High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand.

OBJECTIVES: To develop rapid monitoring tools to detect the F1534C permethrin-resistance mutation in domain IIIS6 of the Aedes aegypti voltage-gated sodium channel gene and determine the frequency and distribution of this mutation in Thailand. METHODS: A TaqMan SNP genotyping and an allele specific PCR (AS-PCR) assay were developed and validated by comparison with DNA sequencing of homozygous susceptible and homozygous resistant laboratory strains, their reciprocal-cross progenies, and field-caught mosquitoes. To determine the resistance phenotype of wild-caught A. aegypti, mosquitoes were exposed to 0.75% permethrin paper. The AS-PCR assay was used to screen 619 individuals from 20 localities throughout Thailand. RESULTS: Overall, both assays gave results consistent with DNA sequencing for laboratory strains of known genotype and for wild-caught A. aegypti. The only slight discrepancy was for the AS-PCR method, which overestimated the mutant allele frequency by 1.8% in wild-caught samples. AS-PCR assays of permethrin-exposed samples show that the mutant C1534 allele is very closely associated with the resistant phenotype. However, 19 permethrin-resistant individuals were homozygous for the wild-type F1534 allele. DNA sequencing revealed all these individuals were homozygous for two other mutations in domain II, V1016G and S989P, which are known to confer resistance (Srisawat et al. 2010). The F1534C mutation is widespread in Thailand with mutant allele frequencies varying among populations from 0.20 to 1.00. CONCLUSIONS: These assays can be used for the rapid detection of the F1534C resistance mutation in A. aegypti populations. The F1534C, and other, mutations underlie an extremely high prevalence of pyrethroid resistance in Thailand.

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

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.

Expression and characterization of three new glutathione transferases, an epsilon (AcGSTE2-2), omega (AcGSTO1-1), and theta (AcGSTT1-1) from Anopheles cracens (Diptera: Culicidae), a major Thai malaria vector.

Glutathione transferases (GSTs) (E.C.2.5.1.18) are multifunctional enzymes involved in the detoxification of many exogenous and endogenous compounds. This study aimed to characterize several new GSTs from Anopheles cracens, a major Thai malaria vector formerly known as Anopheles dirus. The three recombinant enzymes obtained were from the epsilon, theta and omega classes. They showed 80-93% identity to orthologous An. gambiae GSTs. AcGSTE2-2 possessed peroxidase activity that cannot be detected for the An. gambiae AgGSTE2-2. AcGSTT1-1 had high activity toward several substrates that are specific for mammalian theta class. The AcGSTO1-1 can use 1-chloro-2,4-dinitrobenzene, dichloroacetic acid, and hydroxyethyl disulfide substrates. The enzymes bound but did not metabolize the organophosphate temephos. The epsilon AcGSTE2-2 functioned as a peroxidase and DDT metabolizing enzyme. The theta AcGSTT1-1 functioned not only as peroxidase but also acted as a binding protein for organophosphates. The omega GST had thiol transferase activity suggesting a role in oxidative stress response.

Molecular cloning and expression of several new Anopheles cracens epsilon class glutathione transferases.

Glutathione transferases, GSTs, are detoxification proteins that are found in most organisms. The acGSTE3-3 had the ability to conjugate 4-hydroxynonenal, a cytotoxic lipid peroxidation product. Although other Epsilon GSTs showed roles in insecticide metabolism, the acGSTE3-3 appeared to have a major role in detoxifying lipid peroxidation products conferring protection against oxidative damage.

The Aedes aegypti glutathione transferase family.

In this report, we describe the glutathione transferase (GST) gene family in the dengue vector Aedes aegypti and suggest a novel role for a new class of mosquito GSTs. Twenty-six GST genes are present in Ae. aegypti, two of which are alternatively spliced to give a total of 29 transcripts for cytosolic GSTs. The six classes identified in other insect species are all represented and, as in Anopheles gambiae, the majority of the mosquito GSTs belong to the insect-specific Delta and Epsilon classes with eight members each. Sixteen secure 1:1 orthologs were identified between GSTs in Ae. aegypti and An. gambiae, but only four of these have recognisable orthologs in Drosophila melanogaster. Three mosquito-specific GSTs were identified which did not belong to any previously recognised GST classes. One of these, GSTx2, has been previously implicated in conferring 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) resistance in Ae. aegypti from South America. However, we found no evidence for increased levels of this GST protein in DDT/pyrethroid-resistant populations from Thailand. Furthermore, we show that the recombinant GSTX2-2 protein is unable to metabolise DDT. Interestingly, GSTX2-2 showed an affinity for hematin, and this, together with the restricted distribution of this class to haematophagous insects, may indicate a role for these enzymes in protecting mosquitoes against heme toxicity during blood feeding.

