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Insecticides play a crucial role in controlling the transmission of mosquito-borne diseases and the development and spread of insecticide resistance is a major threat to sustainable control. Guidelines developed by the WHO to monitor for insecticide resistance recommend using 1-3 day old, non blood fed female mosquitoes. This standardisation facilitates comparison between different tests, which is important when monitoring for spatial or longitudinal variations in resistance in the field. However, mosquitoes of this age cannot transmit human pathogens. In order to transmit disease, the mosquito must live long enough to pick up the pathogen via a blood meal, survive the extrinsic incubation period and then pass on the pathogen during a subsequent blood meal. Previous studies have reported declines in insecticide resistance with mosquito age. If widely applicable this would have important implications for predictions of the impact of resistance that are based on results from WHO bioassays. This study investigated the impact of senescence and blood feeding on insecticide induced mortality in six different mosquito populations and found higher mortality after insecticide exposure in older mosquitoes in three populations of Aedes aegypti and two Anopheles gambiae populations. Age dependent changes in the expression of a known insecticide detoxification gene, GSTe2, and in the frequency of a target site mutation (kdr 1014F) were investigated in an attempt to explain the results., (Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations., Methodology/principal Findings: We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically., Conclusions/significance: The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.

The Grand Cayman population of Aedes aegypti is highly resistant to DDT and pyrethroid insecticides. Glutathione transferase, cytochrome P450, and esterase levels were increased in the Grand Cayman population relative to a susceptible laboratory strain, but synergist studies did not implicate elevated insecticide detoxification as a major cause of resistance. The role of target site resistance was therefore investigated. Two substitutions in the voltage-gated sodium channel were identified, V1016I in domain II, segment 6 (IIS6) (allele frequency = 0.79) and F1534C in IIIS6 (allele frequency = 0.68). The role of the F1534C mutation in conferring resistance to insecticides has not been previously established and so a tetraplex polymerase chain reaction assay was designed and used to genotype mosquitoes that had been exposed to insecticides. The F1534C mutation was strongly correlated with resistance to DDT and permethrin.

Dengue fever and its more serious complications dengue haemorrhagic fever and dengue shock syndrome are growing public health problems in tropical and subtropical countries. In the absence of a vaccine, most dengue control programmes rely heavily on the use of insecticides to target the Aedes mosquito vectors. As a limited number of insecticides are routinely used in control, monitoring for the presence of resistance is an essential component of dengue prevention programmes. The pyrethroid insecticides target the voltage-gated sodium channel on the insects' neurons. Substitutions at residue 1016 of this protein have been associated with pyrethroid and DDT resistance in Aedes aegypti populations from Latin America and Asia. Here we report on the development of a simple colorimetric assay to detect these mutations in individual mosquitoes. Evaluation of this diagnostic assay on 180 Ae. aegypti individuals from Thailand revealed the presence of high frequencies of the Val1016Gly mutation throughout the country. The assay requires no specialised equipment and will enable monitoring for insecticide resistance associated alleles to be routinely incorporated into dengue surveillance operations.

Pyrethroids are commonly used as mosquito adulticides and evolution of resistance to these compounds is a major threat to public health. 'Knockdown resistance' to pyrethroids (kdr) is frequently caused by nonsynonymous mutations in the voltage-gated sodium channel transmembrane protein (para) that reduce pyrethroid binding. Early detection of kdr is critical to the development of resistance management strategies in mosquitoes including Aedes aegypti, the most prevalent vector of dengue and yellow fever viruses. Brengues et al. described seven novel mutations in hydrophobic segment 6 of domain II of para in Ae. aegypti. Assays on larvae from strains bearing these mutations indicated reduced nerve sensitivity to permethrin inhibition. Two of these occurred in codons Iso1011 and Val1016 in exons 20 and 21 respectively. A transition in the third position of Iso1011 encoded a Met1011 replacement and a transversion in the second position of Val1016 encoded a Gly1016 replacement. We have screened this same region in 1318 mosquitoes in 32 additional strains; 30 from throughout Latin America. While the Gly1016 allele was never detected in Latin America, we found two new mutations in these same codons. A transition in the first position of codon 1011 encodes a Val replacement while a transition in the first position of codon 1016 encodes an Iso replacement. We developed PCR assays for these four mutations that can be read either on an agarose gel or as a melting curve. Selection experiments, one with deltamethrin on a field strain from Santiago de Cuba and another with permethrin on a strain from Isla Mujeres, Mexico rapidly increased the frequency of the Iso1016 allele. Bioassays of F(3) offspring arising from permethrin susceptible Val1016 homozygous parents and permethrin resistant Iso1016 homozygous parents show that Iso1016 segregates as a recessive allele in conferring kdr. Analysis of segregation between alleles at the 1011 and 1016 codons in the F(3) showed a high rate of recombination even though the two codons are only separated by a ~250 bp intron. The tools and information presented provide a means for early detection and characterization of kdr that is critical to the development of strategies for resistance management.