Trend of temephos resistance in Aedes (Stegomyia) mosquitoes in Thailand during 2003-2005

The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae. aegypti mosquitoes of one collection site each in Bangkok, Nakhon Sawan (northcentral), and Nakhon Ratchasrima (northeast) were resistant to temephos, with mortality ranging from 50.5 to 71.4%. Moreover, there was a trend of resistance to temephos among Ae. aegypti populations of all studied districts of Nakorn Ratchasima and most areas of Nakhon Sawan, of which those in one area were susceptible. However, various levels of temephos susceptibility were found in Bangkok populations, including resistance and incipient resistance. In Chonburi Province (eastern), all mosquitoes were susceptible to temephos with an indication of tolerance in one sample. Additionally, mosquitoes from Songkhla (south), Chiang Rai (north), Kanchanaburi (west), and Chanthaburi (east) remained susceptible to temephos during the sample collecting period. Bioassay tests on Aedes albopictus populations collected in this study from Nakhon Sawan, Nakorn Ratchasima, Songkhla, and Kanchanaburi revealed high susceptibility to temephos. Although the use of temephos seems to be potentially effective in many areas of the country, a noticeable trend of resistance indicated that alternative vector control methods should be periodically applied.     

Patterns of insecticide resistance in larval Culex pipiens populations in Israel: dynamics and trends.

Resistance to insecticides of larval Culex pipiens populations in Israel has been monitored for ten years and the results were used for control planning. The insecticides tested were the organophosphates chlorpyrifos, fenthion, and temephos and the pyrethroids permethrin and cypermethrin. Over the years the relative resistance (R/R) values to chlorpyrifos in most populations tested were between 200-400, with records of up to 700 R/R. Practically no susceptible populations were found and the compound has been withdrawn from use, resulting in a decrease in the relative resistance values. In 1996 when fenthion was reintroduced, no resistant populations were found, but in the following years significant resistance appeared in an increasing number of populations with values lower than recorded for chlorpyrifos but still high (up to 100 R/R). All populations tested were found to be susceptible to temephos and accordingly the compound was reintroduced in 2002. Over the ten years, no significant resistance to cypermethrin was found in the majority of the populations tested, while a trend of increased resistance to permethrin was noted with a few records of >1000 R/R. The results demonstrate the importance of continuous monitoring of insecticide resistance for efficient mosquito control. Alternative insecticides and methods of control are discussed.     

Functional and immunohistochemical characterization of CCEae3a,a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus

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.     

Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated.     

The Impact of Selection with Diflubenzuron,a Chitin Synthesis Inhibitor,on the Fitness of Two Brazilian Aedes aegypti Field Populations

Several Aedes aegypti field populations are resistant to neurotoxic insecticides, mainly organophoshates and pyrethroids, which are extensively used as larvicides and adulticides, respectively. Diflubenzuron (DFB), a chitin synthesis inhibitor (CSI), was recently approved for use in drinking water, and is presently employed in Brazil for Ae. aegypti control, against populations resistant to the organophosphate temephos. However, tests of DFB efficacy against field Ae. aegypti populations are lacking. In addition, information regarding the dynamics of CSI resistance, and characterization of any potential fitness effects that may arise in conjunction with resistance are essential for new Ae. aegypti control strategies. Here, the efficacy of DFB was evaluated for two Brazilian Ae. aegypti populations known to be resistant to both temephos and the pyrethroid deltamethrin. Laboratory selection for DFB resistance was then performed over six or seven generations, using a fixed dose of insecticide that inhibited 80% of adult emergence in the first generation. The selection process was stopped when adult emergence in the diflubenzuron-treated groups was equivalent to that of the control groups, kept without insecticide. Diflubenzuron was effective against the two Ae. aegypti field populations evaluated, regardless of their resistance level to neurotoxic insecticides. However, only a few generations of DFB selection were sufficient to change the susceptible status of both populations to this compound. Several aspects of mosquito biology were affected in both selected populations, indicating that diflubenzuron resistance acquisition is associated with a fitness cost. We believe that these results can significantly contribute to the design of control strategies involving the use of insect growth regulators.     

