Insecticide resistance in dengue vectors from hotspots in Selangor,Malaysia

BackgroundIn Malaysia, dengue remains a top priority disease and usage of insecticides is the main method for dengue vector control. Limited baseline insecticide resistance data in dengue hotspots has prompted us to conduct this study. The present study reports the use of a map on the insecticide susceptibility status of Aedes aegypti and Aedes albopictus to provide a quick visualization and overview of the distribution of insecticide resistance.Method and resultsThe insecticide resistance status of Aedes populations collected from 24 dengue hotspot areas from the period of December 2018 until June 2019 was proactively monitored using the World Health Organization standard protocol for adult and larval susceptibility testing was conducted, together with elucidation of the mechanisms involved in observed resistance. For resistance monitoring, susceptibility to three adulticides (permethrin, deltamethrin, and malathion) was tested, as well as susceptibility to the larvicide, temephos. Data showed significant resistance to both deltamethrin and permethrin (pyrethroid insecticides), and to malathion (organophosphate insecticide) in all sampled Aedes aegypti populations, while variable resistance patterns were found in the sampled Aedes albopictus populations. Temephos resistance was observed when larvae were tested using the diagnostic dosage of 0.012mg/L but not at the operational dosage of 1mg/L for both species.ConclusionThe present study highlights evidence of a potential threat to the effectiveness of insecticides currently used in dengue vector control, and the urgent requirement for insecticide resistance management to be integrated into the National Dengue Control Program.     

Insecticide resistance,associated mechanisms and fitness aspects in two Brazilian Stegomyia aegypti (= Aedes aegypti) populations

In Brazil, insecticide resistance in Stegomyia aegypti (= Aedes aegypti) (Diptera: Culicidae) populations to pyrethroids and to the organophosphate (OP) temephos is disseminated. Currently, insect growth regulators (IGRs) and the OP malathion are employed against larvae and adults, respectively. Bioassays with mosquitoes from two northeast municipalities, Crato and Aracaju, revealed, in both populations, susceptibility to IGRs and malathion (RR₉₅ ≤ 2.0), confirming the effectiveness of these compounds. By contrast, temephos and deltamethrin (pyrethroid) resistance levels were high (RR₉₅ > 10), which is consistent with the use of intense chemical control. In Crato, RR₉₅ values were > 50 for both compounds. Knock‐down‐resistant (kdr) mutants in the voltage‐gated sodium channel, the pyrethroid target site, were found in 43 and 32%, respectively, of Aracaju and Crato mosquitoes. Biochemical assays revealed higher metabolic resistance activity (esterases, mixed function oxidases and glutathione‐S‐transferases) at Aracaju. With respect to fitness aspects, mating effectiveness was equivalently impaired in both populations, but Aracaju mosquitoes showed more damaging effects in terms of longer larval development, decreased bloodmeal acceptance, reduced engorgement and lower numbers of eggs laid per female. Compared with mosquitoes in Crato, Aracaju mosquitoes exhibited lower OP and pyrethroid RR₉₅, increased activity of detoxifying enzymes and greater effect on fitness. The potential relationship between insecticide resistance mechanisms and mosquito viability is discussed.     

Changes in enzyme titres with age in four geographical strains of Aedes aegypti and their association with insecticide resistance

Abstract. The enzymes acetylcholinesterase, glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PD), and general esterases were assayed in four strains of Aedes aegypti mosquitoes aged between 1 and 30 days. Microtitre plate methods were used to assay activity in the homogenates of individual mosquitoes. The levels of GST and G6PD declined with the age of the mosquitoes, while the activity for the other enzymes remained constant. Soluble protein content was also found to decline with mosquito age in all the strains. Insecticide bioassays showed that two strains (Trinidad and Virtudes) of Ae. aegypti were resistant to DDT, deltamethrin and malathion, whereas two other strains (Bangkok and Indian) were susceptible to all four classes of insecticides tested. Higher esterase activity levels in the resistant compared to the susceptible strains were assumed to be the cause of organophosphate resistance. The combination of DDT and deltamethrin resistance in two strains with normal GST and G6PD characteristics suggests that a kdr-type nerve insensitivity mechanism may be involved.     

