The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chik... more The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated. Temephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE), but not target site resistance (altered AChE), with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the...
Bendiocarb was introduced for the first time for Indoor Residual Spraying (IRS) in Tanzania in 20... more Bendiocarb was introduced for the first time for Indoor Residual Spraying (IRS) in Tanzania in 2012 as part of the interim national insecticide resistance management plan. This move followed reports of increasingly alarming levels of pyrethroid resistance across the country. This study used the insecticide quantification kit (IQK) to investigate the intra-operational IRS coverage and quality of spraying, and decay rate of bendiocarb on different wall surfaces in Kagera region. To assess intra-operational IRS coverage and quality of spraying, 104 houses were randomly selected out of 161,414 sprayed houses. A total of 509 samples (218 in Muleba and 291 in Karagwe) were obtained by scraping the insecticide samples from wall surfaces. To investigate decay rate, 66 houses (36 in Muleba and 30 in Karagwe) were selected and samples were collected monthly for a period of five months. Laboratory testing of insecticide concentration was done using IQK(TM) [Innovative Vector Control Consortium...
The subclass Acari, comprising mites and ticks, is one of the largest and biologically most diver... more The subclass Acari, comprising mites and ticks, is one of the largest and biologically most diverse groups within the class of the Arachnida, which also includes scorpions, spiders and harvestmen. They are distributed worldwide and have successfully colonised a wide range of terrestrial and aquatic habitats. The majority of mite species living on the aerial parts of higher plants feed
Background: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plas... more Background: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared with three previously described PCR techniques.
The elevated expression of enzymes with insecticide metabolism activity can lead to high levels o... more The elevated expression of enzymes with insecticide metabolism activity can lead to high levels of insecticide resistance in the malaria vector, Anopheles gambiae. In this study, adult female mosquitoes from an insecticide susceptible and resistant strain were dissected into four different body parts. RNA from each of these samples was used in microarray analysis to determine the enrichment patterns of the key detoxification gene families within the mosquito and to identify additional candidate insecticide resistance genes that may have been overlooked in previous experiments on whole organisms. A general enrichment in the transcription of genes from the four major detoxification gene families (carboxylesterases, glutathione transferases, UDP glucornyltransferases and cytochrome P450s) was observed in the midgut and malpighian tubules. Yet the subset of P450 genes that have previously been implicated in insecticide resistance in An gambiae, show a surprisingly varied profile of tiss...
Insecticide resistance poses a serious threat to current malaria control efforts. The Anopheles g... more Insecticide resistance poses a serious threat to current malaria control efforts. The Anopheles gambiae genome will enable identification of new resistance genes and will provide new molecular targets for the design of more effective insecticides.
Primary structural information including the complete nucleotide sequence of the first insect ald... more Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase (AO) was obtained from the common house mosquito Culex quinquefasciatus (Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO. The Culex AO sequence contains a molybdopterin cofactor binding domain and two iron-sulfur centres. A comparison of the partial sequences of AO from insecticide resistant and susceptible strains of C. quinquefasciatus shows two distinct alleles of this enzyme, one of which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases. The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae. The elevated enzyme can be seen as a separate band on polyacrylamide gel electrophoresis. The role of AO in xenobiotic oxidation in mammals and the partial inhibition of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance.
Proceedings of the National Academy of Sciences, 2005
Metabolic pathways play an important role in insecticide resistance, but the full spectra of the ... more Metabolic pathways play an important role in insecticide resistance, but the full spectra of the genes involved in resistance has not been established. We constructed a microarray containing unique fragments from 230 Anopheles gambiae genes putatively involved in insecticide metabolism [cytochrome P450s (P450s), GSTs, and carboxylesterases and redox genes, partners of the P450 oxidative metabolic complex, and various controls]. We used this detox chip to monitor the expression of the detoxifying genes in insecticide resistant and susceptible An. gambiae laboratory strains. Five genes were strongly up-regulated in the dichlorodiphenyltrichloroethane-resistant strain ZAN͞U. These genes included the GST GSTE2, which has previously been implicated in dichlorodiphenyltrichloroethane resistance, two P450s, and two peroxidase genes. GSTE2 was also elevated in the pyrethroidresistant RSP strain. In addition, the P450 CYP325A3, belonging to a class not previously associated with insecticide resistance, was expressed at statistically higher levels in this strain. The applications of this detox chip and its potential contribution to malaria vector insecticide resistance management programs are discussed. mosquito ͉ cytochrome P450 ͉ GST ͉ carboxylesterase This paper was submitted directly (Track II) to the PNAS office.
Insecticide resistance is a worldwide problem with major impact on agriculture and human health. ... more Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w 2 ]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over-and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ,1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results.
Background: Monitoring of insect vector populations with respect to their susceptibility to one o... more Background: Monitoring of insect vector populations with respect to their susceptibility to one or more insecticides is a crucial element of the strategies used for the control of arthropod-borne diseases. This management task can nowadays be achieved more efficiently when assisted by IT (Information Technology) tools, ranging from modern integrated databases to GIS (Geographic Information System). Here we describe an application ontology that we developed de novo, and a specially designed database that, based on this ontology, can be used for the purpose of controlling mosquitoes and, thus, the diseases that they transmit.
Background: Pyrethroid insecticides are widely utilized in dengue control. However, the major vec... more Background: Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance.
Background: Insecticide-based methods represent the most effective means of blocking the transmis... more 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.
We have recently identified two resistance-associated point mutations of organophosphate (OP)-ins... more We have recently identified two resistance-associated point mutations of organophosphate (OP)-insensitive acetylcholinesterase in the olive fruit fly Bactrocera oleae, the most important olive orchard pest world-wide. We have developed simple PCR-restriction fragment length polymorphism assays for each mutation, utilising an AccI restriction site created by Ile214Val, and a BssHII restriction site destroyed by a neutral change always accompanying the second mutation Gly488Ser. Samples from Greece homozygous for both mutations proved the most insensitive to dimethoate. The frequencies of these mutations in field-collected samples from several countries were investigated. Ninety-three percent of samples from Greece and Albania, where OPs have been extensively used in B. oleae control, were homozygous for both mutations. Resistance-associated alleles were detected at lower frequencies, but still with both mutations in conjunction in the majority of cases, in western Mediterranean countries with limited use of OPs. Samples from South Africa, however, did not have either of the resistance-associated mutations. The double mutation haplotype clearly confers a strong selective advantage in field populations of B. oleae exposed to OPs.
... PJ Skouras, JT Margaritopoulos, NA Seraphides, IM Ioannides, EG Kakani, KD Mathiopoulos, JA T... more ... PJ Skouras, JT Margaritopoulos, NA Seraphides, IM Ioannides, EG Kakani, KD Mathiopoulos, JA Tsitsipis, Organophosphate resistance in olive fruit fly, Bactrocera oleae, populations in Greece and Cyprus, Pest Manag Sci. 63 (2007) 42-48. 21. PS Orphanidis, P. Kalmoukos, B ...