A survey of dengue viral infection in Aedes aegypti and Aedes albopictus from re-epidemic areas in the north of Thailand using nucleic acid sequence based amplification assay.

Immature stages of Aedes aegypti and Ae. albopictus were collected from 17 dengue re-epidemic areas in Chiang Mai and Lampang Provinces, in the north of Thailand. They were reared to adults and tested for dengue viral RNA by a nucleic acid sequence based amplification assay (NASBA). Of a total of 9,825 Ae. aegypti and 150 Ae. albopictus examined, none of them were found positive for the virus, suggesting that transovarial transmission may be very low in the vector populations and may not play a significant role in the epidemiology of dengue infection in Thailand.

Identification, characterization and structure of a new Delta class glutathione transferase isoenzyme.

The insect GST (glutathione transferase) supergene family encodes a varied group of proteins belonging to at least six individual classes. Interest in insect GSTs has focused on their role in conferring insecticide resistance. Previously from the mosquito malaria vector Anopheles dirus, two genes encoding five Delta class GSTs have been characterized for structural as well as enzyme activities. We have obtained a new Delta class GST gene and isoenzyme from A. dirus, which we name adGSTD5-5. The adGSTD5-5 isoenzyme was identified and was only detectably expressed in A. dirus adult females. A putative promoter analysis suggests that this GST has an involvement in oogenesis. The enzyme displayed little activity for classical GST substrates, although it possessed the greatest activity for DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] observed for Delta GSTs. However, GST activity was inhibited or enhanced in the presence of various fatty acids, suggesting that the enzyme may be modulated by fatty acids. We obtained a crystal structure for adGSTD5-5 and compared it with other Delta GSTs, which showed that adGSTD5-5 possesses an elongated and more polar active-site topology.

Intra-subunit residue interactions from the protein surface to the active site of glutathione S-transferase AdGSTD3-3 impact on structure and enzyme properties.

Structural residues are one of the major factors that modulate the catalytic specificity as well as having a role in stability of the glutathione S-transferases (GST). To understand how residues remote from the active site can affect enzymatic properties, four mutants, His144Ala, Val147Leu, Val147Ala and Arg96Ala, were generated. The selected residues appear to be in a putative intra-subunit interaction pathway from the exterior Asp150 to the active site Arg66 of AdGSTD3-3. The analysis of the four mutants suggested that the interaction formed between Asp150 and His144 is required for the packing of the hydrophobic core in domain 2. Mutations of both Asp150 and His144 impacted upon enzymatic properties. Two Val147 mutants also showed contribution to packing and support of the N-capping box motif by demonstrating shorter half-lives. The planar guanidinium of Arg96 is in a stacked geometry with the face of the aromatic ring of Phe140 in a cation-pi interaction. The Arg96 also interacts with several other residues one of which, Asp100, is in the active site. These interactions restrict movement of the residues in this region and as the data demonstrates when Arg96 is changed have dramatic impact on stability and enzyme properties. These findings indicate the significance of the roles played by residue interactions which can cause conformational changes and thereby influence the catalytic activity and stability of an enzyme.

Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti.

Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. In the mosquito, Anopheles gambiae, increased expression of an Epsilon class GST, GSTE2, confers resistance to DDT. We have identified eight GST genes in the dengue vector, Aedes aegypti. Four of these belong to the insect specific GST classes Delta and Epsilon and three are from the more ubiquitously distributed Theta and Sigma classes. The expression levels of the two Epsilon genes, a Theta GST and a previously identified Ae. aegypti GST [Grant and Hammock, 1992. Molecular and General Genetics 234, 169-176] were established for an insecticide susceptible and a resistant strain. We show that the putative ortholog of GSTe2 in Ae. aegypti (AaGSTe2) is over expressed in mosquitoes that are resistant to the insecticides DDT and permethrin. Characterisation of recombinant AaGSTE2-2 confirmed the role of this enzyme in DDT metabolism. In addition, unlike its Anopheles ortholog, AaGSTE2-2 also exhibited glutathione peroxidase activity.

Trypsin and aminopeptidase activities in blood-fed females Anopheles dirus (Diptera: Culicidae) of differing susceptibility to Plasmodium yoelii nigeriensis.

Midgut proteolytic enzymes contribute to the success or failure of Plasmodium infection of the mosquito. The present study investigated trypsin and aminopeptidase activities in the midgut of two strains of Anopheles dirus selected for susceptibility and refractoriness to Plasmodium yoelii nigeriensis. At intervals of 6 hours following a bloodmeal, the midguts of fully engorged female mosquitos were dissected, homogenized, and assayed for enzyme activity. No differences trypsin activity (nmole/min) were observed between the two strains throughout the course of blood digestion. By contrast, the aminopeptidase activity measured at 0 to 18 hours post-feeding was the same for the two strains, but at 24, 30 and 36 hours significantly less activity was observed in the refractory females. The results suggest neither trypsin nor aminopeptidase plays a role in the limitation of parasite development.