Resistance of Aedes aegypti (L.) (Diptera: Culicidae) populations to organophosphates temephos in the Paraíba State, Brazil

Rumors of Aedes aegypti (L.) resistance to temephos in diferent Brazilian states justified this research, whose objective was to verify and characterize the resistance to temephos in A. aegypti populations from Paraíba State. The temephos resistance was evaluated and characterized through a diagnostic dose of 0.012 mg/l and concentration-mortality curves. The mortality data of multiple concentrations were submitted to Probit analysis, and the resistance ratios (RR) were figured out from the CL50s of the survived population and CL50 a laboratory susceptible population. All the A. aegypti populations showed resistance to temephos. The Sítio Piabas population with RR = 4.0, showed lower resistance, the Campina Grande with RR = 6.0, Lagoa do Mato with RR = 9.3 and Capim de Cheiro with RR = 9.0, showed a moderate resistance, and Boqueir?o with RR = 11.0, Brejo dos Santos with RR = 16.6 and Itaporanga with RR= 15.6, showed intermediate levels of resistance to temephos. These results confirm the need of a continuous monitoring and managing program of A. aegypti resistance in Paraíba State.     

Insecticide-Driven Patterns of Genetic Variation in the Dengue Vector Aedes aegypti in Martinique Island

Effective vector control is currently challenged worldwide by the evolution of resistance to all classes of chemical insecticides in mosquitoes. In Martinique, populations of the dengue vector Aedes aegypti have been intensively treated with temephos and deltamethrin insecticides over the last fifty years, resulting in heterogeneous levels of resistance across the island. Resistance spreading depends on standing genetic variation, selection intensity and gene flow among populations. To determine gene flow intensity, we first investigated neutral patterns of genetic variability in sixteen populations representative of the many environments found in Martinique and experiencing various levels of insecticide pressure, using 6 microsatellites. Allelic richness was lower in populations resistant to deltamethrin, and consanguinity was higher in populations resistant to temephos, consistent with a negative effect of insecticide pressure on neutral genetic diversity. The global genetic differentiation was low, suggesting high gene flow among populations, but significant structure was found, with a pattern of isolation-by-distance at the global scale. Then, we investigated adaptive patterns of divergence in six out of the 16 populations using 319 single nucleotide polymorphisms (SNPs). Five SNP outliers displaying levels of genetic differentiation out of neutral expectations were detected, including the kdr-V1016I mutation in the voltage-gated sodium channel gene. Association tests revealed a total of seven SNPs associated with deltamethrin resistance. Six other SNPs were associated with temephos resistance, including two non-synonymous substitutions in an alkaline phosphatase and in a sulfotransferase respectively. Altogether, both neutral and adaptive patterns of genetic variation in mosquito populations appear to be largely driven by insecticide pressure in Martinique.     

Temephos resistance and esterase activity in the mosquito Aedes aegypti in Havana, Cuba increased dramatically between 2006 and 2008

Aedes aegypti (L.) (Diptera: Culicidae) control programmes in Cuba rely on the application of the organophosphate temephos for larval control. Hence, the monitoring of resistance to this insecticide is an essential component of such programmes. Here, 15 field populations from different municipalities of Havana City were assayed for resistance to temephos. High levels of resistance were detected in all strains and resistance ratios were highly correlated with esterase activity (P = 0.00001). Populations from three municipalities were tested in both 2006 and 2008; resistance and esterase activities both significantly increased during this 2-year period. Synergist studies demonstrated that neither glutathione transferases nor monooxygenases were associated with the increase in resistance to temephos in this period. The duration of the efficacy of commercial formulations of temephos in controlling Ae. aegypti populations in Havana City was reduced by the high level of temephos resistance observed; hence these data are of clear operational significance for the dengue control programme in Cuba. New integrated strategies to avoid further increases in temephos resistance in Cuba are necessary.     

Identification of Carboxylesterase Genes Implicated in Temephos Resistance in the Dengue Vector Aedes aegypti

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.     