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.     

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.     

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.     

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.     

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.     

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.     

Pyrethroid resistance reduces the efficacy of space sprays for dengue control on the island of Martinique (Caribbean)

Background

Dengue fever is reemerging on the island of Martinique and is a serious threat for the human population. During dengue epidemics, adult Aedes aegypti control with pyrethroid space sprays is implemented in order to rapidly reduce transmission. Unfortunately, vector control programs are facing operational challenges with the emergence of pyrethroid resistant Ae. aegypti populations.

Methodology/Principal Findings

To assess the impact of pyrethroid resistance on the efficacy of treatments, applications of deltamethrin and natural pyrethrins were performed with vehicle-mounted thermal foggers in 9 localities of Martinique, where Ae. aegypti populations are strongly resistant to pyrethroids. Efficacy was assessed by monitoring mortality rates of naturally resistant and laboratory susceptible mosquitoes placed in sentinel cages. Before, during and after spraying, larval and adult densities were estimated. Results showed high mortality rates of susceptible sentinel mosquitoes treated with deltamethrin while resistant mosquitoes exhibited very low mortality. There was no reduction of either larval or adult Ae. aegypti population densities after treatments.

Conclusions/Significance

This is the first documented evidence that pyrethroid resistance impedes dengue vector control using pyrethroid-based treatments. These results emphasize the need for alternative tools and strategies for dengue control programs.     

Dengue infection increases the locomotor activity of Aedes aegypti females

Background

Aedes aegypti is the main vector of the virus causing Dengue fever, a disease that has increased dramatically in importance in recent decades, affecting many tropical and sub-tropical areas of the globe. It is known that viruses and other parasites can potentially alter vector behavior. We investigated whether infection with Dengue virus modifies the behavior of Aedes aegypti females with respect to their activity level.

Methods/Principal Findings

We carried out intrathoracic Dengue 2 virus (DENV-2) infections in Aedes aegypti females and recorded their locomotor activity behavior. We observed an increase of up to ∼50% in the activity of infected mosquitoes compared to the uninfected controls.

Conclusions

Dengue infection alters mosquito locomotor activity behavior. We speculate that the higher levels of activity observed in infected Aedes aegypti females might involve the circadian clock. Further studies are needed to assess whether this behavioral change could have implications for the dynamics of Dengue virus transmission.     

Improved filter paper test for detecting and quantifying increased esterase activity in organophosphate-resistant mosquitoes (Diptera: Culicidae)

A previously described filter paper test procedure for detecting of esterases involved in organophosphate insecticide resistance in the Culex pipiens L. complex was modified to permit quantification of esterase activity and resistance in single insects. The new procedure, FP/Est test, was used to survey organophosphate resistance in 11 field collections from seven states. Clear discrimination of increased activity was possible by visual inspection and by densitometric analysis. The proportion of insects with susceptible-like esterase activity was strongly correlated with (and often was not significantly different from) the proportion found to be susceptible by bioassay with chlorpyrifos, temephos, fenthion, and malathion, indicating that the FP/Est test is a reliable method for detecting and monitoring of organophosphate resistance. In addition, the 90th percentile of esterase activity in each collection was significantly correlated with the LC90 of each of the four insecticides, suggesting that the FP/Est test also can be used as a rough estimate of resistance levels. Application of the FP/Est test to monitor resistance caused by increased esterase activity in mosquitoes and agricultural pests is discussed.     

