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William C. Black - One of the best experts on this subject based on the ideXlab platform.
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QTL mapping of genome regions controlling Temephos resistance in larvae of the mosquito Aedes aegypti.
PLoS neglected tropical diseases, 2014Co-Authors: Guadalupe Reyes-solis, Karla Saavedra-rodriguez, Adriana Elizabeth Flores Suarez, William C. BlackAbstract:Introduction: The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Temephos is an organophosphate insecticide used globally to suppress Ae. aegypti larval populations but resistance has evolved in many locations. Methodology/Principal Findings: Quantitative Trait Loci (QTL) controlling Temephos survival in Ae. aegypti larvae were mapped in a pair of F3 advanced intercross lines arising from Temephos resistant parents from Solidaridad, Mexico and Temephos susceptible parents from Iquitos, Peru. Two sets of 200 F3 larvae were exposed to a discriminating dose of Temephos and then dead larvae were collected and preserved for DNA isolation every two hours up to 16 hours. Larvae surviving longer than 16 hours were considered resistant. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 23 single copy genes and 26 microsatellite loci of known physical positions in the Ae. aegypti genome. In both reciprocal crosses, Multiple Interval Mapping identified eleven QTL associated with time until death. In the Solidaridad6Iquitos (SLD6Iq) cross twelve were associated with survival but in the reciprocal IqxSLD cross, only six QTL were survival associated. Polymorphisms at acetylcholine esterase (AchE) loci 1 and 2 were not associated with either resistance phenotype suggesting that target site insensitivity is not an organophosphate resistance mechanism in this region of Mexico. Conclusions/Significance: Temephos resistance is under the control of many metabolic genes of small effect and dispersed throughout the Ae. aegypti genome.
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differential transcription profiles in aedes aegypti detoxification genes after Temephos selection
Insect Molecular Biology, 2014Co-Authors: Karla Saavedrarodriguez, Hilary Ranson, Clare Strode, Adriana E. Flores, Janet Hemingway, Selene M Garcialuna, Guadalupe Reyessolis, William C. BlackAbstract:The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide Temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti ‘Detox Chip’ microarray during five generations of Temephos selection. We selected for Temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations. Uniform upregulation was seen in the epsilon class glutathione-S-transferase (eGST) genes in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated after five generations of Temephos selection. While expression of many carboxyl/cholinesterase esterase (CCE) genes increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 monooxygenase (CYP) genes and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using glutathione-S-transferase (GST), CCE and CYP inhibitors suggest that various CCEs instead of GSTs are the main metabolic mechanism conferring resistance to Temephos. We show that Temephos-selected strains show no cross resistance to permethrin and that genes associated with Temephos selection are largely independent of those selected with permethrin in a previous study.
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Differential transcription profiles inAedes aegyptidetoxification genes after Temephos selection: Aedes aegypti, Temephos selection
Insect molecular biology, 2013Co-Authors: Karla Saavedra-rodriguez, Hilary Ranson, Clare Strode, Adriana E. Flores, Selene M. Garcia-luna, Guadalupe Reyes-solis, Janet Hemingway, William C. BlackAbstract:The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide Temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti ‘Detox Chip’ microarray during five generations of Temephos selection. We selected for Temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations (LC50). Uniform upregulation was seen in the epsilon class glutathione-S-transferase genes (eGSTs) in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated following five generations of Temephos selection. While expression of many esterase genes (CCE) increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 genes (CYP) and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using GST, CCE and CYP inhibitors suggest that various CCE instead of GSTs are the main metabolic mechanism conferring resistance to Temephos. We show that Temephos selected strains show no cross resistance to permethrin and that genes associated with Temephos selection are largely independent of those selected with permethrin in a previous study.
John Vontas - One of the best experts on this subject based on the ideXlab platform.
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Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with Temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus
Insect biochemistry and molecular biology, 2016Co-Authors: Linda Grigoraki, Hilary Ranson, Vassileia Balabanidou, Christos Meristoudis, Antonis Miridakis, Luc Swevers, John VontasAbstract:Temephos is a major organophosphate (OP) larvicide that has been used extensively for the control of Aedes albopictus and Aedes aegypti, the major vectors for viral diseases, such as dengue fever, zika and chikungunya. Resistance to Temephos has been recently detected and associated with the upregulation of carboxylesterases (CCEs) through gene amplification, in both species. Here, we expressed the CCEae3a genes which showed the most striking up-regulation in resistant Aedes strains, using the baculovirus system. All CCEae3a variants encoded functional enzymes, with high activity and preference for p-nitrophenyl butyrate, a substrate that was shown capable to differentiate Temephos resistant from susceptible Aedes larvae. Enzyme kinetic studies showed that CCEae3as from both Ae. aegypti and Ae. albopictus (CCEae3a_aeg and CCEae3a_alb, respectively) strongly interact with Temephos oxon and slowly released the OP molecule, indicating a sequestration resistance mechanism. No difference was detected between resistant and susceptible CCEae3a_aeg variants (CCEae3a_aegR and CCEae3a_aegS, respectively), indicating that previously reported polymorphism is unlikely to play a role in Temephos resistance. HPLC/MS showed that CCEae3as were able to metabolize Temephos oxon to the Temephos monoester [(4-hydroxyphenyl) sulfanyl] phenyl O,O-dimethylphosphorothioate. Western blot and immunolocalization studies, based on a specific antibody raised against the CCEae3a_alb showed that the enzyme is expressed at higher levels in resistant insects, primarily in malpighian tubules (MT) and nerve tissues.
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transcriptome profiling and genetic study reveal amplified carboxylesterase genes implicated in Temephos resistance in the asian tiger mosquito aedes albopictus
PLOS Neglected Tropical Diseases, 2015Co-Authors: Linda Grigoraki, Jacques Lagnel, Ilias Kioulos, Anastasia Kampouraki, Evangelia Morou, Pierrick Labbe, Mylene Weill, John VontasAbstract:Background 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. Principal findings 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 most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in Temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and Temephos resistance, by demonstrating a strong association between survival to Temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins,
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Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus
PLoS Neglected Tropical Diseases, 2015Co-Authors: Linda Grigoraki, Jacques Lagnel, Ilias Kioulos, Anastasia Kampouraki, Evangelia Morou, Pierrick Labbe, Mylene Weill, John VontasAbstract:Background: 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. Principal findings: 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 most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in Temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and Temephos resistance, by demonstrating a strong association between survival to Temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance. Significance: The identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic resources that were produced will be useful to the community, to study relevant aspects of Ae. albopictus biology.
Hilary Ranson - One of the best experts on this subject based on the ideXlab platform.
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Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with Temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus
Insect biochemistry and molecular biology, 2016Co-Authors: Linda Grigoraki, Hilary Ranson, Vassileia Balabanidou, Christos Meristoudis, Antonis Miridakis, Luc Swevers, John VontasAbstract:Temephos is a major organophosphate (OP) larvicide that has been used extensively for the control of Aedes albopictus and Aedes aegypti, the major vectors for viral diseases, such as dengue fever, zika and chikungunya. Resistance to Temephos has been recently detected and associated with the upregulation of carboxylesterases (CCEs) through gene amplification, in both species. Here, we expressed the CCEae3a genes which showed the most striking up-regulation in resistant Aedes strains, using the baculovirus system. All CCEae3a variants encoded functional enzymes, with high activity and preference for p-nitrophenyl butyrate, a substrate that was shown capable to differentiate Temephos resistant from susceptible Aedes larvae. Enzyme kinetic studies showed that CCEae3as from both Ae. aegypti and Ae. albopictus (CCEae3a_aeg and CCEae3a_alb, respectively) strongly interact with Temephos oxon and slowly released the OP molecule, indicating a sequestration resistance mechanism. No difference was detected between resistant and susceptible CCEae3a_aeg variants (CCEae3a_aegR and CCEae3a_aegS, respectively), indicating that previously reported polymorphism is unlikely to play a role in Temephos resistance. HPLC/MS showed that CCEae3as were able to metabolize Temephos oxon to the Temephos monoester [(4-hydroxyphenyl) sulfanyl] phenyl O,O-dimethylphosphorothioate. Western blot and immunolocalization studies, based on a specific antibody raised against the CCEae3a_alb showed that the enzyme is expressed at higher levels in resistant insects, primarily in malpighian tubules (MT) and nerve tissues.
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differential transcription profiles in aedes aegypti detoxification genes after Temephos selection
Insect Molecular Biology, 2014Co-Authors: Karla Saavedrarodriguez, Hilary Ranson, Clare Strode, Adriana E. Flores, Janet Hemingway, Selene M Garcialuna, Guadalupe Reyessolis, William C. BlackAbstract:The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide Temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti ‘Detox Chip’ microarray during five generations of Temephos selection. We selected for Temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations. Uniform upregulation was seen in the epsilon class glutathione-S-transferase (eGST) genes in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated after five generations of Temephos selection. While expression of many carboxyl/cholinesterase esterase (CCE) genes increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 monooxygenase (CYP) genes and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using glutathione-S-transferase (GST), CCE and CYP inhibitors suggest that various CCEs instead of GSTs are the main metabolic mechanism conferring resistance to Temephos. We show that Temephos-selected strains show no cross resistance to permethrin and that genes associated with Temephos selection are largely independent of those selected with permethrin in a previous study.
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Identification of carboxylesterase genes implicated in Temephos resistance in the dengue vector Aedes aegypti.
PLoS neglected tropical diseases, 2014Co-Authors: Rodolphe Poupardin, Wannaporn Srisukontarat, Cristina Yunta, Hilary RansonAbstract:Background Thailand is currently experiencing one of its worst dengue outbreaks in decades. As in most countries where this disease is endemic, dengue control in Thailand is largely reliant on the use of insecticides targeting both immature and adult stages of the Aedes mosquito, with the organophosphate insecticide, Temephos, being the insecticide of choice for attacking the mosquito larvae. Resistance to Temephos was first detected in Aedes aegypti larvae in Thailand approximately 25 years ago but the mechanism responsible for this resistance has not been determined. Principal Findings Bioassays on Ae. aegypti larvae from Thailand detected Temephos resistance ratios ranging from 3.5 fold in Chiang Mai to nearly 10 fold in Nakhon Sawan (NS) province. Synergist and biochemical assays suggested a role for increased carboxylesterase (CCE) activities in conferring Temephos resistance in the NS population and microarray analysis revealed that the CCE gene, CCEae3a, was upregulated more than 60 fold in the NS population compared to the susceptible population. Upregulation of CCEae3a was shown to be partially due to gene duplication. Another CCE gene, CCEae6a, was also highly regulated in both comparisons. Sequencing and in silico structure prediction of CCEae3a showed that several amino acid polymorphisms in the NS population may also play a role in the increased resistance phenotype. Significance Carboxylesterases have previously been implicated in conferring Temephos resistance in Ae aegypti but the specific member(s) of this family responsible for this phenotype have not been identified. The identification of a strong candidate is an important step in the development of new molecular diagnostic tools for management of Temephos resistant populations and thus improved control of dengue.
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Differential transcription profiles inAedes aegyptidetoxification genes after Temephos selection: Aedes aegypti, Temephos selection
Insect molecular biology, 2013Co-Authors: Karla Saavedra-rodriguez, Hilary Ranson, Clare Strode, Adriana E. Flores, Selene M. Garcia-luna, Guadalupe Reyes-solis, Janet Hemingway, William C. BlackAbstract:The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide Temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti ‘Detox Chip’ microarray during five generations of Temephos selection. We selected for Temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations (LC50). Uniform upregulation was seen in the epsilon class glutathione-S-transferase genes (eGSTs) in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated following five generations of Temephos selection. While expression of many esterase genes (CCE) increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 genes (CYP) and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using GST, CCE and CYP inhibitors suggest that various CCE instead of GSTs are the main metabolic mechanism conferring resistance to Temephos. We show that Temephos selected strains show no cross resistance to permethrin and that genes associated with Temephos selection are largely independent of those selected with permethrin in a previous study.
