Figure 4.

Real time analysis to measure steady state levels of 3-HKT transcript among surviving/dead An. stephensi larvae and pupae reared on transgenic Chlamydomonas expressing 3-HKT dsRNA. In each experiment 3-HKT expression was compared and normalised to An. stephensi actin transcript levels. The data shown in each figure is the average of three biological replicates. Each biological replication involved five technical replications for each treatment and control. The mean ΔΔCT values from three biological replicates were subjected to analysis through single factor ANOVA. Asterisks indicate the treatments that had significantly lower 3-HKT transcript levels relative to the parental control, determined by single factor ANOVA (P<0.05). RNA extracted from larvae/pupae reared on nontransgenic Chlamydomonas was used as the control in all experiments. In all experiments A, B, C (as in 108-13A1) etc. refer to the wells from which the larvae/pupae were sourced for analysis and the numbers 1, 2 refer to the first or second larvae/pupae used for analysis from a single well. a) 3-HKT transcript levels observed among surviving larvae reared on clones 108-13 and 108-15. Except for larvae 108-15C1, which did not differ significantly from the control larvae, all other treatments showed significant reduction in 3-HKT transcript levels compared to the parental control. b) 3-HKT transcript levels observed among dead pupae from larvae reared on 108-13. c) 3-HKT transcript levels observed among dead pupae from larvae reared on the 108-15 transgenic Chlamydomonas clone.