The Vector Population Monitoring Tool (VPMT): High-Throughput DNA-Based Diagnostics for the Monitoring of Mosquito Vector Populations

Regular monitoring of mosquito vector populations is an integral component of most vector control programmes. Contemporary data on mosquito species composition, infection status, and resistance to insecticides are a prerequisite for effective intervention. For this purpose we, with funding from the Innovative Vector Control Consortium (IVCC), have developed a suite of high-throughput assays based on a single “closed-tube” platform that collectively comprise the “Vector Population Monitoring Tool” (VPMT). The VPMT can be used to screen mosquito disease vector populations for a number of traits including Anopheles gambiae s.l. and Anopheles funestus species identification, detection of infection with Plasmodium parasites, and identification of insecticide resistance mechanisms. In this paper we focus on the Anopheles-specific assays that comprise the VPMT and include details of a new assay for resistance todieldrin Rdl detection. The application of these tools, general and specific guidelines on their use based on field testing in Africa, and plans for further development are discussed

Molecular analysis of multiple cytochrome P450s from the maleria vector Anopheles gambiae

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

An adult-specific CYP6P450 gene is overexpressed in a pyrethroid-resistant strain of the malaria vector, Anopheles gambiae

Many malaria control programmes are based on insecticide application as adulticides, often in the form of pyrethroid-impregnated bed nets. However, the efficacy of this control measure can be reduced by genetic changes in vector insecticide susceptibility. Pyrethroid resistance has been detected in the major African malaria vector, Anopheles gambiae, and has been attributed to a combination of target site insensitivity and increased oxidative metabolism of the insecticide, catalysed by cytochrome P450s. An adult-specific cytochrome P450 monooxygenase 6 (CYP6) P450 gene, CYP6Z1, located within a large cluster of cytochrome P450 genes in chromosome arm 3R of An. gambiae, is expressed approximately 11-fold higher in males and 4.5-fold in females from a pyrethroid-resistant strain than in a susceptible strain from the same geographical area. In both strains, CYP6Z1 expression is higher in males than females. Southern blot analysis discounted gene amplification as a cause of this overexpression. The isolation of An. gambiae cDNAs encoding cytochrome b(5) and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-cytochrome P450 reductase cDNAs is also reported. (C) 2003 Elsevier B.V. All rights reserved

Short Report: A Multiplex PCR Assay for Simultaneous Genotyping of kdr and ace-1 Loci in Anopheles gambiae

The selection of insecticide-resistant genotypes in Anopheles gambiae, the most important malaria vector in Africa, makes disease control problematic in several endemic areas. The early detection and monitoring of resistance associated mutations in field mosquito populations is essential for the application of successful insecticide-based control interventions. Currently, the surveillance of these mutations is performed using individual assays, some of which require sophisticated and expensive equipment. Here we describe a novel multiplex polymerase chain reaction-based assay for detecting simultaneously the five single nucleotide polymorphisms in the voltage-gated sodium channel and the ace-l genes, which have been associated with the mosquito response to most commonly used insecticides

New developments in Belgian childcare policy and practice

In this article recent evolutions in the French and Flemish communty of Belgium are critically analysed. In the French Community of Belgium the importance of the pedagogical function of childcare has increased, while the policy of the Flemish Community focused on the social function. In both parts the was also a diffrent evolution concerning the professionalisation process. While the situation in the French Community did not changed, in the flemish Community a process of deprofessionalisation has been going on since the beginning of the new millenium

Pinpointing P450s associated with pyrethroid metabolism in the dengue vector, Aedes aegypti: developing new tools to combat insecticide resistance. PLoS Negl Trop Dis. 2012; 6(3):e1595. Epub 2012/04/06. doi: 10.1371/journal.pntd.0001595 PMID

Abstract Background: Pyrethroids are increasingly used to block the transmission of diseases spread by Aedes aegypti such as dengue and yellow fever. However, insecticide resistance poses a serious threat, thus there is an urgent need to identify the genes and proteins associated with pyrethroid resistance in order to produce effective counter measures. In Ae. aegypti, overexpression of P450s such as the CYP9J32 gene have been linked with pyrethroid resistance. Our aim was to confirm the role of CYP9J32 and other P450s in insecticide metabolism in order to identify potential diagnostic resistance markers

Molecular diagnostics for detecting pyrethroid and organophosphate resistance mutations in the Q biotype of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

Pyrethroid and organophosphate resistance-associated mutations have been recently reported in the whitefly Bemisia tabaci (Gennadius), a major pest of protected and outdoor crops worldwide. Here, we developed simple PCR-agarose gel visualization based assays for reliably monitoring the L9251 and T929V pyrethroid resistance mutations in the B, tabaci para-type voltage gated sodium channel and the iAChE F331W organophosphate resistance mutation in the acetylcholinesterase enzyme ace1. PCR-RFLP assays were developed for detecting the L9251 and the F331W resistance mutations. A highly specific PASA was developed for detecting the T929V mutation. The molecular diagnostic tools were used to monitor the frequency of the resistance mutations in a large number of field caught Q biotype B. tabaci from Crete (Greece), where both organophosphates and pyrethroids are extensively used. The F331W mutation was fixed in all field individuals examined. The pyrethroid resistance mutations were detected in high frequencies: 0.38 and 0.54 for L9251 and T929V, respectively. The simple diagnostics are accurate and robust, to be used alongside classical bioassays to prevent ineffective insecticide applications, and for early identification of the spreading of resistant Q biotype populations into new regions around the globe. (C) 2009 Elsevier Inc. All rights reserved

Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.

A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens