ABSTRACTMECHANISMS OF PYRETHRUM AND PYRETHROID REPELLENCYBy Elizabeth Bandason Pyrethrum is a natural insecticide, extracted from the flower heads of Chrysanthemum cinerariifolium. Pyrethroids are synthetic compounds structurally derived from pyrethrins. Besides their insecticidal activity, it is well-documented from behavioral assays that pyrethrum and pyrethroids induce repellency. However, the molecular basis of pyrethrum/pyrethroid repellency is unknown. The aim of this study was to elucidate the mechanism of pyrethrum and pyrethroid repellency in mosquitoes and crop pests.To investigate repellency against mosquitoes, a previously established Hand in cage assay was performed. We recorded mosquito landing rate on a hand in response to vapor from a mesh treated with pyrethrum or pyrethroids (transfluthrin, deltamethrin and permethrin).Three insecticide-susceptible Aedes aegypti strains, Waco, Orlando and Rock, two pyrethroid-resistant Ae. aegypti strains, Puerto Rico (PR) and Isokdr, an Ae. aegypti anosmic Orco (olfactory receptor co-receptor) mutant, and an insecticide-susceptible Anopheles gambiae strain, Kisumu were used. All Hand in cage experiments used serial dilutions of the test compounds to generate dose response curves. Different cohorts (50 per cohort) of nulliparous 6-8 day-old female mosquitoes were used per trial. Throughout the investigation, significant levels of repellency by pyrethrum and pyrethroids were observed in the insecticide-susceptible mosquito strains although the magnitude varied with test compounds. The repellent effects by pyrethrum and pyrethroids were reduced in the two resistant strains, PR and Isokdr, compared to the susceptible strains, Waco and Rock. Pretreatment of mosquitoes with piperonyl butoxide (PBO), a P450 inhibitor, enhanced transfluthrin repellency in both PR and Waco mosquitoes, but did not abolish the difference in repellency between them. Furthermore, repellency to DEET was not significantly different between susceptible and resistant mosquito strains. Repellency against susceptible Aedes and Anopheles mosquitoes was also evaluated using two types of insecticide treated nets, PermaNet 2.0 and Olyset. Repellency of PermaNet2.0 on pyrethroid-susceptible Ae. aegypti Rock and Waco mosquitoes, and An. gambiae Kisumu mosquitoes was observed, but was reduced in pyrethroid-resistant Peurto Rico (PR) and Isokdr and anosmic orco mosquitoes. Repellency effect of the Olyset net on An. gambiae Kisumu mosquitoes was evident, but not on Ae. aegypti mosquitoes. Electroantennograph (EAG) recordings were conducted from antenna of adult Ae. aegypti. Pyrethrum and pyrethroids elicited EAG responses in Rock and Orlando mosquito strains. No EAG responses were recorded in Orco knockout mosquitoes.Studies on the diamondback moth and maize grain borer using T-maze assays and feeding choice tests revealed that pyrethroids also evoke repellency in the maize grain borer and diamondback moth. The larvae of diamondback moth preferred eating a leaf in an untreated arena than the treated one. Adults of maize grain borer preferred the untreated control arm in the T-maze assay.This study began to uncover the enigma of repellency of the one of the most important class of insecticides used globally to combat vector-borne human diseases. We confirmed repellency by pyrethrum/pyrethroids in both disease vectors and crop pests, and demonstrated that Ae. aegypti mosquitoes can sense pyrethrum/pyrethroids via an authentic Orco-dependent olfactory pathway. The repellency is likely mediated by dual activation of both sodium channels and/or olfactory receptors. Collectively, our study provides a conceptual framework for understanding of the modes of action of pyrethrum/pyrethroids as an important group of insect repellents.
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