The codling moth Cydia pomonella L. is controlled mostly with chemical insecticides in Greece and... more The codling moth Cydia pomonella L. is controlled mostly with chemical insecticides in Greece and control failures have been reported. However, there are no insecticide resistance studies in the country as yet. We examined the insecticide resistance status of 33 and 38 populations of fifth-instar non-diapausing and diapausing larvae, respectively by applying bioassays, biochemical and DNA diagnostics. Diagnostic concentrations of
Glutathione S-transferase (GST) activity assays in insects are usually performed by spectrophotom... more Glutathione S-transferase (GST) activity assays in insects are usually performed by spectrophotometric kinetic measurements of conjugated product formation with substrates such as reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). This requires a spectrophotometer that can measure absorbance in the UV range and microcentrifugation to remove the particulates from crude homogenates which absorb light at 340 nm. Such an assay is not ideal for detecting elevated levels of GST activity in insects under field conditions, which is a requirement in, for example, insecticide resistance management programs. We have developed a simple quantitative assay for visually determining GST activity in individual insects. The substrates GSH and CDNB are used in this assay. After the linear enzyme reaction has run for a fixed time, free GSH is determined stoichiometrically by iodometric titration. The results can be determined visually from the discrete color change. We demonstrate the equivalence of this iodometric end point assay and the standard kinetic assay for a five-fold range of purified recombinant Anopheles gambiae agGST1-6 enzyme concentrations and for crude homogenates of individual insects. Results of the application of this test in the diagnosis of GST-based insecticide resistance are presented, demonstrating its practicality for field use.
An organophosphate-resistant strain of the olive fruit fly Bactrocera oleae, the most important p... more An organophosphate-resistant strain of the olive fruit fly Bactrocera oleae, the most important pest for olive orchards worldwide, was obtained by laboratory selection with dimethoate. Resistance mechanisms were investigated in comparison with the colonized parental strain and a field population collected from the same area after 12 years of continuous dimethoate-based insecticide pressure. Combined biochemical and bioassay data suggested that, although esterase and/or glutathione S-transferase metabolic pathways were present and active against dimethoate, they were not selected for and did not have a major role in resistance. There was no evidence of increased oxidase activity in the resistant strains or significant synergism of dimethoate toxicity by piperonyl butoxide; thus, oxidative metabolism was not a major component of resistance. An altered acetylcholinesterase (AChE) with poorer catalytic efficiency for the substrate acetylthiocholine iodide and 5-to 16-fold lower sensitivity to inhibition by omethoate was the major resistance mechanism. Dimethoate selected the insensitive AChE allele(s) in the resistant insects, which were also insensitive to paraoxon, but the altered AChE mechanism conferred negative cross-resistance to the carbamate propoxur. ᭧
The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chik... more The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated. Temephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE), but not target site resistance (altered AChE), with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the...
Bendiocarb was introduced for the first time for Indoor Residual Spraying (IRS) in Tanzania in 20... more Bendiocarb was introduced for the first time for Indoor Residual Spraying (IRS) in Tanzania in 2012 as part of the interim national insecticide resistance management plan. This move followed reports of increasingly alarming levels of pyrethroid resistance across the country. This study used the insecticide quantification kit (IQK) to investigate the intra-operational IRS coverage and quality of spraying, and decay rate of bendiocarb on different wall surfaces in Kagera region. To assess intra-operational IRS coverage and quality of spraying, 104 houses were randomly selected out of 161,414 sprayed houses. A total of 509 samples (218 in Muleba and 291 in Karagwe) were obtained by scraping the insecticide samples from wall surfaces. To investigate decay rate, 66 houses (36 in Muleba and 30 in Karagwe) were selected and samples were collected monthly for a period of five months. Laboratory testing of insecticide concentration was done using IQK(TM) [Innovative Vector Control Consortium...
The subclass Acari, comprising mites and ticks, is one of the largest and biologically most diver... more The subclass Acari, comprising mites and ticks, is one of the largest and biologically most diverse groups within the class of the Arachnida, which also includes scorpions, spiders and harvestmen. They are distributed worldwide and have successfully colonised a wide range of terrestrial and aquatic habitats. The majority of mite species living on the aerial parts of higher plants feed
Background: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plas... more Background: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared with three previously described PCR techniques.
