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.     

Chikungunya Virus Transmission Potential by Local Aedes Mosquitoes in the Americas and Europe

Background

Chikungunya virus (CHIKV), mainly transmitted in urban areas by the mosquitoes Aedes aegypti and Aedes albopictus, constitutes a major public health problem. In late 2013, CHIKV emerged on Saint-Martin Island in the Caribbean and spread throughout the region reaching more than 40 countries. Thus far, Ae. aegypti mosquitoes have been implicated as the sole vector in the outbreaks, leading to the hypothesis that CHIKV spread could be limited only to regions where this mosquito species is dominant.

Methodology/Principal Findings

We determined the ability of local populations of Ae. aegypti and Ae. albopictus from the Americas and Europe to transmit the CHIKV strain of the Asian genotype isolated from Saint-Martin Island (CHIKV_SM) during the recent epidemic, and an East-Central-South African (ECSA) genotype CHIKV strain isolated from La Réunion Island (CHIKV_LR) as a well-characterized control virus. We also evaluated the effect of temperature on transmission of CHIKV_SM by European Ae. albopictus. We found that (i) Aedes aegypti from Saint-Martin Island transmit CHIKV_SM and CHIKV_LR with similar efficiency, (ii) Ae. aegypti from the Americas display similar transmission efficiency for CHIKV_SM, (iii) American and European populations of the alternative vector species Ae. albopictus were as competent as Ae. aegypti populations with respect to transmission of CHIKV_SM and (iv) exposure of European Ae. albopictus to low temperatures (20°C) significantly reduced the transmission potential for CHIKV_SM.

Conclusions/Significance

CHIKV strains belonging to the ECSA genotype could also have initiated local transmission in the new world. Additionally, the ongoing CHIKV outbreak in the Americas could potentially spread throughout Ae. aegypti- and Ae. albopictus-infested regions of the Americas with possible imported cases of CHIKV to Ae. albopictus-infested regions in Europe. Colder temperatures may decrease the local transmission of CHIKV_SM by European Ae. albopictus, potentially explaining the lack of autochthonous transmission of CHIKV_SM in Europe despite the hundreds of imported CHIKV cases returning from the Caribbean.     

Deltamethrin Resistance Mechanisms in Aedes aegypti Populations from Three French Overseas Territories Worldwide

Background

Aedes aegypti is a cosmopolite mosquito, vector of arboviruses. The worldwide studies of its insecticide resistance have demonstrated a strong loss of susceptibility to pyrethroids, the major class of insecticide used for vector control. French overseas territories such as French Guiana (South America), Guadeloupe islands (Lesser Antilles) as well as New Caledonia (Pacific Ocean), have encountered such resistance.

Methodology/Principal Findings

We initiated a research program on the pyrethroid resistance in French Guiana, Guadeloupe and New Caledonia. Aedes aegypti populations were tested for their deltamethrin resistance level then screened by an improved microarray developed to specifically study metabolic resistance mechanisms. Cytochrome P450 genes were implicated in conferring resistance. CYP6BB2, CYP6M11, CYP6N12, CYP9J9, CYP9J10 and CCE3 genes were upregulated in the resistant populations and were common to other populations at a regional scale. The implication of these genes in resistance phenomenon is therefore strongly suggested. Other genes from detoxification pathways were also differentially regulated. Screening for target site mutations on the voltage-gated sodium channel gene demonstrated the presence of I1016 and C1534.

Conclusion /significance

This study highlighted the presence of a common set of differentially up-regulated detoxifying genes, mainly cytochrome P450 genes in all three populations. GUA and GUY populations shared a higher number of those genes compared to CAL. Two kdr mutations well known to be associated to pyrethroid resistance were also detected in those two populations but not in CAL. Different selective pressures and genetic backgrounds can explain such differences. These results are also compared with those obtained from other parts of the world and are discussed in the context of integrative research on vector competence.     

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.     

Molecular Differentiation of the African Yellow Fever Vector Aedes bromeliae (Diptera: Culicidae) from Its Sympatric Non-vector Sister Species,Aedes lilii

Introduction

Yellow fever continues to be a problem in sub-Saharan Africa with repeated epidemics occurring. The mosquito Aedes bromeliae is a major vector of yellow fever, but it cannot be readily differentiated from its non-vector zoophilic sister species Ae. lilii using morphological characters. Genetic differences have been reported between anthropophilic Ae. bromeliae and zoophilic Ae. lilii and between forest and domestic populations. However, due to the application of different molecular markers and non-overlapping populations employed in previous studies, interpretation of species delimitation is unclear.

