Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality

Abstract

Background: Although larviciding can reduce the number of outdoor biting malaria vector mosquitoes, which may help to prevent residual malaria transmission, the current larvicide repertoire is faced with great challenges to sustainability. The identification of new effective, economical, and biorational larvicides could facilitate maintenance and expansion of the practice of larviciding in integrated malaria vector mosquito control programmes. Interfering RNA molecules represent a novel class of larvicides with untapped potential for sustainable mosquito control. This investigation tested the hypothesis that short interfering RNA molecules can be used as mosquito larvicides.

Results: A small interfering RNA (siRNA) screen for larval lethal genes identified siRNAs corresponding to the Anopheles gambiae suppressor of actin (Sac1), leukocyte receptor complex member (lrc), and offtrack (otk) genes. Saccharomyces cerevisiae (baker's yeast) was engineered to produce short hairpin RNAs (shRNAs) for silencing of these genes. Feeding larvae with the engineered yeasts resulted in silenced target gene expression, a severe loss of neural synapses in the larval brain, and high levels of larval mortality. The larvicidal activities of yeast interfering RNA larvicides were retained following heat inactivation and drying of the yeast into user-friendly tablet formulations that induced up to 100% larval mortality in laboratory trials.

Conclusions: Ready-to-use dried inactivated yeast interfering RNA larvicide tablets may someday be an effective and inexpensive addition to malaria mosquito control programmes and a valuable, biorational tool for addressing residual malaria transmission.

Keywords: Brain; Larvae; Larviciding; Malaria; Mosquito; Pesticide; RNAi; Saccharomyces cerevisiae; Synapse; Vector.

Fig. 1

Larval mortality is induced by interfering RNA larvicides with target sites in the Sac1, lrc, and otk genes. The Sac1.1, lrc.2, and otk.16 siRNAs were identified in a screen for larval lethal genes in which siRNAs were evaluated by microinjection of third instar larvae (a) and through brief soaking treatment of first instar larvae (b). In the screen, an siRNA with no known target in Anopheles gambiae served as the control (a, b). The screen was performed in duplicate (see “Methods” for further details). For each replicate, 30 animals per treatment were microinjected, and 20 animals/treatment were soaked. Data were analysed with Fisher’s exact test [22]. c The larvicidal activity of these interfering RNAs was further confirmed when significant mortality was observed in larvae fed with heat-inactivated E. coli expressing dsRNA corresponding to the Sac1.1, lrc.2, and otk.16 target sites. Animals fed with bacteria expressing dsRNA corresponding to GFP served as the control in these experiments. Data were compiled from replicate experiments (n = 240 control-treated larvae, 160 Sac1.1-treated larvae, 240 lrc.2-treated larvae, and 160 otk.16-treated larvae) and assessed by two-way ANOVA with Tukey’s multiple comparison test. ***p < 0.001 in comparison to control-fed larvae; **p < 0.05 in comparison to control-fed larvae; error bars denote standard errors of the mean (SEMs)

Fig. 2

Yeast interfering RNA larvicides induce significant mortality. Significant larval death was observed in larvae fed with gel-coated live (a) or heat-inactivated (b) yeast engineered to express shRNA corresponding to the Sac1.1 and otk.16 siRNA target sequences (as compared to control yeast expressing shRNA corresponding to the control siRNA sequence). Data were compiled from replicate experiments, with 120 larvae evaluated per condition for live yeast and 180 larvae assessed per condition for inactive yeast (see “Methods” for details). Data were statistically assessed by ANOVA with Tukey’s multiple comparison test. No statistically significant differences were observed in the larvicidal capacity of live vs. heat-inactivated gel-coated yeast interfering RNA larvicides. ***p < 0.001 in comparison to control-fed larvae; error bars denote SEMs

Fig. 3

Dried inactivated yeast interfering RNA tablets induce significant larval death. Dried inactivated yeast interfering RNA larvicide tablets (a; penny shown for scale) were prepared and fed to 20 larvae. Significant larval death was observed in larvae fed with yeast expressing shRNA hairpins corresponding to the Sac1.1, lrc.51, and otk.16 target sequences as compared to larvae fed control yeast interfering RNA tablets (b). Data were compiled from three biological replicate experiments (total n = 240 larvae/condition; see “Methods” for details) and analysed by ANOVA with Tukey’s multiple comparison test. ***p < 0.001 in comparison to control-fed larvae; error bars denote SEMs

Fig. 4

Confirmed silencing of the Sac1, lrc, and otk genes in the larval brain by dried inactivated yeast interfering RNA tablets. Significantly lower levels of Sac1 (a1–a3), lrc (b1–b3), and otk (c1–c3) transcripts were detected in the L4 brains of larvae fed with the Sac1.1 (a2), lrc.51 (b2), and otk.16 (c2) dried inactivated yeast interfering RNA larvicides vs. animals fed with control yeast (a1, b1, c1). For each probe, results from three biological replicate experiments were compiled (n = 85 Sac1.1-treated brains, n = 80 lrc.51-treated brains, and n = 80 otk.16-treated brains; n = 40 control-treated brains/per experiment). Data were evaluated by t test (***p < 0.001 in comparison to control-fed larvae). The brains are oriented dorsal upward in this figure. LAL, larval antennal lobe; OF, olfactory foramen; OL, optic lobe; SOG, sub-oesophageal ganglion; SuEG, supra-oesophageal ganglion

Fig. 5

Neural defects observed in larvae treated with yeast interfering RNA larvicides Sac1.1, lrc.51, and otk.16. L4 larval brains were labeled with mAb3C11 (white in a1–d1; red in a2–d2), which labels expression of Synapsin, a marker for the neuropil and synaptic active zones. TO-PRO was used to counter-stain nuclei in the brain (blue in a2–d2). The brains of larvae fed with yeast expressing shRNAs Sac1.1 (b1, b2), lrc.51 (c1, c2), and otk.16 (d1, d2) yeast interfering RNA tablets show loss of staining in the synaptic neuropil regions when compared with animals fed with control yeast (a1, a2). Three biological replicate experiments were performed. The data shown are representative of the results from 25 brains evaluated per condition. LAL, larval antennal lobe; OF, olfactory foramen; OL, optic lobe; SOG, sub-oesophageal ganglion; SuEG, supra-oesophageal ganglion