Insecticide susceptibility tests of Anopheles minimus s.l., Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus in northern Thailand.

The susceptibility of Anopheles minimus s.l., Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus to insecticide in northern Thailand was monitored by using the WHO standard susceptibility test. One- to two-day old female mosquitos, which were reared from wild caught females or immature stages, were exposed to discriminating dosages of insecticides for recommended exposure periods, and the 24-hour mortality recorded. The results revealed that, in general, An. minimus s.l. was still susceptible to DDT and permethrin, except in some areas where a slight increase in tolerance to DDT was observed. Ae. aegypti and Ae. albopictus were both highly resistant to DDT, but in some areas the former was also resistant to permethrin and deltamethrin. Cx. quinquefasciatus was resistant to DDT and etofenprox, with a slight increase in tolerance to permethrin, deltamethrin, malathion and fenitrothion. No resistance to lambda-cyhalothrin was detected in any of the species studied.

The role of the Aedes aegypti Epsilon glutathione transferases in conferring resistance to DDT and pyrethroid insecticides.

The Epsilon glutathione transferase (GST) class in the dengue vector Aedes aegypti consists of eight sequentially arranged genes spanning 53,645 bp on super contig 1.291, which maps to chromosome 2. One Epsilon GST, GSTE2, has previously been implicated in conferring resistance to DDT. The amino acid sequence of GSTE2 in an insecticide susceptible and a DDT resistant strain differs at five residues two of which occur in the putative DDT binding site. Characterization of the respective recombinant enzymes revealed that both variants have comparable DDT dehydrochlorinase activity although the isoform from the resistant strain has higher affinity for the insecticide. GSTe2 and two additional Epsilon GST genes, GSTe5 and GSTe7, are expressed at elevated levels in the resistant population and the recombinant homodimer GSTE5-5 also exhibits low levels of DDT dehydrochlorinase activity. Partial silencing of either GSTe7 or GSTe2 by RNA interference resulted in an increased susceptibility to the pyrethroid, deltamethrin suggesting that these GST enzymes may also play a role in resistance to pyrethroid insecticides.

A survey of dengue viral infection in Aedes aegypti and Aedes albopictus from re-epidemic areas in the north of Thailand using nucleic acid sequence based amplification assay.

Immature stages of Aedes aegypti and Ae. albopictus were collected from 17 dengue re-epidemic areas in Chiang Mai and Lampang Provinces, in the north of Thailand. They were reared to adults and tested for dengue viral RNA by a nucleic acid sequence based amplification assay (NASBA). Of a total of 9,825 Ae. aegypti and 150 Ae. albopictus examined, none of them were found positive for the virus, suggesting that transovarial transmission may be very low in the vector populations and may not play a significant role in the epidemiology of dengue infection in Thailand.

Trypsin and aminopeptidase activities in blood-fed females Anopheles dirus (Diptera: Culicidae) of differing susceptibility to Plasmodium yoelii nigeriensis.

Midgut proteolytic enzymes contribute to the success or failure of Plasmodium infection of the mosquito. The present study investigated trypsin and aminopeptidase activities in the midgut of two strains of Anopheles dirus selected for susceptibility and refractoriness to Plasmodium yoelii nigeriensis. At intervals of 6 hours following a bloodmeal, the midguts of fully engorged female mosquitos were dissected, homogenized, and assayed for enzyme activity. No differences trypsin activity (nmole/min) were observed between the two strains throughout the course of blood digestion. By contrast, the aminopeptidase activity measured at 0 to 18 hours post-feeding was the same for the two strains, but at 24, 30 and 36 hours significantly less activity was observed in the refractory females. The results suggest neither trypsin nor aminopeptidase plays a role in the limitation of parasite development.

Insecticide susceptibility tests of Anopheles minimus s.l., Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus in northern Thailand.

The susceptibility of Anopheles minimus s.l., Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus to insecticide in northern Thailand was monitored by using the WHO standard susceptibility test. One- to two-day old female mosquitos, which were reared from wild caught females or immature stages, were exposed to discriminating dosages of insecticides for recommended exposure periods, and the 24-hour mortality recorded. The results revealed that, in general, An. minimus s.l. was still susceptible to DDT and permethrin, except in some areas where a slight increase in tolerance to DDT was observed. Ae. aegypti and Ae. albopictus were both highly resistant to DDT, but in some areas the former was also resistant to permethrin and deltamethrin. Cx. quinquefasciatus was resistant to DDT and etofenprox, with a slight increase in tolerance to permethrin, deltamethrin, malathion and fenitrothion. No resistance to lambda-cyhalothrin was detected in any of the species studied.