The elevated expression of enzymes with insecticide metabolism activity can lead to high levels o... more The elevated expression of enzymes with insecticide metabolism activity can lead to high levels of insecticide resistance in the malaria vector, Anopheles gambiae. In this study, adult female mosquitoes from an insecticide susceptible and resistant strain were dissected into four different body parts. RNA from each of these samples was used in microarray analysis to determine the enrichment patterns of the key detoxification gene families within the mosquito and to identify additional candidate insecticide resistance genes that may have been overlooked in previous experiments on whole organisms. A general enrichment in the transcription of genes from the four major detoxification gene families (carboxylesterases, glutathione transferases, UDP glucornyltransferases and cytochrome P450s) was observed in the midgut and malpighian tubules. Yet the subset of P450 genes that have previously been implicated in insecticide resistance in An gambiae, show a surprisingly varied profile of tiss...
Insecticide resistance poses a serious threat to current malaria control efforts. The Anopheles g... more Insecticide resistance poses a serious threat to current malaria control efforts. The Anopheles gambiae genome will enable identification of new resistance genes and will provide new molecular targets for the design of more effective insecticides.
Primary structural information including the complete nucleotide sequence of the first insect ald... more Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase (AO) was obtained from the common house mosquito Culex quinquefasciatus (Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO. The Culex AO sequence contains a molybdopterin cofactor binding domain and two iron-sulfur centres. A comparison of the partial sequences of AO from insecticide resistant and susceptible strains of C. quinquefasciatus shows two distinct alleles of this enzyme, one of which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases. The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae. The elevated enzyme can be seen as a separate band on polyacrylamide gel electrophoresis. The role of AO in xenobiotic oxidation in mammals and the partial inhibition of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance.
Proceedings of the National Academy of Sciences, 2005
Metabolic pathways play an important role in insecticide resistance, but the full spectra of the ... more Metabolic pathways play an important role in insecticide resistance, but the full spectra of the genes involved in resistance has not been established. We constructed a microarray containing unique fragments from 230 Anopheles gambiae genes putatively involved in insecticide metabolism [cytochrome P450s (P450s), GSTs, and carboxylesterases and redox genes, partners of the P450 oxidative metabolic complex, and various controls]. We used this detox chip to monitor the expression of the detoxifying genes in insecticide resistant and susceptible An. gambiae laboratory strains. Five genes were strongly up-regulated in the dichlorodiphenyltrichloroethane-resistant strain ZAN͞U. These genes included the GST GSTE2, which has previously been implicated in dichlorodiphenyltrichloroethane resistance, two P450s, and two peroxidase genes. GSTE2 was also elevated in the pyrethroidresistant RSP strain. In addition, the P450 CYP325A3, belonging to a class not previously associated with insecticide resistance, was expressed at statistically higher levels in this strain. The applications of this detox chip and its potential contribution to malaria vector insecticide resistance management programs are discussed. mosquito ͉ cytochrome P450 ͉ GST ͉ carboxylesterase This paper was submitted directly (Track II) to the PNAS office.
Insecticide resistance is a worldwide problem with major impact on agriculture and human health. ... more Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w 2 ]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over-and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ,1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results.
Background: Monitoring of insect vector populations with respect to their susceptibility to one o... more Background: Monitoring of insect vector populations with respect to their susceptibility to one or more insecticides is a crucial element of the strategies used for the control of arthropod-borne diseases. This management task can nowadays be achieved more efficiently when assisted by IT (Information Technology) tools, ranging from modern integrated databases to GIS (Geographic Information System). Here we describe an application ontology that we developed de novo, and a specially designed database that, based on this ontology, can be used for the purpose of controlling mosquitoes and, thus, the diseases that they transmit.
Background: Pyrethroid insecticides are widely utilized in dengue control. However, the major vec... more Background: Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance.
Background: Insecticide-based methods represent the most effective means of blocking the transmis... more 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.
