Mode of toxicity of the ß-triketone, leptospermone, to Aedes aegypti mosquitoes

Authors: MCCOMIC, SARAH - University Of Florida; GELDENHUYS, WERNER - West Virginia University; Cantrell, Charles; CHEN, RUI - University Of Florida; MISHRA, SHOVA - University Of Florida; BURGESS IV, EDWIN - University Of Florida; SWALE, DANIEL - University Of Florida; ANDERSON, TROY - University Of Nebraska

Submitted to: Pesticide Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2025
Publication Date: 4/1/2025
Citation: Mccomic, S.E., Geldenhuys, W.J., Cantrell, C.L., Chen, R., Mishra, S., Burgess Iv, E.R., Swale, D.R., Anderson, T.D. 2025. Mode of toxicity of the ß-triketone, leptospermone, to Aedes aegypti mosquitoes. Pesticide Biochemistry and Physiology. 210(2025):106401. https://doi.org/10.1016/j.pestbp.2025.106401.
DOI: https://doi.org/10.1016/j.pestbp.2025.106401

Interpretive Summary: Mosquito- and tick-vectored pathogens that cause human disease are emerging or re-emerging throughout the world and thus, there is a need to develop new products that kill arthropod vectors or otherwise prevent human mosquito or tick bites. In addition to the baseline requirements of high toxicity and affordable, new insecticides are required to meet increasingly stringent regulatory requirements that has challenged the development of novel chemistry and target sites by increasing the time to commercialization and exponentially increasing cost of development. To address gaps in chemical availability for development of HPPD-directed insecticides, we aimed to test the ß-triketone-rich essential oil of manuka oil (Leptospermum scoparium). A major component of manuka oil is the ß-triketone leptospermone that is known to inhibit plant HPPD and thus, we aimed to test the toxicological profile of leptospermone to blood-fed mosquitoes to identify chemical leads of natural origin that can be repurposed as novel mosquitocides. Unexpectedly, data suggests the mechanism of toxicity for leptospermone in mosquitoes is unlikely to be HPPD and thus, the goal of this study was to utilize classic toxicological methods, electrophysiology, and molecular modeling to define the mechanism of toxicity of leptospermone. The strong correlation of leptospermone structural similarity to other established carbonic anhydrase (CA) inhibitors prompted CA enzyme activity testing with Aedes aegypti and leptospermone. These studies provide evidence that leptospermone elicits a different mode of action in arthropods than other ß-triketones, opening the door for a potential novel product that will be highly selective towards arthropods yet remaining non-toxic to mammals.

Technical Abstract: Inhibitors of the HPPD enzyme in plants have previously been shown to also elicit HPPD inhibition and induce toxicity to blood-fed hematophagous arthropods. However, leptospermone, a natural ß-triketone isolated from Callistemon citrinus has not provided the same toxicity or physiological responses to arthropods, and blood- or non-blood fed status was not a factor. The objective of this study was to unravel the potential mechanism of action, toxicity, and physiological responses of leptospermone to different arthropod species. Topical exposure of leptospermone showed no significant differences in toxicity between sugar- or blood-fed Ae. aegypti with LD50 of 398.3 ng/mosquito (95% CI: 320.7-491.0 ng/mosquito, Hillslope: 2.4, r2: 0.96) and 616.4 ng/mosquito (95% CI: 483.6-794.7 ng/mosquito, Hillslope: 1.8, r2: 0.84), respectively. Additionally, headless fourth instar Ae. agypti in the presence of leptospermone in water produced fewer movements and showed paralytic activity compared to controls. Extracellular CNS recordings with D. melanogaster provided inhibitory nerve firing responses similar to the effect of GABA agonists, but intracellular patch clamp studies ruled out any possibility of GABA agonism by leptospermone. Molecular modeling of leptospermone showed high structural similarity to known carbonic anhydrase (CA) inhibitors, leading us to perform an enzyme activity assay to determine if leptospermone inhibits CA. Leptospermone significantly inhibited activity from midgut samples of Ae. aegypti but not H. sapiens or B. taurus purified CA. Further, no inhibition was seen with acetazolamide, a known CA inhibitor, to Ae. aegypti CA leading us to develop a purification assay of Ae. aegypti CA. Significant inhibition still occurred with leptospermone but not with acetazolamide, suggesting allosteric site differences between mammals and arthropod CAs. Taken together, these studies provide evidence that leptospermone elicits a different mode of action in arthropods than other ß-triketones, opening the door for a potential novel product that will be highly selective towards arthropods yet remaining non-toxic to mammals.