Enzymes-based resistant mechanism in pyrethroid resistant and susceptible Aedes aegypti strains from northern Thailand

Abstract

Previous studies have shown that permethrin resistance in our selected PMD-R strain of Aedes aegypti from Chiang Mai, Thailand, was associated with a homozygous mutation in the knockdown resistance (kdr) gene and other mechanisms. In this study, we investigated the metabolic mechanism of resistance of this strain compared to the PMD strain which is susceptible to permethrin. The permethrin susceptibility of larvae was determined by a dose-response bioassay. Two synergists, namely piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate (BNPP), were also added to determine if the resistance is conferred by oxidase or esterase enzymes, respectively. The LC(50) value for PMD-R (25.42 ppb) was ∼25-fold higher than for PMD (1.02 ppb). The LC(50) was reduced 3.03-fold in PMD-R and 2.27-fold in PMD when the oxidase inhibitor (PBO) was added, but little or no reduction was observed in the presence of BNPP, indicating that oxidative enzymes play an important role in resistance. However, the LC(50) previously observed in the heterozygous mutation form was reduced ∼eightfold, indicating that metabolic resistance is inferior to kdr. The levels of cytochrome P450 (P450) extracted from fourth instar larvae were similar in both strains and were about 2.3-fold greater in microsomal fractions than in crude supernatant and cytosol fractions. Microsome oxidase activities were determined by incubation with each of three substrates, i.e., permethrin, phenoxybenzyl alcohol (PBOH), and phenoxybenzaldehyde (PBCHO), in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD(+)), PBO, and BNPP. It is known that hydrolysis of permethrin produces PBOH which is further oxidized to PBCHO by alcohol dehydrogenase (ADH) and then to phenoxybenzoic acid (PBCOOH) by aldehyde dehydrogenase (ALDH). When incubated with permethrin, a small amount of PBCOOH was detected in both strains (about 1.1-1.2 nmol/min/mg protein), regardless of the addition of NADPH. The addition of PBO resulted in about 70% and 50% reduction of PBCOOH in PMD and PMD-R, respectively. The addition of BNPP reduced PBCOOH about 50% and 35% in PMD and PMD-R, respectively. Using PBOH as substrate increased PBCOOH ∼16-fold and ∼40-fold in PMD and PMD-R, respectively. Using PBCHO as substrate increased PBCOOH ∼26-fold and ∼50-fold in PMD and PMD-R, respectively. The addition of NADPH, and particularly NAD(+), increased the level of PBCOOH. Together, the results have indicated the presence of a metabolic metabolism involving P450, ADHs, and ALDHs in both PMD and PMD-R strains, with greater enzyme activity in the latter.

Identification and Characterisation of Aedes aegypti Aldehyde Dehydrogenases Involved in Pyrethroid Metabolism

“…In mammals, the oxidation of pyrethroids was catalysed by ALDH [16]. A study in insecticide metabolism revealed the important role of ALDH in the detoxification of pyrethroid in mosquito [20]. Multiple detoxification enzymes were identified as a target of pyrethroid activity-based probes in rat proteome, including P450s, UDP-glucuronosyltransferases, Flavin-containing monooxygenase and ALDH [45].…”

“…aegypti , it has been reported that parental permethrin can be hydrolysed in vitro. Our previous study demonstrated that the formation of PBacid was decreased in the presence of an esterase inhibitor, BNPP, suggesting the function of esterases in permethrin metabolism [20]. The importance of particular CEs in pyrethroid detoxification has not yet been studied.…”

“…In the mosquito Anopheles gambiae , the up-regulation of ALDH after exposure to permethrin has been reported [19]. Enzyme-based metabolite assays also indicated that the catalytic activity of P450s, ADHs and ALDHs were increased in microsomal fractions of a DDT/permethrin-resistant strain (PMD-R) of Aedes aegypti from Thailand [20]. In our preliminary study using a proteomic approach, crude homogenates of 4 th instar larvae of Aedes mosquitoes were partially purified using glutathione agarose columns.…”

Characterization of metabolic detoxifying enzymes in an insecticide resistant strain of <i>Aedes aegypti</i> harboring homozygous S989P and V1016G <i>kdr</i> mutations

“…Two ALDH genes, ALDH9948 and ALDH14080 were upregulated in the PMD-R strain and both recombinant proteins showed oxidase activity against phenoxybenzyl aldehyde, an aldehyde moiety of pyrethroid (Lumjuan et al, 2014). The addition of piperonyl butoxide (PBO), an oxidase inhibitor, to larval bioassays of both PMD-R and F/C 1534 heterozygous individuals reduced permethrin resistance by ~3 fold and ~8 fold, respectively (Yanola et al, 2010;Somwang et al, 2011). Permethrin resistance in the PMD-R strain is conferred mainly by the F1534C mutation with oxidative enzymes playing a partial role in overall resistance.…”

MiR-278-3p regulates pyrethroid resistance in Culex pipiens pallens

“…Unfortunately, excessive and continuous application of pyrethroids has caused the development of pyrethroid resistance, which has become the major obstacle to control the mosquito borne diseases. The mechanisms responsible for pyrethroid resistance are mainly associated with target site modification and metabolic resistance (Bouvier et al 2002; Somwang et al 2011). Overexpression and/or elevated activity of P450s may enhance biodegradation of the pyrethroids (Lertkiatmongkol et al 2011).…”