Competitiveness and life‐history characteristics of Daphnia with respect to susceptibility to a bacterial pathogen

Costs of resistance, i.e. trade‐offs between resistance to parasites or pathogens and other fitness components, may prevent the fixation of resistant genotypes and therefore explain the maintenance of genetic polymorphism for resistance in the wild. Using two approaches, the cost of resistance to a sterilizing bacterial pathogen were tested for in the crustacean Daphnia magna. First, groups of susceptible and resistant hosts from each of four natural populations were compared in terms of their life‐history characteristics. Secondly, we examined the competitiveness of nine clones from one population for which more detailed information on genetic variation for resistance was known. In no case did the results show that competitiveness or life history characteristics of resistant Daphnia systematically differed from susceptible ones. These results suggest that costs of resistance are unlikely to explain the maintenance of genetic variation in D. magna populations. We discuss methods for measuring fitness and speculate on which genetic models of host‐parasite co‐evolution may apply to the Daphnia‐microparasite system.     

QTL Mapping of Genome Regions Controlling Temephos Resistance in Larvae of the Mosquito Aedes aegypti

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, México 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 Solidaridad×Iquitos (SLD×Iq) 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 México.

Conclusions/Significance

Temephos resistance is under the control of many metabolic genes of small effect and dispersed throughout the Ae. aegypti genome.     

Resistance of Aedes aegypti from the state of São Paulo,Brazil, to organophosphates insecticides

Since the reintroduction of Aedes aegypti in the state of S o Paulo, in the middle of the 1980-decade, organophosphate insecticides are being used to control the dengue vector. In 1996, an annual program for monitoring the susceptibility of Ae. aegypti to the insecticides was implemented. Some of the results of this monitoring program are presented. Ae. aegypti populations from ten localities have been submitted to bioassays with the diagnostic dose of temephos and fenitrothion. Only two (Mar lia and Presidente Prudente) remain susceptible to both insecticides and one (Santos) exhibits true resistance. Ae. aegypti from the remaining localities showed an incipient altered susceptibility. Resistance ratios varied from 1.2 to 2.9 for temephos and from 1.5 to 3.2 to fenitrothion, indicating moderate levels of resistance. Biochemical assays did not detect alterations in the enzyme acetilcholinesterase, but indicated that resistance is associated with esterases.     

Effects of Resistance in White Clover (Trifolium repens) on Heterodera trifolii

Clones of two partially resistant and two susceptible white clover, Trifolium repens, genotypes were exposed to eggs of Heterodera trifolii and nematode development in stained roots measured at 2, 4, 7, 11, 18, 23, and 37 days after inoculation. The differences in development between nematode populations in resistant and susceptible genotypes showed that resistance operated after infection during feeding and development. At 7 days after inoculation, counts of second-stage juveniles did not differ between genotypes, whereas at 37 days more adults had developed in the susceptible than in the resistant genotypes. In a separate experiment, cysts hosted by susceptible genotypes were larger and contained more eggs than those on resistant genotypes so that the product of the values for cysts per plant and for eggs per cyst resulted in a more sensitive measure of resistance than from using cysts per plant alone.     

ANALYSIS ON LIFE TABLE OF ORGANOPHOSPHATE-RESISTANT STRAINS OF CULEX PIPIENS PALLENS

Abstract  Effects of dipterex, temephos and chlorpyrifos resistance on the relative fitness in Culex pipiens pallens were evaluated in terms of developmental and reproductive characteristics. Life tables of Dip-R (dipterex), Tem-R (temephos), Chl-R (chlorpyrifos) resistant strains and susceptible strain (S) were also constructed to determine the relative fitness by the net reproductive rates (r). The results indicated that three OP-resistant strains possessed reproductive and developmental disadvantages involving in bloodsucking ratio, average oviposition rate and survival ratio relative to the S strain. The Dip-R, Tem-R and Chl-R strains had the relative fitness value of 0. 542, 0. 631 and 0.618, respectively.     