Methodology/Principle Findings

DNA sequences were generated from specimens of Ae. simpsoni s.l. from the Republic of Benin, Tanzania and Uganda for two nuclear genes apolipophorin 2 (apoLp2) and cytochrome p450 (CYPJ92), the ribosomal internal transcribed spacer region (ITS) and the mitochondrial cytochrome c oxidase (COI) barcoding region. Nuclear genes apoLp2 and CYPJ92 were unable to differentiate between species Ae. bromeliae and Ae. lilii due to ancestral lineage sorting, while ITS sequence data provided clear topological separation on a phylogeny. The standard COI barcoding region was shown to be subject to species introgression and unable to clearly distinguish the two taxa. Here we present a reliable direct PCR-based method for differentiation of the vector species Ae. bromeliae from its isomorphic, sympatric and non-biomedically important sister taxon, Ae. lilii, based on the ITS region. Using molecular species verification, we describe novel immature habitats for Ae. lilii and report both sympatric and allopatric populations. Whereas only Ae. lilii is found in the Republic of Benin and only Ae. bromeliae in Tanzania, both species are sympatric in Uganda.

Conclusions/Significance

Our accurate identification method will allow informed distribution and detailed ecological studies that will facilitate assessment of arboviral disease risk and development of future targeted vector control.     

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.     

Aedes aegypti abundance and insecticide resistance profiles in the Applying Wolbachia to Eliminate Dengue trial

The Applying Wolbachia to Eliminate Dengue (AWED) trial was a parallel cluster randomised trial that demonstrated Wolbachia (wMel) introgression into Ae. aegypti populations reduced dengue incidence. In this predefined substudy, we compared between treatment arms, the relative abundance of Ae. aegypti and Ae. albopictus before, during and after wMel-introgression. Between March 2015 and March 2020, 60,084 BG trap collections yielded 478,254 Ae. aegypti and 17,623 Ae. albopictus. Between treatment arms there was no measurable difference in Ae. aegypti relative abundance before or after wMel-deployments, with a count ratio of 0.96 (95% CI 0.76, 1.21) and 1.00 (95% CI 0.85, 1.17) respectively. More Ae. aegypti were caught per trap per week in the wMel-intervention arm compared to the control arm during wMel deployments (count ratio 1.23 (95% CI 1.03, 1.46)). Between treatment arms there was no measurable difference in the Ae. albopictus population size before, during or after wMel-deployment (overall count ratio 1.10 (95% CI 0.89, 1.35)). We also compared insecticide resistance phenotypes of Ae. aegypti in the first and second years after wMel-deployments. Ae. aegypti field populations from wMel-treated and untreated arms were similarly resistant to malathion (0.8%), permethrin (1.25%) and cyfluthrin (0.15%) in year 1 and year 2 of the trial. In summary, we found no between-arm differences in the relative abundance of Ae. aegypti or Ae. albopictus prior to or after wMel introgression, and no between-arm difference in Ae. aegypti insecticide resistance phenotypes. These data suggest neither Aedes abundance, nor insecticide resistance, confounded the epidemiological outcomes of the AWED trial.     

Genetic Diversity and Phylogeny of Aedes aegypti,the Main Arbovirus Vector in the Pacific

Background

The Pacific region is an area unique in the world, composed of thousands of islands with differing climates and environments. The spreading and establishment of the mosquito Aedes aegypti in these islands might be linked to human migration. Ae. aegypti is the major vector of arboviruses (dengue, chikungunya and Zika viruses) in the region. The intense circulation of these viruses in the Pacific during the last decade led to an increase of vector control measures by local health authorities. The aim of this study is to analyze the genetic relationships among Ae. aegypti populations in this region.

Methodology/Principal Finding

We studied the genetic variability and population genetics of 270 Ae. aegypti, sampled from 9 locations in New Caledonia, Fiji, Tonga and French Polynesia by analyzing nine microsatellites and two mitochondrial DNA regions (CO1 and ND4). Microsatellite markers revealed heterogeneity in the genetic structure between the western, central and eastern Pacific island countries. The microsatellite markers indicate a statistically moderate differentiation (F_ST = 0.136; P < = 0.001) in relation to island isolation. A high degree of mixed ancestry can be observed in the most important towns (e.g. Noumea, Suva and Papeete) compared with the most isolated islands (e.g. Ouvea and Vaitahu). Phylogenetic analysis indicated that most of samples are related to Asian and American specimens.

