A genetically enhanced sterile insect technique against the fruit fly, Bactrocera dorsalis (Hendel) by feeding adult double-stranded RNAs

Abstract

RNAi based sterile insect technique (SIT) is an authentic insect management approach but requires proper target genes. During this study, spermless males were developed by interfering with germ cell differentiation and azoospermia related genes. Data demonstrates significant reductions in the target genes expressions (boul, zpg, dsx ^M , fzo and gas8) after oral dsRNAs administration. Knock down of target genes significantly affected the reproductive ability of males and reduced egg-hatching as compared to the control group. Furthermore, different combinations of selected gene dsRNAs (boul + zpg, boul + dsx ^M and zpg + dsx ^M ) were made, which resulted up to 85.40% of male sterility. The most effective combination was selected to prepare different concentrations of dsRNA, 250, 500, 750 and 1000 ng/μl, that caused 18.97%, 38.68%, 58.02% and 85.40% male sterility, respectively. Subsequently, 1000 ng/μl of the same combination of ds-RNAs was used against differently aged adult flies (1, 5, 7, 10 days) which lead to 85.40%, 31.42%, 21.76% and 9.90% male sterility, respectively. SIT developed in this study showed that, boul + zpg combination of dsRNA feeding for 6 hours significantly reduced the number of spermatozoa and viability of sperm in 1-day-old B. dorsalis flies. In short, this study provides an effective SIT technique for long-term B. dorsalis management.

Figure 1

Differential gene expression in various body parts of Bactrocera dorsalis adults including testis, ovary, fat body, gut, head, and malpighian tubules, in response to selective genes (Boul, Zpg, dsx ^M, Fzo and Gas8). Bars and different letters indicate significant differences in the expression level at p < 0.05 according to Tukey’s test.

Figure 2

Effects of RNAi silencing of target genes on five consecutive days at different concentrations. Effect of orally administered dsRNAs against target genes (boul, zpg, dsx ^M, fzo and gas8) (A–E) at different concentrations (250, 500, 1000 & 2000 ng/ul) were analyzed. Normalized target gene expression is reported relative to the expression of the ds-EGFP control, which was set to 1-fold. All error bars depict the SE of the mean of three independent replicates. Treatments were compared with their respective controls using ANOVA (Dunnett-test, P < 0.05). *, **, *** and ns indicates P < 0.05, P < 0.01, P < 0.001 and non-significant, respectively.

Figure 3

Egg laying and hatching capacity of B. dorsalis against dsRNA of target genes (boul, zpg, dsx ^M, fzo and gas8) with EGFP as a control.

Figure 4

Egg hatching rate per day in response to a synergistic effect of two dsRNAs. The average number of eggs hatched per day between candidate gene dsRNA combinations and ds-EGFP treated flies (A) boul + zpg (B) boul + dsx ^M & (C) zpg + dsx ^M). Three biological replicates were performed and significant differences in egg hatching rates were found.

Figure 5

Egg hatching rate in response to individual and combinations of target gene dsRNA compared with ds-EGFP treated flies. Three biological replicates were performed, *** indicates significant difference. One way ANOVA indicate difference in average number of hatchings between all candidate genes dsRNA (P < 0.0001, Tukey test).

Figure 6

Egg hatching rate in response to different concentrations of dsRNA for the boul + zpg genes. (A) The average number of eggs hatched per day between target gene dsRNA and ds-EGFP at different concentrations. (B) One way ANOVA indicates a difference in the accumulative number of eggs hatched at different concentrations compared with the control group. Different letters indicate the significant differences between all treatments. Three biological replicates were performed (P < 0.0001, Tukey test).

Figure 7

Egg hatching rate in response to 1000 ng/μl of dsRNA (boul + zpg) genes in different ages of adult males. (A) The average number of eggs hatched per day between target gene dsRNA and ds-EGFP at different concentrations were noted. (B) One way ANOVA indicates a difference in the accumulative number of eggs hatched after feeding 1000 ng/μl dsRNA to males of different ages at compared with the control group (ds-EGFP). Different letters indicated the significant differences between all treatments. Three biological replicates were performed (P < 0.0001, Tukey test).

Figure 8

Average number of spermatozoa percentage of dead and live sperms. (A) Average number of spermatozoa in seminal vesicles, (B) percentage of live sperms per male was determined in seminal vesicles of 20 males per treatment, the effect of treatments was analyzed using T-test. ** indicates P < 0.01. (C) Microscopic picture of testis (200 μm) revealing the dead sperm (100 μm) (red) and live sperm (100 μm) (green).