Insecticide resistance and genetic structure of Aedes aegypti populations from Rio de Janeiro State,Brazil

Vector control largely relies on neurotoxic chemicals, and insecticide resistance (IR) directly threatens their effectiveness. In some cases, specific alleles cause IR, and knowledge of the genetic diversity and gene flow among mosquito populations is crucial to track their arrival, rise, and spread. Here we evaluated Aedes aegypti populations’ susceptibility status, collected in 2016 from six different municipalities of Rio de Janeiro state (RJ), to temephos, pyriproxyfen, malathion, and deltamethrin. We collected eggs of Ae. aegypti in Campos dos Goytacazes (Cgy), Itaperuna (Ipn), Iguaba Grande (Igg), Itaboraí (Ibr), Mangaratiba (Mgr), and Vassouras (Vsr). We followed the World Health Organization (WHO) guidelines and investigated the degree of susceptibility/resistance of mosquitoes to these insecticides. We used the Rockefeller strain as a susceptible positive control. We genotyped the V1016I and F1534C knockdown resistance (kdr) alleles using qPCR TaqMan SNP genotyping assay. Besides, with the use of Ae. aegypti SNP-chip, we performed genomic population analyses by genotyping more than 15,000 biallelic SNPs in mosquitoes from each population. We added previous data from populations from other countries to evaluate the ancestry of RJ populations.All RJ Ae. aegypti populations were susceptible to pyriproxyfen and malathion and highly resistant to deltamethrin. The resistance ratios for temephos was below 3,0 in Cgy, Ibr, and Igg populations, representing the lowest rates since IR monitoring started in this Brazilian region. We found the kdr alleles in high frequencies in all populations, partially justifying the observed resistance to pyrethroid. Population genetics analysis showed that Ae. aegypti revealed potential higher migration among some RJ localities and low genetic structure for most of them. Future population genetic studies, together with IR data in Ae aegypti on a broader scale, can help us predict the gene flow within and among the Brazilian States, allowing us to track the dynamics of arrival and changes in the frequency of IR alleles, and providing critical information to improving vector control program.     

Temephos Resistance in Aedes aegypti in Colombia Compromises Dengue Vector Control

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 15× higher 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.     

Direct Delivery of Inoculum to Shoot Tissue Interferes with Genotypic Resistance to Ralstonia solanacearum in Tomato Seedlings

Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 10⁸ CFU/ml^?1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed‐soaking inoculation, seed‐sowing followed by inoculum drenching, or at 2‐week stage through petiole‐excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root‐injury. Seed‐based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings. Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within 7–10 days. Root‐injury inoculation at 2‐week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding.     

Organophosphate Resistance in Aedes aegypti: Study from Dengue Hemorrhagic Fever Endemic Subdistrict in Riau,Indonesia

Background:Dengue hemorrhagic fever (DHF) is a significant health problem. The high number of cases requires preventions, including controlling the dengue vector, Aedes aegypti mosquito. One of the control methods is the use of insecticides containing organophosphate. This study aims to detect organophosphate resistance in Aedes aegypti from DHF endemic subdistrict, Riau, Indonesia by a sensitivity test of temephos and 5% malathion and measuring the activity of non-specific alpha and beta esterase enzymes.Methods:This observational study determined Aedes aegypti resistance from larvae to adult in one DHF endemic subdistrict in Riau, Indonesia. The bioassay was used for temephos sensitivity of Aedes aegypti larvae. The LC99 value was analyzed using probit and compared with the diagnostic value from WHO. The WHO susceptibility test was conducted to determine 5% malathion resistance from adult mosquitoes. The mortality of less than 90% was declared as resistant. Measurement of alpha and beta esterase levels used Lee''s microplate assay technique based on visual identification and absorbance value (AV).Results:The results showed that Aedes aegypti were resistant to temephos. It also showed that adult mosquitoes were resistant to 5% malathion. Based on the alpha esterase activity test, it was found that most of the mosquitoes showed very sensitive meanwhile, based on the beta esterase activity test, most of the mosquitoes were moderate resistance.Conclusion:This study suggests that Aedes aegypti population from DHF endemic subdistrict in Riau, Indonesia are indicated to develop resistance to organophosphate.Key Words: Aedes aegypti, Dengue Hemorrhagic fever, Organophosphate, Resistance