Evaluation of  resistance to Ichthyophthirius multifiliis in CECROPIN TRANSGENIC channel catfish Ictalurus punctatus and channel catfish female X blue catfish male Ictalurus furcatus hybrids.

The current study was conducted to compare cecropin transgenic channel catfish and channel catfish female X blue catfish male (CB) hybrid catfish to non-transgenic channel catfish and CB hybrid catfish for resistance to Ichthyophthirius multifiliis. Cecropin B gene was driven by cytomegalovirus (CMV) promoter. Seventy six one-year-old catfish were randomly chosen for this experiment from 4 genetic groups, including F₃ cecropin transgenic channel catfish, cecropin transgenic CB hybrid catfish, non-transgenic channel catfish and non-transgenic CB hybrid catfish. Fish were challenged communally in the same tank in a cohabitation challenge. Dead fish were collected twice per day, morphologically identified as channel catfish or CB hybrid catfish and weighed. The anal fin was sampled for DNA analysis. Dead fish were necropsied to confirm the cause of death.

DNA was extracted using proteinase K digestion followed by ethanol precipitation. Channel catfish and CB hybrid catfish were identified based on the shape of the swim bladder and the polymorphism of the follistatin (Fst) and hepcidin antimicrobial (Hamp) genes. To identify cecropin transgenic catfish, primers amplifying a partial segment of CMV promoter in a 2-step nested PCR reaction were used. Negative controls included DNA from non-transgenic channel catfish and non-transgenic CB hybrid catfish in addition to a no-DNA control. PCR products were resolved in 1.5% agarose gel and visualized with a Molecular Imager® Gel Doc™ XR+ System using Image Lab™ software (Bio-Rad Laboratories, Inc). PCR products were gel purified and sequenced to confirm the PCR results.

Cumulative % mortality was calculated for each group (Figure 1). When Cox's proportional hazards model for time to death was used to investigate the effects of species, cecropin, body weight and their interactions to the second level on the survival time, no differences were detected. However, LD 50 for days to death was 2 for non-transgenic channel catfish, 3 for cecropin transgenic channel catfish and 4 for both cecropin transgenic and non-transgenic CB hybrid catfish, possibly indicating slightly longer survival for some groups, which would give more time for rescue in a practical situation where treatment would be applied.  Further statistical analyses will be performed to draw a final conclusion.