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Temephos Resistance in Aedes aegypti in Colombia Compromises Dengue Vector Control
PLoS neglected tropical diseases, 2013Co-Authors: Nelson Grisales, Hilary Ranson, Rodolphe Poupardin, Santiago Alejandro Gallón Gómez, Idalyd Fonseca-gonzález, Audrey LenhartAbstract:Background: Control and prevention of dengue relies heavily on the application of insecticides to control dengue vector mosquitoes. In Colombia, application of the larvicide Temephos to the aquatic breeding sites of Aedes aegypti is a key part of the dengue control strategy. Resistance to Temephos was recently detected in the dengue-endemic city of Cucuta, leading to questions about its efficacy as a control tool. Here, we characterize the underlying mechanisms and estimate the operational impact of this resistance. Methodology/Principal Findings: Larval bioassays of Ae. aegypti larvae from Cucuta determined the Temephos LC50 to be 0.066 ppm (95% CI 0.06‐0.074), approximately 156higher than the value obtained from a susceptible laboratory colony. The efficacy of the field dose of Temephos at killing this resistant Cucuta population was greatly reduced, with mortality rates ,80% two weeks after application and ,50% after 4 weeks. Neither biochemical assays nor partial sequencing of the ace-1 gene implicated target site resistance as the primary resistance mechanism. Synergism assays and microarray analysis suggested that metabolic mechanisms were most likely responsible for the Temephos resistance. Interestingly, although the greatest synergism was observed with the carboxylesterase inhibitor, DEF, the primary candidate genes from the microarray analysis, and confirmed by quantitative PCR, were cytochrome P450 oxidases, notably CYP6N12, CYP6F3 and CYP6M11. Conclusions/Significance: In Colombia, resistance to Temephos in Ae. aegypti compromises the duration of its effect as a vector control tool. Several candidate genes potentially responsible for metabolic resistance to Temephos were identified. Given the limited number of insecticides that are approved for vector control, future chemical-based control strategies should take into account the mechanisms underlying the resistance to discern which insecticides would likely lead to the greatest control efficacy while minimizing further selection of resistant phenotypes.
Adriana E. Flores - One of the best experts on this subject based on the ideXlab platform.
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differential transcription profiles in aedes aegypti detoxification genes after Temephos selection
Insect Molecular Biology, 2014Co-Authors: Karla Saavedrarodriguez, Hilary Ranson, Clare Strode, Adriana E. Flores, Janet Hemingway, Selene M Garcialuna, Guadalupe Reyessolis, William C. BlackAbstract:The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide Temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti ‘Detox Chip’ microarray during five generations of Temephos selection. We selected for Temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations. Uniform upregulation was seen in the epsilon class glutathione-S-transferase (eGST) genes in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated after five generations of Temephos selection. While expression of many carboxyl/cholinesterase esterase (CCE) genes increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 monooxygenase (CYP) genes and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using glutathione-S-transferase (GST), CCE and CYP inhibitors suggest that various CCEs instead of GSTs are the main metabolic mechanism conferring resistance to Temephos. We show that Temephos-selected strains show no cross resistance to permethrin and that genes associated with Temephos selection are largely independent of those selected with permethrin in a previous study.
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Susceptibility status of Aedes aegypti (L.) (Diptera: Culicidae) to Temephos in Venezuela.