We have recently identified two resistance-associated point mutations of organophosphate (OP)-ins... more We have recently identified two resistance-associated point mutations of organophosphate (OP)-insensitive acetylcholinesterase in the olive fruit fly Bactrocera oleae, the most important olive orchard pest world-wide. We have developed simple PCR-restriction fragment length polymorphism assays for each mutation, utilising an AccI restriction site created by Ile214Val, and a BssHII restriction site destroyed by a neutral change always accompanying the second mutation Gly488Ser. Samples from Greece homozygous for both mutations proved the most insensitive to dimethoate. The frequencies of these mutations in field-collected samples from several countries were investigated. Ninety-three percent of samples from Greece and Albania, where OPs have been extensively used in B. oleae control, were homozygous for both mutations. Resistance-associated alleles were detected at lower frequencies, but still with both mutations in conjunction in the majority of cases, in western Mediterranean countries with limited use of OPs. Samples from South Africa, however, did not have either of the resistance-associated mutations. The double mutation haplotype clearly confers a strong selective advantage in field populations of B. oleae exposed to OPs.
... PJ Skouras, JT Margaritopoulos, NA Seraphides, IM Ioannides, EG Kakani, KD Mathiopoulos, JA T... more ... PJ Skouras, JT Margaritopoulos, NA Seraphides, IM Ioannides, EG Kakani, KD Mathiopoulos, JA Tsitsipis, Organophosphate resistance in olive fruit fly, Bactrocera oleae, populations in Greece and Cyprus, Pest Manag Sci. 63 (2007) 42-48. 21. PS Orphanidis, P. Kalmoukos, B ...
The codling moth Cydia pomonella L. is controlled mostly with chemical insecticides in Greece and... more The codling moth Cydia pomonella L. is controlled mostly with chemical insecticides in Greece and control failures have been reported. However, there are no insecticide resistance studies in the country as yet. We examined the insecticide resistance status of 33 and 38 populations of fifth-instar non-diapausing and diapausing larvae, respectively by applying bioassays, biochemical and DNA diagnostics. Diagnostic concentrations of
Glutathione S-transferase (GST) activity assays in insects are usually performed by spectrophotom... more Glutathione S-transferase (GST) activity assays in insects are usually performed by spectrophotometric kinetic measurements of conjugated product formation with substrates such as reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). This requires a spectrophotometer that can measure absorbance in the UV range and microcentrifugation to remove the particulates from crude homogenates which absorb light at 340 nm. Such an assay is not ideal for detecting elevated levels of GST activity in insects under field conditions, which is a requirement in, for example, insecticide resistance management programs. We have developed a simple quantitative assay for visually determining GST activity in individual insects. The substrates GSH and CDNB are used in this assay. After the linear enzyme reaction has run for a fixed time, free GSH is determined stoichiometrically by iodometric titration. The results can be determined visually from the discrete color change. We demonstrate the equivalence of this iodometric end point assay and the standard kinetic assay for a five-fold range of purified recombinant Anopheles gambiae agGST1-6 enzyme concentrations and for crude homogenates of individual insects. Results of the application of this test in the diagnosis of GST-based insecticide resistance are presented, demonstrating its practicality for field use.
An organophosphate-resistant strain of the olive fruit fly Bactrocera oleae, the most important p... more An organophosphate-resistant strain of the olive fruit fly Bactrocera oleae, the most important pest for olive orchards worldwide, was obtained by laboratory selection with dimethoate. Resistance mechanisms were investigated in comparison with the colonized parental strain and a field population collected from the same area after 12 years of continuous dimethoate-based insecticide pressure. Combined biochemical and bioassay data suggested that, although esterase and/or glutathione S-transferase metabolic pathways were present and active against dimethoate, they were not selected for and did not have a major role in resistance. There was no evidence of increased oxidase activity in the resistant strains or significant synergism of dimethoate toxicity by piperonyl butoxide; thus, oxidative metabolism was not a major component of resistance. An altered acetylcholinesterase (AChE) with poorer catalytic efficiency for the substrate acetylthiocholine iodide and 5-to 16-fold lower sensitivity to inhibition by omethoate was the major resistance mechanism. Dimethoate selected the insensitive AChE allele(s) in the resistant insects, which were also insensitive to paraoxon, but the altered AChE mechanism conferred negative cross-resistance to the carbamate propoxur. ᭧
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