Absence of knockdown resistance suggests metabolic resistance in the main malaria vectors of the Mekong region

<p>Abstract</p> <p>Background</p> <p>As insecticide resistance may jeopardize the successful malaria control programmes in the Mekong region, a large investigation was previously conducted in the Mekong countries to assess the susceptibility of the main malaria vectors against DDT and pyrethroid insecticides. It showed that the main vector, <it>Anopheles epiroticus</it>, was highly pyrethroid-resistant in the Mekong delta, whereas <it>Anopheles minimus sensu lato </it>was pyrethroid-resistant in northern Vietnam. <it>Anopheles dirus sensu stricto </it>showed possible resistance to type II pyrethroids in central Vietnam. <it>Anopheles subpictus </it>was DDT- and pyrethroid-resistant in the Mekong Delta. The present study intends to explore the resistance mechanisms involved.</p> <p>Methods</p> <p>By use of molecular assays and biochemical assays the presence of the two major insecticide resistance mechanisms, knockdown and metabolic resistance, were assessed in the main malaria vectors of the Mekong region.</p> <p>Results</p> <p>Two FRET/MCA assays and one PCR-RFLP were developed to screen a large number of <it>Anopheles </it>populations from the Mekong region for the presence of knockdown resistance (<it>kdr</it>), but no <it>kdr </it>mutation was observed in any of the study species. Biochemical assays suggest an esterase mediated pyrethroid detoxification in <it>An. epiroticus </it>and <it>An. subpictus </it>of the Mekong delta. The DDT resistance in <it>An. subpictus </it>might be conferred to a high GST activity. The pyrethroid resistance in <it>An. minimus s.l</it>. is possibly associated with increased detoxification by esterases and P450 monooxygenases.</p> <p>Conclusion</p> <p>As different metabolic enzyme systems might be responsible for the pyrethroid and DDT resistance in the main vectors, each species may have a different response to alternative insecticides, which might complicate the malaria vector control in the Mekong region.</p

Trends in DDT and pyrethroid resistance in Anopheles gambiae s.s. populations from urban and agro-industrial settings in southern Cameroon

Background: Pyrethroid insecticides are widely used for insect pest control in Cameroon. In certain insect species, particularly the malaria vector Anopheles gambiae, resistance to this class of insecticides is a source of great concern and needs to be monitored in order to sustain the efficacy of vector control operations in the fields. This study highlights trends in DDT and pyrethroid resistance in wild An. gambiae populations from South Cameroon. Methods: Mosquitoes were collected between 2001 and 2007 in four sites in South Cameroon, where insecticides are used for agricultural or personal protection purposes. Insecticide use was documented in each site by interviewing residents. Batches of 2-4 days old adult female mosquitoes reared from larval collections were tested for susceptibility to DDT, permethrin and deltamethrin using standard WHO procedures. Control, dead and survivors mosquitoes from bioassays were identified by PCR-RFLP and characterized for the kdr mutations using either the AS-PCR or the HOLA method. Results: Four chemical insecticide groups were cited in the study sites: organochlorines, organophosphates, carbamates and pyrethroids. These chemicals were used for personal, crop or wood protection. In the four An. gambiae populations tested, significant variation in resistance levels, molecular forms composition and kdr frequencies were recorded in the time span of the study. Increases in DDT and pyrethroid resistance, as observed in most areas, were generally associated with an increase in the relative frequency of the S molecular form carrying the kdr mutations at higher frequencies. In Mangoum, however, where only the S form was present, a significant increase in the frequency of kdr alleles between 2003 to 2007 diverged with a decrease of the level of resistance to DDT and pyrethroids. Analyses of the kdr frequencies in dead and surviving mosquitoes showed partial correlation between the kdr genotypes and resistance phenotypes, suggesting that the kdr mechanism may act with certain cofactors to be identified. Conclusion: These results demonstrate the ongoing spread of kdr alleles in An. gambiae in Central Africa. The rapid evolution of insecticide resistance in this highly dynamic and genetically polymorphic species remains a challenge for its control

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. (c) 2005 Elsevier Ltd. All rights reserved

The role of gene splicing, gene amplification and regulation in mosquito insecticide resistance

The primary routes of insecticide resistance in all insects are alterations in the insecticide target sites or changes in the rate at which the insecticide is detoxified. Three enzyme systems, glutathione S–transferases, esterases and monooxygenases, are involved in the detoxification of the four major insecticide classes. These enzymes act by rapidly metabolizing the insecticide to non–toxic products, or by rapidly binding and very slowly turning over the insecticide (sequestration). In Culex mosquitoes, the most common organophosphate insecticide resistance mechanism is caused by co–amplification of two esterases. The amplified esterases are differentially regulated, with three times more Estβ2 1 being produced than Estα2 1 . Cis –acting regulatory sequences associated with these esterases are under investigation. All the amplified esterases in different Culex species act through sequestration. The rates at which they bind with insecticides are more rapid than those for their non–amplified counterparts in the insecticide–susceptible insects. In contrast, esterase–based organophosphate resistance in Anopheles is invariably based on changes in substrate specificities and increased turnover rates of a small subset of insecticides. The up–regulation of both glutathione S–transferases and monooxygenases in resistant mosquitoes is due to the effect of a single major gene in each case. The products of these major genes up–regulate a broad range of enzymes. The diversity of glutathione S–transferases produced by Anopheles mosquitoes is increased by the splicing of different 5' ends of genes, with a single 3' end, within one class of this enzyme family. The trans –acting regulatory factors responsible for the up–regulation of both the monooxygenase and glutathione S–transferases still need to be identified, but the recent development of molecular tools for positional cloning in Anopheles gambiae now makes this possible. </jats:p

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. (C) 2007 Elsevier Ltd. All rights reserved