title: Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus.
abstract: Temephos is a major organophosphate (OP) larvicide that has been used extensively for the control of Aedes albopictus and Aedes aegypti, the major vectors for viral diseases, such as dengue fever, zika and chikungunya. Resistance to temephos has been recently detected and associated with the upregulation of carboxylesterases (CCEs) through gene amplification, in both species. Here, we expressed the CCEae3a genes which showed the most striking up-regulation in resistant Aedes strains, using the baculovirus system. All CCEae3a variants encoded functional enzymes, with high activity and preference for p-nitrophenyl butyrate, a substrate that was shown capable to differentiate temephos resistant from susceptible Aedes larvae. Enzyme kinetic studies showed that CCEae3as from both Ae. aegypti and Ae. albopictus (CCEae3a_aeg and CCEae3a_alb, respectively) strongly interact with temephos oxon and slowly released the OP molecule, indicating a sequestration resistance mechanism. No difference was detected between resistant and susceptible CCEae3a_aeg variants (CCEae3a_aegR and CCEae3a_aegS, respectively), indicating that previously reported polymorphism is unlikely to play a role in temephos resistance. HPLC/MS showed that CCEae3as were able to metabolize temephos oxon to the temephos monoester [(4-hydroxyphenyl) sulfanyl] phenyl O,O-dimethylphosphorothioate. Western blot and immunolocalization studies, based on a specific antibody raised against the CCEae3a_alb showed that the enzyme is expressed at higher levels in resistant insects, primarily in malpighian tubules (MT) and nerve tissues.
authors.affiliation: Department of Biology, University of Crete, Vassilika Vouton, 73100 Heraklion, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, 73100 Heraklion, Greece.
authors.forename: Linda
authors.initials: L
authors.lastname: Grigoraki
authors.affiliation: Department of Biology, University of Crete, Vassilika Vouton, 73100 Heraklion, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, 73100 Heraklion, Greece.
authors.forename: Vassileia
authors.initials: V
authors.lastname: Balabanidou
authors.affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research 'Demokritos', Athens, Greece.
authors.forename: Christos
authors.initials: C
authors.lastname: Meristoudis
authors.affiliation: Environmental Chemical Processes Laboratory (ECPL), Department of Chemistry, University of Crete, 71003 Heraklion, Greece.
authors.forename: Antonis
authors.initials: A
authors.lastname: Myridakis
authors.affiliation: Department of Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK.
authors.forename: Hilary
authors.initials: H
authors.lastname: Ranson
authors.affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research 'Demokritos', Athens, Greece.
authors.forename: Luc
authors.initials: L
authors.lastname: Swevers
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, 73100 Heraklion, Greece; Pesticide Science Laboratory, Department of Crop Science, Agricultural University of Athens, 11855 Athens, Greece. Electronic address: vontas@imbb.forth.gr.
authors.forename: John
authors.initials: J
authors.lastname: Vontas
mesh_terms: D000330:Aedes / Q000201:enzymology / Q000235:genetics*; D000818:Animals; D043182:Carboxylesterase / Q000235:genetics* / Q000378:metabolism; D007305:Insecticide Resistance; D007306:Insecticides / Q000494:pharmacology*; D007700:Kinetics; D000002:Temefos / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: D007306:Insecticides; D043182:Carboxylesterase; D000002:Temefos
keywords: Aedes; Arbovirus; Detoxification; Diagnostics; Insecticide resistance