Conclusions/Significance

Our results suggest a link between human migrations in the Pacific region and the origin of Ae. aegypti populations. The genetic pattern observed might be linked to the island isolation and to the different environmental conditions or ecosystems.     

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.     

Copulation Activity,Sperm Production and Conidia Transfer in Aedes aegypti Males Contaminated by Metarhizium anisopliae: A Biological Control Prospect

Background

Dengue is the most prevalent arboviral disease transmitted by Aedes aegypti worldwide, whose chemical control is difficult, expensive, and of inconsistent efficacy. Releases of Metarhizium anisopliae—exposed Ae. aegypti males to disseminate conidia among female mosquitoes by mating represents a promising biological control approach against this important vector. A better understanding of fungus virulence and impact on reproductive parameters of Ae. aegypti, is need before testing auto-dissemination strategies.

Methodology/Principal Findings

Mortality, mating competitiveness, sperm production, and the capacity to auto-disseminate the fungus to females up to the 5^thcopulation, were compared between Aedes aegypti males exposed to 5.96 x 10⁷ conidia per cm² of M. anisopliae and uninfected males. Half (50%) of fungus-exposed males (FEMs) died within the first 4 days post-exposure (PE). FEMs required 34% more time to successively copulate with 5 females (165 ± 3 minutes) than uninfected males (109 ± 3 minutes). Additionally, fungus infection reduced the sperm production by 87% at 5 days PE. Some beneficial impacts were observed, FEMs were able to successfully compete with uninfected males in cages, inseminating an equivalent number of females (about 25%). Under semi-field conditions, the ability of FEMs to search for and inseminate females was also equivalent to uninfected males (both inseminating about 40% females); but for the remaining females that were not inseminated, evidence of tarsal contact (transfer of fluorescent dust) was significantly greater in FEMs compared to controls. The estimated conidia load of a female exposed on the 5^th copulation was 5,200 mL^-1 which was sufficient to cause mortality.

Conclusion/Significance

Our study is the first to demonstrate auto-dissemination of M. anisopliae through transfer of fungus from males to female Ae. aegypti during mating under semi-field conditions. Our results suggest that auto-dissemination studies using releases of FEMs inside households could successfully infect wild Ae. aegypti females, providing another viable biological control tool for this important the dengue vector.     

Evaluation of the human IgG antibody response to Aedes albopictus saliva as a new specific biomarker of exposure to vector bites

Background

The spread of Aedes albopictus, a vector for re-emergent arbovirus diseases like chikungunya and dengue, points up the need for better control strategies and new tools to evaluate transmission risk. Human antibody (Ab) responses to mosquito salivary proteins could represent a reliable biomarker for evaluating human-vector contact and the efficacy of control programs.

Methodology/Principal Findings

We used ELISA tests to evaluate specific immunoglobulin G (IgG) responses to salivary gland extracts (SGE) in adults exposed to Aedes albopictus in Reunion Island. The percentage of immune responders (88%) and levels of anti-SGE IgG Abs were high in exposed individuals. At an individual level, our results indicate heterogeneity of the exposure to Aedes albopictus bites. In addition, low-level immune cross-reactivity between Aedes albopictus and Aedes aegypti SGEs was observed, mainly in the highest responders.

Conclusion/Significance

Ab responses to saliva could be used as an immuno-epidemiological tool for evaluating exposure to Aedes albopictus bites. Combined with entomological and epidemiological methods, a “salivary” biomarker of exposure to Aedes albopictus could enhance surveillance of its spread and the risk of arbovirus transmission, and could be used as a direct tool for the evaluation of Aedes albopictus control strategies.     

Modeling intra-mosquito dynamics of Zika virus and its dose-dependence confirms the low epidemic potential of Aedes albopictus

