effective in inhibiting microbial growth than other AMPs previously characterized. The ability of these two cathelicidins along with the peptides, cecropin and pleurocidin, to protect channel catfish (Ictalurus punctatus, Rafinesque) and hybrid catfish (I. punctatus ♀ × blue catfish, Ictalurus furcatus, Valenciennes ♂) against Edwardsiella ictaluri, one of the most prevalent pathogens affecting commercial catfish industry, was investigated. Cathelicidin-injected fish (50 µg ml-1  fish-1 ) that were simultaneously challenged with E. ictaluri through bath immersion at a concentration of ~1 × 106 CFU/ml had increased survival rates compared with other peptide treatments and the infected control. Bacterial numbers were also reduced in the liver and kidney of channel catfish and hybrid catfish in the cathelicidin treatments 24 hr post-infection. After 8 days of challenge, serum was collected to determine immune-related parameters such as bactericidal activity, lysozyme, serum protein, albumin and globulin. These immune-related parameters were significantly elevated in fish injected with the two cathelicidins as compared to other peptide treatments. These results indicate that cathelicidins derived from alligator and sea snake can stimulate immunity and enhance the resistance to E. ictaluri infection in channel catfish and hybrid catfish.