Pest management science, 2013Co-Authors: Leslie C Alvarez, Gustavo Ponce, Milagros Oviedo, Beatriz Lopez, Adriana E. FloresAbstract:BACKGROUND Temephos is an insecticide widely used in Venezuela to control the proliferation of the larvae of Aedes aegypti (L.), the principal vector of dengue virus. The aim of this study was to identify the susceptibility to Temephos of Ae. aegypti in four locations in western Venezuela: Lara, Tres Esquinas, Urena and Pampanito. Larval bioassays were conducted on samples collected in 2008 and 2010, and the levels of α- and β-esterases, mixed-function oxidases, glutathione-S-transferase and insensitive acethyl cholinesterase were determined. RESULTS Larval populations from western Venezuela obtained during 2008 and 2010 were found to be susceptible to Temephos, with low resistance ratios and without overexpression of enzymes. CONCLUSIONS The low RR values reveal the effectiveness of Temephos in controlling the larval populations of Ae. aegypti. Control strategies must be vigorously monitored to maintain the susceptibility to Temephos of these populations of Ae. aegypti. © 2013 Society of Chemical Industry
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Differential transcription profiles inAedes aegyptidetoxification genes after Temephos selection: Aedes aegypti, Temephos selection
Insect molecular biology, 2013Co-Authors: Karla Saavedra-rodriguez, Hilary Ranson, Clare Strode, Adriana E. Flores, Selene M. Garcia-luna, Guadalupe Reyes-solis, Janet Hemingway, William C. BlackAbstract:The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide Temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti ‘Detox Chip’ microarray during five generations of Temephos selection. We selected for Temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations (LC50). Uniform upregulation was seen in the epsilon class glutathione-S-transferase genes (eGSTs) in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated following five generations of Temephos selection. While expression of many esterase genes (CCE) increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 genes (CYP) and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using GST, CCE and CYP inhibitors suggest that various CCE instead of GSTs are the main metabolic mechanism conferring resistance to Temephos. We show that Temephos selected strains show no cross resistance to permethrin and that genes associated with Temephos selection are largely independent of those selected with permethrin in a previous study.
Linda Grigoraki - One of the best experts on this subject based on the ideXlab platform.
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Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with Temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus
Insect biochemistry and molecular biology, 2016Co-Authors: Linda Grigoraki, Hilary Ranson, Vassileia Balabanidou, Christos Meristoudis, Antonis Miridakis, Luc Swevers, John VontasAbstract:Temephos is a major organophosphate (OP) larvicide that has been used extensively for the control of Aedes albopictus and Aedes aegypti, the major vectors for viral diseases, such as dengue fever, zika and chikungunya. Resistance to Temephos has been recently detected and associated with the upregulation of carboxylesterases (CCEs) through gene amplification, in both species. Here, we expressed the CCEae3a genes which showed the most striking up-regulation in resistant Aedes strains, using the baculovirus system. All CCEae3a variants encoded functional enzymes, with high activity and preference for p-nitrophenyl butyrate, a substrate that was shown capable to differentiate Temephos resistant from susceptible Aedes larvae. Enzyme kinetic studies showed that CCEae3as from both Ae. aegypti and Ae. albopictus (CCEae3a_aeg and CCEae3a_alb, respectively) strongly interact with Temephos oxon and slowly released the OP molecule, indicating a sequestration resistance mechanism. No difference was detected between resistant and susceptible CCEae3a_aeg variants (CCEae3a_aegR and CCEae3a_aegS, respectively), indicating that previously reported polymorphism is unlikely to play a role in Temephos resistance. HPLC/MS showed that CCEae3as were able to metabolize Temephos oxon to the Temephos monoester [(4-hydroxyphenyl) sulfanyl] phenyl O,O-dimethylphosphorothioate. Western blot and immunolocalization studies, based on a specific antibody raised against the CCEae3a_alb showed that the enzyme is expressed at higher levels in resistant insects, primarily in malpighian tubules (MT) and nerve tissues.
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transcriptome profiling and genetic study reveal amplified carboxylesterase genes implicated in Temephos resistance in the asian tiger mosquito aedes albopictus
PLOS Neglected Tropical Diseases, 2015Co-Authors: Linda Grigoraki, Jacques Lagnel, Ilias Kioulos, Anastasia Kampouraki, Evangelia Morou, Pierrick Labbe, Mylene Weill, John VontasAbstract:Background 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. Principal findings 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 most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in Temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and Temephos resistance, by demonstrating a strong association between survival to Temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins,
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Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus
PLoS Neglected Tropical Diseases, 2015Co-Authors: Linda Grigoraki, Jacques Lagnel, Ilias Kioulos, Anastasia Kampouraki, Evangelia Morou, Pierrick Labbe, Mylene Weill, John VontasAbstract:Background: 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. Principal findings: 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 most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in Temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and Temephos resistance, by demonstrating a strong association between survival to Temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance. Significance: The identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic resources that were produced will be useful to the community, to study relevant aspects of Ae. albopictus biology.
