Mosquito mutations F290V and F331W expressed in acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): Biochemical properties and inhibitor sensitivity

Authors: Temeyer, Kevin; TONG, FAN - University Of Florida; Schlechte, Kristie; CHEN, QIAO-HONG - University Of Florida; CARLIER, PAUL - Virginia Tech; Perez De Leon, Adalberto; BLOOMQUIST, JEFFREY - University Of Florida

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2025
Publication Date: 2/17/2025
Citation: Temeyer, K.B., Tong, F., Schlechte, K.G., Chen, Q., Carlier, P.R., Perez De Leon, A.A., Bloomquist, J.R. 2025. Mosquito mutations F290V and F331W expressed in acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): Biochemical properties and inhibitor sensitivity. Parasites & Vectors. volume 18, page 57. https://doi.org/10.1186/s13071-025-06691-5.
DOI: https://doi.org/10.1186/s13071-025-06691-5

Interpretive Summary: The Old-World sand fly, Phlebotomus papatasi, is a major vector for transmission of cutaneous leishmaniasis to humans and animals in Southern Europe, Southwestern Asia, the Middle East, and Northern Africa. There have been sporadic reports of sand fly resistance to insecticides, but few have been experimentally confirmed, and none have been characterized at the molecular level. Our previous research yielded a recombinant enzyme from sand flies, that contained a specific mutation (G119S) found in African mosquito acetylcholinesterase known to cause high level mosquito resistance to organophosphate (OP) insecticides. The recombinant sand fly enzyme expressing the mosquito resistance mutation was also highly resistant to OP insecticides. This recombinant enzyme was then used to screen novel synthetic insecticidal chemicals, and successfully identified some that were effective at inactivating the OP-resistant recombinant enzyme. These new chemicals could be used to control major mosquito vectors of malaria in Africa, and sand flies. However, the malaria mosquitoes evolved a new acetylcholinesterase gene with the new mutation (F290V) that helped them survive exposure to insecticides with anticholinesterase activity. The additional mutation (F331W) developed in Asian mosquitoes also produced resistance to OP insecticides. New research reported herein, documents construction of recombinant sand fly acetylcholinesterase containing each of the OP resistance mutations (G119S, F290V, and F331W) expressed alone, or in various combinations. Each of the mutations, when expressed alone, resulted in production of an OP resistant enzyme. When the mutations were combined in the same recombinant enzyme, it resulted in a complete loss of acetylcholinesterase activity, indicating that the mutations were not capable of generating even higher resistance when combined. Each of the individual mutations expressed in recombinant sand fly acetylcholinesterase were used to screen and identify novel synthetic carbamate chemicals effective at inactivating the OP-resistant enzyme constructs. In addition to sandflies and mosquitoes, these novel synthetic carbamates with improved specificity and safety, provide new capabilities to control other important insect and tick disease vectors resistant to OP and existing carbamate pesticides.

Technical Abstract: Background: The Old-World sand fly, Phlebotomus papatasi (Scopoli), a vector of zoonotic cutaneous leishmaniasis, is usually controlled by insecticides, including anticholinesterases. Previous studies revealed 85% amino acid sequence identity of recombinant P. papatasi acetylcholinesterase (rPpAChE1) to mosquito AChE and identified synthetic carbamates that selectively inhibited rPpAChE1 and circumvented the G119S mutation responsible for high level resistance to anticholinesterases. This study reports the construction, baculovirus expression, and biochemical properties of rPpAChE1 containing the F290V and F331W orthologous mutations from mosquitoes. Methods: Recombinant PpAChE1 enzymes with or without the F290V, F331W, and G119S orthologous mosquito mutations were expressed in Sf21cells utilizing the baculoviral system. Ellman assays determined changes in catalytic properties and inhibitor sensitivity resulting from wild type and mutant rPpAChE1 containing single or combinations of orthologous mosquito mutations. Results: Each of the orthologous mutations (F290V, F331W, and G119S) from mosquito AChE significantly reduced inhibition sensitivity to organophosphate or carbamate pesticides, and catalytic activity was lost when they were expressed in combination. Novel synthetic carbamates were identified that significantly inhibited the rPpAChEs expressing each of the single orthologous mosquito mutations. Conclusions: These novel carbamates could be developed as efficacious insecticides with improved specificity and safety for use in sand fly or mosquito populations expressing the mutant AChEs.