RNAi-based bioinsecticide for Aedes mosquito control

Abstract

Zika virus infection and dengue and chikungunya fevers are emerging viral diseases that have become public health threats. Their aetiologic agents are transmitted by the bite of genus Aedes mosquitoes. Without effective therapies or vaccines, vector control is the main strategy for preventing the spread of these diseases. Increased insecticide resistance calls for biorational actions focused on control of the target vector population. The chitin required for larval survival structures is a good target for biorational control. Chitin synthases A and B (CHS) are enzymes in the chitin synthesis pathway. Double-stranded RNA (dsRNA)-mediated gene silencing (RNAi) achieves specific knockdown of target proteins. Our goal in this work, a new proposed RNAi-based bioinsecticide, was developed as a potential strategy for mosquito population control. DsRNA molecules that target five different regions in the CHSA and B transcript sequences were produced in vitro and in vivo through expression in E. coli HT115 and tested by direct addition to larval breeding water. Mature and immature larvae treated with dsRNA targeting CHS catalytic sites showed significantly decreased viability associated with a reduction in CHS transcript levels. The few larval and adult survivors displayed an altered morphology and chitin content. In association with diflubenzuron, this bioinsecticide exhibited insecticidal adjuvant properties.

Figure 1

Survival curve of all experimental groups of larvae (1^st-instar) treated with 0.2 µg/mL of each purified dsRNA (400 ng dsRNA/2 mL) (a) and treated with 2 × 10⁻² µg/mL of E. coli HT115 lysate expressing each dsRNA (~4000 ng dsRNA/2 mL) from the PL4440 plasmid: pCHSA_1064 (dsCHSA_1064), pCHSA_1550 (dsCHSA_1550), pCHSB_693 (dsCHSB_693), pCHSB_1205 (dsCHSB_1205) and pCHSA_1928 (dsCHSA_1928) (d); both treatments target different regions in CHSA and B: dsRNA2718_1 (dsCHSA_1064), dsRNA2718_2 (dsCHSA_1550), dsRNA5618_1 (dsCHSB_693), dsRNA5618_2 (dsCHSB_1205) and dsRNA2718_3 (dsCHSA_1928). Relative expression of CHSA (b,e) and CHSB (c,f) transcripts in larvae (3^rd-instar) after treatment. For the in vitro dsRNA experiment, the following controls were used: no addition of dsRNA or addition of dsRNA targeting the MalE gene, E. coli maltose-binding protein, an unrelated gene (dsMalE). As controls of the E. coli HT115 lysates expressing each dsRNA, the following treatments were used: PL4440 (E. coli HT115 lysate with empty PL4440 plasmid) or no dsRNA (control). The experiments were performed with three biological replicates. The RPS6 gene was used as an endogenous control to normalize the expression of CHS transcript levels. Bars represent the means ± SEM. All asterisks indicate significantly different values from those of the controls (ANOVA followed by Tukey’s test, P <0.05).

Figure 2

Insecticidal adjuvant effect of the bioinsecticide with the diflubenzuron insecticide (DFB) at a concentration of 10⁻⁴ mg/L (LC₅₀) after six days of treatment. Control: not treated with bioinsecticide or DFB; DFB10⁻⁴ mg/L: 3^rd-instar larvae treated with DFB 10⁻⁴ mg/L previously dissolved in acetone; pCHSA_1928: larvae treated with bioinsecticide (2 × 10⁻² μg/mL); pCHSA_1928 + DFB10⁻⁴: larvae treated with 2 × 10⁻² μg/mL bioinsecticide in the 1^st-instar and DFB 10⁻⁴ mg/L in the 3^rd-instar; Solvent control: larvae treated with acetone solvent at the same volume used in the DFB treatment groups.

Figure 3

(a) Survival curves of experimental groups of larvae treated at the 4^th-instar stage with 2 × 10⁻² µg/mL of E. coli HT115 expressing dsCHSA_1928 lysed with chlorhexidine solution (bioinsecticide). Control groups: larvae without dsRNA addition (control), larvae with E. coli HT115 with empty PL4440 plasmid lysed with chlorhexidine (pL4440) and larvae exposed to E. coli HT115 lysed with chlorhexidine (chlorhexidine control). Relative expression of (b) CHSA and (c) CHSB in larvae treated at the 4^th-instar with bacterial lysate expressing dsCHSA_1928 (pCHSA_1928), PL4440 (bacterial lysate with empty plasmid) or no dsRNA (control) (three biological replicates, three technical replicates). The RPS6 gene was used as an endogenous control to normalize the level of expression. Bars represent the means ± SEM. All asterisks indicate values significantly different from those of the controls (ANOVA followed by Tukey’s test, P < 0.05).

Figure 4

(a) Percentages of pupal moulting and (b) adult emergence of larvae treated at the 4^th-instar with 2 × 10⁻² µg/mL E. coli HT115 induced by lactose expressing dsCHSA_1928 lysed with chlorhexidine solution (bioinsecticide). Control groups: larvae with no dsRNA addition (control), to E. coli HT115 lysed with chlorhexidine at a concentration of 2 × 10⁻² µg/mL. and larvae exposed to E. coli HT115 with empty PL4440 plasmid lysed with chlorhexidine (pL4440). Bars represent the means ± SEM. All asterisks indicate values significantly different from those of the controls (ANOVA followed by Tukey’s test, P < 0.05). (c) Male adult insects from larvae treated at the 4^th-instar with E. coli HT115 expressing dsCHSA_1928 lysed with chlorhexidine solution (bioinsecticide treatment) and not treated with dsRNA (control). Each division on the scale is equivalent to 1 mm.

Figure 5

(a) Survival curve of experimental groups of larvae treated at the 1^st-instar with different concentrations of bioinsecticide: 2 × 10⁻² µg/mL, 2 × 10⁻³ µg/mL and 2 × 10⁻⁴ µg/mL. Control groups: larvae without dsRNA addition (control) and larvae with E. coli HT115 with empty PL4440 plasmid lysed with chlorhexidine (pL4440) (five biological replicates). Relative expression of (b) CHSA and (c) CHSB in larvae treated at the first instar with the three concentrations described above and untreated with dsRNA (control) (three biological replicates, five technical replicates). The RPS6 gene was used as an endogenous control to normalize the level of expression. Bars represent the means ± SEM. All different letters indicate values significantly different from those of the controls (ANOVA followed by Tukey’s test, P < 0.05).

Figure 6

Representative images obtained using optical microscopy of larvae without dsRNA treatment: (a) control and (b) treated at the 1^st-instar with 2 × 10⁻² µg/mL of bioinsecticide. Scale bars = 1 mm. White arrows indicate malformations in larval morphology.

Figure 7

Representative images obtained using fluorescence microscopy of larvae of 4^th-instar. Larvae of the 1^th-instar were treated with three different concentrations of bioinsecticide (pCHSA_1928) and analysed by fluorescence microscopy at 4^th-instar. A FITC-WGA probe was used to detect the chitin polymer, which is indicated by the green FITC-WGA fluorescence signal. Control group without addition of bioinsecticide (a) and larvae treated with 2 × 10⁻⁴ µg/mL (b) 2 × 10⁻³ µg/mL (c) or 2 × 10⁻² µg/mL (d) of bioinsecticide. In all groups, various segments of the insect body were photographed: head (Hd), thorax (Th), intestine (In), respiratory siphon (RS), chitinous bristles (Br), and anal papillae (AP). Scale bars = 1 mm.