Disruption of Embryonic Development in Channel Catfish, Ictalurus Punctatus, Using 'Sterile-Feral' Gene Constructs

Abstract

Channel catfish, Ictalurus punctatus, embryos were electroporated with a sterile feral 3 (SF3), sterile feral 4 (SF4), glutamate decarboxylase (GAD) or CAB constructs and a blank control. Doxycycline was applied to some of the SF3 and SF4 replicates, at 50, 100 and 150 ppm, from 4 hours post fertilization to first hatch. The developing embryos were observed from fertilization up through hatch. Dead and deformed embryos were removed during this period. Mortality rates were computed for different time intervals during development as well as for the entire development period. Deformity rates were also analyzed. Objectives were to determine the efficacy of the sterile feral constructs to disrupt embryonic development, the optimal concentration of doxycycline to prevent embryonic disruption and the time interval most crucial for application of doxycycline to prevent the expression of the developmental disrupter.

Embryos electroporated with the SF3 construct consistently demonstrated a higher mean mortality than that of the control groups. The overall mean mortality of the SF3 groups for all three experiments was 35.9% higher than the overall mean mortality of the three blank control groups. Similar results were obtained for the SF4 construct, whose overall mean mortality for the three experiments was 27.2% higher than that of the overall mean mortality for the three experiments with the blank control groups. The GAD construct produced an overall mean mortality that was 34.5% lower than that of the mean mortality of SF3 and 25.6% lower than that of SF4. The single mortality rate produce by the CAB construct was 82.3% lower than that of the mean mortality of SF3 and 79.9% lower than SF4. These consistently lower mortality rates produced by both CAB and GAD demonstrated that the introduction of exogenous DNA was not responsible for the larger mortality rates observed in the SF4 group and SF3 groups, but the action of the SF3 and SF4 constructs. The concentration of doxycycline most optimal for blocking the expression of the blocker in the SF3 construct was 100 ppm and 150 ppm for blocking the developmental disrupter of the SF4 construct. Some portion of the 18-61 hour post fertilization time period appears to be the time interval in which it is most crucial to apply doxycycline to prevent embryonic disruption.