Mode of toxicity of the β-triketone leptospermone to Aedes aegypti mosquitoes

Leptospermone, a natural β-triketone and major constituent of manuka oil (Leptospermum scoparium), is an established inhibitor of plant HPPD and was identified to induce rapid knockdown and induce high toxicity to Aedes aegypti adults via topical and tarsal contact exposure with LD₅₀ values of 150 ng/mg of mosquito and 357 ng/cm², respectively. Although toxic to mosquitoes, leptospermone was non-toxic to ticks, the honey bee, or the fruit fly indicating a high degree of insect specificity. Importantly, leptospermone was equally toxic to non-blood fed and blood-fed mosquitoes suggesting the mode of action is not via HPPD inhibition. Molecular modeling suggested high structural similarities between leptospermone and mammalian sulfonamide carbonic anhydrase (CA) inhibitors. In vitro potency assays with mosquito midgut homogenate or purified CA verify leptospermone inhibits Ae. aegypti CA, but not mammalian CAs. CAs are metalloenzymes that regulate the pH of tissues and ubiquitously expressed throughout insect tissues but are abundantly expressed in the mosquito midgut and, thus, we tested leptospermone to alter pH regulation in the mosquito midgut. Indeed, leptospermone significantly reduced the pH of Ae. aegypti midguts when compared to control mosquitoes which further supports the notion that leptospermone mode of action in insects is via inhibition of CA. These data verify leptospermone is an effective mosquitocide that induces rapid knockdown and toxicity to Ae. aegypti at doses that approach natural pyrethrins against pyrethroid-resistant mosquito strains. Further, the data indicate leptospermone mode of action is CA inhibition, which is a novel mosquitocide target and is different when compared to the mode of action in plants.