Originating from African forests, Zika virus (ZIKV) has now emerged worldwide in urbanized areas, mainly transmitted by Aedes aegypti mosquitoes. Although Aedes albopictus can transmit ZIKV experimentally and was suspected to be a ZIKV vector in Central Africa, the potential of this species to sustain virus transmission was yet to be uncovered until the end of 2019, when several autochthonous transmissions of the virus vectored by Ae. albopictus occurred in France. Aside from these few locally acquired ZIKV infections, most territories colonized by Ae. albopictus have been spared so far. The risk level of ZIKV emergence in these areas remains however an open question. To assess Ae. albopictus’ vector potential for ZIKV and identify key virus outbreak predictors, we built a complete framework using the complementary combination of (i) dose-dependent experimental Ae. albopictus exposure to ZIKV followed by time-dependent assessment of infection and systemic infection rates, (ii) modeling of intra-human ZIKV viremia dynamics, and (iii) in silico epidemiological simulations using an Agent-Based Model. The highest risk of transmission occurred during the pre-symptomatic stage of the disease, at the peak of viremia. At this dose, mosquito infection probability was estimated to be 20%, and 21 days were required to reach the median systemic infection rates. Mosquito population origin, either temperate or tropical, had no impact on infection rates or intra-host virus dynamic. Despite these unfavorable characteristics for transmission, Ae. albopictus was still able to trigger and yield large outbreaks in a simulated environment in the presence of sufficiently high mosquito biting rates. Our results reveal a low but existing epidemic potential of Ae. albopictus for ZIKV, that might explain the absence of large scale ZIKV epidemics so far in territories occupied only by Ae. albopictus. They nevertheless support active surveillance and eradication programs in these territories to maintain the risk of emergence to a low level.     

Detección de Aedes (Stegomyia) albopictus (Skuse) en ovitrampas en Mérida,México

IntroducciónEl programa de enfermedades transmitidas por vectores en México tiene una red establecida de ovitrampas para la vigilancia entomológica de Aedes spp. Los servicios de salud del estado de Yucatán, en respuesta a reportes de Aedes albopictus en la periferia de Mérida, capital del estado, incrementaron la especificidad de dicha vigilancia.ObjetivoDescribir la presencia y distribución de Ae. albopictus en Mérida y su abundancia relativa comparada con Aedes aegypti, en ovitrampas del programa de control de vectores.Materiales y métodosDurante octubre de 2019, se seleccionaron al azar 91 ovitrampas en 31 barrios de Mérida. Los mosquitos adultos se obtuvieron del insectario de la Unidad Colaborativa para Bioensayos Entomológicos de la Universidad Autónoma de Yucatán a partir de huevos recolectados en campo. Se determinó la abundancia relativa de individuos adultos de cada especie identificada y por barrios evaluados.ResultadosEn el 32% de los barrios muestreados, se detectó Ae. albopictus y, en todos ellos, Ae. aegypti. Se recolectaron 28 adultos de Ae. albopictus (10 hembras y 18 machos) en las ovitrampas. No se observó correlación entre la abundancia de adultos ni de hembras Ae. aegypti y Ae. albopictus por barrio (p>0,05).ConclusionesLos resultados confirmaron que Ae. albopictus estaba coexistiendo con Ae. aegypti en Mérida en el momento del estudio. La baja abundancia relativa sugiere que Ae. albopictus se encontraba en la fase inicial de invasión.Palabras clave: Aedes, mosquitos vectores, control de vectores, enfermedades transmitidas por vectores, México     

Organochlorines and organophosphates susceptibility of Aedes albopictus Skuse larvae from agricultural and non-agricultural localities in Peninsular Malaysia

Aedes albopictus larvae obtained from different types of agricultural and non-agricultural localities in Peninsular Malaysia were subjected to several larvicides at World Health Organization (WHO) recommended dosages. Upon 24 h of WHO larval bioassay using two organochlorines and six organophosphates, high resistance against dichlorodiphenyltrichloroethane (DDT), temephos, chlorpyrifos and bromophos were demonstrated among all larval populations. Aedes albopictus larvae from both paddy growing areas (92.33% mortality) and rubber estates (97.00% mortality) were moderately resistant to dieldrin while only Ae. albopictus larvae from dengue prone residential areas (89.00% mortality) showed high resistance against dieldrin. All Ae. albopictus larval populations also developed either incipient or high resistance to both malathion (33.67%–95.33% mortality) and fenitrothion (73.00%–92.67% mortality). Only Ae. albopictus larvae from fogging-free residential areas that were tolerant to fenthion (97.33% mortality), whereas Ae. albopictus larvae from dengue prone residential areas were highly resistant to the same organophosphate (88.33% mortality). Cross resistance between intraclass and interclass larvicides of organochlorines and organophosphates were also exhibited in this study. The present study provided baseline data on various susceptibility levels of Ae. albopictus larval populations from different types of agricultural and non-agricultural localities against organochlorines and organophosphates at WHO recommended dosages. Nevertheless, further susceptibility investigations are suggested using revised doses of larvicides established from the local reference strain of Ae. albopictus to prevent the underestimation or overestimation of insecticide resistance level among Ae. albopictus field strains of larvae.