Characterization of a novel RNAi yeast insecticide that silences mosquito 5-HT1 receptor genes

Abstract

G protein-coupled receptors (GPCRs), which regulate numerous intracellular signaling cascades that mediate many essential physiological processes, are attractive yet underexploited insecticide targets. RNA interference (RNAi) technology could facilitate the custom design of environmentally safe pesticides that target GPCRs in select target pests yet are not toxic to non-target species. This study investigates the hypothesis that an RNAi yeast insecticide designed to silence mosquito serotonin receptor 1 (5-HTR1) genes can kill mosquitoes without harming non-target arthropods. 5-HTR.426, a Saccharomyces cerevisiae strain that expresses an shRNA targeting a site specifically conserved in mosquito 5-HTR1 genes, was generated. The yeast can be heat-inactivated and delivered to mosquito larvae as ready-to-use tablets or to adult mosquitoes using attractive targeted sugar baits (ATSBs). The results of laboratory and outdoor semi-field trials demonstrated that consumption of 5-HTR.426 yeast results in highly significant mortality rates in Aedes, Anopheles, and Culex mosquito larvae and adults. Yeast consumption resulted in significant 5-HTR1 silencing and severe neural defects in the mosquito brain but was not found to be toxic to non-target arthropods. These results indicate that RNAi insecticide technology can facilitate selective targeting of GPCRs in intended pests without impacting GPCR activity in non-targeted organisms. In future studies, scaled production of yeast expressing the 5-HTR.426 RNAi insecticide could facilitate field trials to further evaluate this promising new mosquito control intervention.

Figure 1

5-HTR.426 shRNA-expressing yeast induces mortality under laboratory and semi-field conditions. (a1) In laboratory experiments, yeast expressing 5-HTR.426 shRNA induced dose-dependent mortality in the A. aegypti LVP-IB12 laboratory strain. The LD₅₀ (for treatment of 20 larvae) was 27 mg. (b1) When the same yeast was applied to mosquito strains collected from the field and tested in semi-field conditions in Trinidad, it eliminated all the larvae in the experiment (orange bars). (a2) In laboratory trials, 5-HTR.426 yeast induced dose-dependent mortality in A. aegypti LVP-IB12 adult females (LC₅₀ = 2.014 ug/ul). (b2) In outdoor semi-field trials conducted in Trinidad, 5-HTR.426 yeast also induced significant mortality in field strains of Aedes and Culex mosquitoes. The data in (b1) and (b2) are presented as mean percentage mortality, with error bars representing standard error of the mean. *** indicates a statistically significant difference from the control group (P < 0.001). ASB, attractive sugar bait alone; control refers to treatments with control RNAi yeast.

Figure 2

Yeast expressing 5-HTR.426 shRNA results in high levels of adult mortality. Female mosquitoes of five different species were fed with 5-HTR.426 shRNA expressing yeast either using the lab sugar bait system (laboratory trials shown in (a1–e1)) or commercial bait system (simulated field trials shown in (a2–e2)). The yeast induced high levels of mortality over 6 days post feeding. Individuals fed with yeast mixed with commercial bait die 24–36 h earlier than those fed with lab sugar bait with yeast. ASB, attractive sugar bait alone; control refers to treatments with control RNAi yeast.

Figure 3

5-HTR.426 yeast results in silencing of the target transcript. Consumption of yeast expressing 5-HTR.426 throughout larval life results in significant reduction of 5-HTR1 transcripts in A. aegypti larvae (a1–c1). A. aegypti adults fed with ASB (lab bait) containing 5-HTR.426 yeast induces a similar reduction in the transcript levels when compared with individuals fed with the control yeast (control; a2–c2). *** = P < 0.001 vs. control yeast; data were analyzed with Student’s t-test. Scale Bar = 100 μm.

Figure 4

Feeding A. aegypti larvae with 5-HTR.426 yeast causes neural defects. Larval brains were labeled with three antibodies: mAbnc82 (an active synapse marker; white in (a1, a2); green in (c1, c2)), anti-HRP (neural marker; white in (b1, b2); red in (c1, c2)), and TO-PRO (nuclear marker; blue in (c1, c2)). The levels of nc82, a protein involved in neural development, were significantly reduced in the synaptic neuropil of larvae fed with 5-HTR.426 yeast (a2, c2) compared to control yeast-treated (control) larvae (a1, c1). The data are presented as average mean gray values, with error bars denoting standard error of the mean. ***Indicates a statistically significant difference from the control group (P < 0.001). Representative larval brains are oriented dorsal upward in this figure. LAL, larval antennal lobe; OL, optic lobe; SOG, sub-esophageal ganglion; SuEG, supraesophageal ganglion. Scale Bar = 100 μm.

Figure 5

5-HTR.426 yeast induces neural defects in A. aegypti adult females. Adult brains of female A. aegypti mosquitoes that were fed with either control yeast (a1–c1) or 5-HTR.426 yeast (a2–c2), were immunolabeled with mAbnc82 (an active synapse marker; white in (a1, a2); green in (c1, c2)), anti-HRP antibodies (a neural marker; white in (b1, b2); red in (c1, c2)), and TO-PRO dye (nuclear stain; blue in (c1, c2)). The levels of nc82 were significantly reduced in the synaptic neuropil of adults fed with 5-HTR.426 (a2, c2 vs. a1, c1; d1) yeast compared to control yeast-treated (control) adults (P < 0.001) while the levels of TO-PRO remained the same in both cases (d2). Representative adult brains are oriented dorsal upward in this figure. AL, larval antennal lobe; OL, optic lobe; SOG, sub-esophageal ganglion; SuEG, supraesophageal ganglion. Scale Bar = 100 μm.

Figure 6

5-HTR.426 yeast induces serotonergic neural defects in the larval brain of 5-HTR.426-treated mosquitoes. In control yeast-fed (control; white in (a1); red in (b1)) L4 larvae, serotonergic neurons marked by the anti-5-HT antibody (arrowheads) show normal patterns of innervation throughout the larval brain. Individuals treated with 5-HTR.426 insecticidal yeast (a2–a5) displayed the phenotypes represented here and quantified in Table 7. In ~ 50% of individuals, there is a complete absence of 5-HT neurons (a2, b2); 36% of treated individuals show 5-HT expression in 2–3 cell bodies (arrowheads) without any dendrite expression (a3, b3), and the remaining 12% of labeled individuals show expression in 3–4 cell bodies (arrowheads) with variable expression in dendrites (a4, b4). Representative adult brains are oriented dorsal upward in this figure. AL, larval antennal lobe; OL, optic lobe; SOG, sub-esophageal ganglion; SuEG, supraesophageal ganglion. Yellow arrow heads indicate cell bodies, and yellow asterisks indicate remnant dendrites. Scale bar = 25 mm.