Characterization of a novel esterase conferring insecticide resistance in the mosquito Culex tarsalis

Resistance to the organophosphate insecticide, malathion, in a strain of Culex tarsalis mosquitoes is due to increased activity of a malathion carboxylesterase (MCE). To determine whether resistance was due to a qualitative or quantitative change in the MCE, the enzyme was purified from both malathion-resistant and -susceptible mosquitoes. Enzyme kinetic measurements revealed that the two strains have one MCE in common, but resistant mosquitoes also have a unique MCE which hydrolyses malathion 18 times faster. Interestingly, this MCE does not hydrolyse α-naphthyl acetate, a substrate commonly used to detect increased levels of esterases in other organophosphate-resistant insects. Unlike the over-produced esterase of some related mosquito species, each MCE in C. tarsalis accounts for only a small fraction (0.015%) of the total extractable protein in either strain. Therefore, resistance in these insects is due to the presence of a qualitatively different enzyme, and not to a quantitative increase of a non-specific esterase. This study therefore demonstrates that the underlying biochemical mechanisms of insecticide resistance in one insect cannot necessarily be predicted from those of another, even closely related species. © 1995 Wiley-Liss, Inc.     

Comparative analysis of midgut bacterial communities in three aedine mosquito species from dengue‐endemic and non‐endemic areas of Rajasthan,India

Dengue viruses are transmitted to humans through the bites of infected female aedine mosquitoes. Differences in the composition and structure of bacterial communities in the midguts of mosquitoes may affect the vector's ability to transmit the disease. To investigate and analyse the role of midgut bacterial communities in viral transmission, midgut bacteria from three species, namely Stegomyia aegypti (= Aedes aegypti), Fredwardsius vittatus (= Aedes vittatus) and Stegomyia albopicta (= Aedes albopictus) (all: Diptera: Culicidae), from dengue‐endemic and non‐endemic areas of Rajasthan, India were compared. Construction and analyses of six 16S rRNA gene libraries indicated that Serratia spp.‐related phylotypes dominated all clone libraries of the three mosquito species from areas in which dengue is not endemic. In dengue‐endemic areas, phylotypes related to Aeromonas, Enhydrobacter spp. and uncultivated bacterium dominated the clone libraries of S. aegypti, F. vittatus and S. albopicta, respectively. Diversity indices analysis and real‐time TaqMan polymerase chain reaction assays showed bacterial diversity and abundance in the midguts of S. aegypti to be higher than in the other two species. Significant differences observed among midgut bacterial communities of the three mosquito species from areas in which dengue is and is not endemic, respectively, may be related to the vectorial capacity of mosquitoes to carry dengue viruses and, hence, to the prevalence of disease in some areas.     

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.     

Molecular surveillance of resistance to pyrethroids insecticides in Colombian Aedes aegypti populations

IntroductionIn Colombia, organochloride, organophosphate, carbamate, and pyrethroid insecticides are broadly used to control Aedes aegypti populations. However, Colombian mosquito populations have shown variability in their susceptibility profiles to these insecticides, with some expressing high resistance levels.Materials and methodsIn this study, we analyzed the susceptibility status of ten Colombian field populations of Ae. aegypti to two pyrethroids; permethrin (type-I pyrethroid) and lambda-cyhalothrin (type-II pyrethroid). In addition, we evaluated if mosquitoes pressured with increasing lambda-cyhalothrin concentrations during some filial generations exhibited altered allelic frequency of these kdr mutations and the activity levels of some metabolic enzymes.ResultsMosquitoes from all field populations showed resistance to lambda-cyhalothrin and permethrin. We found that resistance profiles could only be partially explained by kdr mutations and altered enzymatic activities such as esterases and mixed-function oxidases, indicating that other yet unknown mechanisms could be involved. The molecular and biochemical analyses of the most pyrethroid-resistant mosquito population (Acacías) indicated that kdr mutations and altered metabolic enzyme activity are involved in the resistance phenotype expression.ConclusionsIn this context, we propose genetic surveillance of the mosquito populations to monitor the emergence of resistance as an excellent initiative to improve mosquito-borne disease control measures.