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Nolden, Melanie (2023) Characterization of cytochrome P450-mediated pyrethroid resistance in Anopheles funestus with special reference to transfluthrin, Thesis (Doctoral), Liverpool School of Tropical Medicine.
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M Nolden_120997_PhD_thesis_revised_V2_final.pdf - Accepted Version Download (13MB) | Preview |
Abstract
Vector control tools that target malaria transmitting mosquitoes of the Anopheles genus are responsible for averting 80 % of malaria cases between 2000 and 2015. However, 88 % of all WHO-recommended products for vector control products rely on pyrethroids. Pyrethroids are a class of synthetic insecticides that act on the voltage gated sodium channel. Frequent application has led to a selection of alleles conferring pyrethroid resistance. In the major malaria mosquito Anopheles funestus, pyrethroid resistance is driven by metabolic resistance associated with upregulated P450-monooxygenases (P450s), in particular CYP6P9a and CYP6P9b. The polyfluorinated pyrethroid transfluthrin has been shown to control the highly pyrethroid-resistant laboratory An. funestus FUMOZ-R strain. This volatile molecule is increasingly used in materials for vector control such as eave ribbons, chairs, or sandals. However, to date transfluthrin metabolism by P450s has not been elucidated. Synergists that inhibit P450s can also restore pyrethroid-susceptibility. Piperonylbutoxide (PBO) is the only synergist used in vector control, however there is recent evidence that mosquitoes develop resistance towards PBO.
The toxicology of common pyrethroids and transfluthrin towards pyrethroid resistant An. funestus FUMOZ-R and a susceptible FANG strain were evaluated using glazed-tile contact bioassays. Bioassays to examine synergistic effects of PBO and azole fungicides were
conducted to evaluate the involvement of P450s in pyrethroids metabolism in An. funestus FUMOZ-R and to assess the feasibility of using azoles as an alternative to PBO in vector control products. Baculovirus recombinant expression of CYP6P9a and CYP6P9b in insect cells in the presence and absence of cytochrome b5 was used to examine the role of this redox partner in pyrethroid metabolism. Finally, incubation of CYP6P9a and CYP6P9b with deltamethrin, permethrin and transfluthrin with subsequent UPLC-MS/MS analysis were conducted to examine the breakdown of these compounds.
We have shown that cytochrome b5 is not necessary in deltamethrin metabolism using recombinant baculovirus/high-5 cell expression, however it can enhance the metabolism of fluorescent model substrates. Site directed mutagenesis of valine to isoleucine residue at
position 310 of CYP6P9b, which is part of the substrate recognition site (SRS) 4 and 9.8 Å distanced from the Heme revealed enhanced metabolism towards fluorescent model substrates and towards permethrin. This mutation is present in permethrin resistant
populations in Benin, confirming a potential resistance marker for permethrin resistance. Furthermore, we have revealed in-vivo the synergism of the azole fungicides 1-ABT and triflumizole in combination with different pyrethroids towards resistant An. funestus and
shown the nanomolar inhibition of triflumizole, prochloraz and ketoconazole towards recombinant CYP6P9a and CYP6P9b. Finally, we have shown that transfluthrin is protected from P450 oxidation due to the fluorination of common sites of attack on the benzyl ring and
the lack of the phenoxybenzylether. The cleavage of the phenoxybenzylether as well as hydroxylation of 4’-para- position is a preferred route of metabolism of the P450 enzymes
CYP6P9a and CYP6P9b conferring high level of deltamethrin and permethrin resistance. This work emphasizes the resistant breaking potential of transfluthrin and suggests the further evaluation of azole fungicides in vector control products to extend the lifetime of
pyrethroids.
| Item Type: | Thesis (Doctoral) | ||||||||
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| Subjects: | QX Parasitology > QX 20 Research (General) QX Parasitology > Insects. Other Parasites > QX 515 Anopheles |
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| Faculty: Department: | Biological Sciences > Vector Biology Department | ||||||||
| Depositing User: | Lynn Roberts-Maloney | ||||||||
| Date Deposited: | 22 Mar 2023 15:16 | ||||||||
| Last Modified: | 22 Jun 2023 01:11 | ||||||||
| URI: | https://archive.lstmed.ac.uk/id/eprint/22171 |
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