Molecular analysis of targeted insecticide resistance gene mutations in field Aedes aegypti and Culex quinquefasciatus mosquito vectors of arboviruses from Saudi Arabia

Abstract Background: Gene mutations on target sites can be a valuable reference to the status of insecticide resistance. Jeddah, a global commercial and major port-of-entry city, is bearing the most (>70%) dengue disease burden and the population of the dengue vector Aedes aegypti in Saudi Arabia. Culex quinquefasciatus is a second example as one of the major arboviral vectors in the region. However, the status of insecticide resistance and targeted site mutations on the responsible genes are not fully characterized.Methods: We randomly sampled both mosquito species, Ae. aegypti and Cx. quinquefasciatus across Jeddah by daily mosquito surveillance in 2016 to detect the resistance-associated target site mutations on the voltage gated sodium channel (VGSC) and acetylcholinesterase 1 (ace-1) genes. Results: Our findings showed that Ae. aegypti resistance-associated VGSC gene mutations revealed polymorphic mutations on the 989, 1016, and 1534 sites. Additionally, we documented two types of introns between exons 20 and 21, however, the I1011M point mutation was undetected. Linkage disequilibrium associations were shown between V1016G with S989P, V1016G with F1534, and V1016G with type A intron. Furthermore, no mutation on ace-1 was identified in Ae. aegypti. In Cx. quinquefasciatus, homozygous L1014F/L1014F (95.23%) on the VGSC and heterozygous G119/G119S (100%) on ace-1 were widely distributed in the samples studied. Analysis of intron sequences obtained in our study and homologous sequences retrieved from GenBank showed that the intron type was significantly associated with 1016 (P = 0.000) allele type, and may reflect the history of insecticide treatment in different continents.Conclusions: To the best of our knowledge, this is the first record of the intron types between exons 20 and 21 on VGSC of Ae. aegypti populations, and the first report of insecticide resistance gene mutation being present in field caught Cx. quinquefasciatus in Jeddah, Saudi Arabia. High prevalence of insecticide resistance gene mutations in local primary mosquito vector species alert the urgent need to carry out a comprehensive insecticide resistance gene mutation surveillance and monitoring to guide sustained and effective integrated vector management planning and innovative guidelines in local predominate mosquito populations and mosquito-borne disease control and elimination in Saudi Arabia.

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Molecular analysis of targeted insecticide resistance gene mutations in field Aedes aegypti and Culex quinquefasciatus mosquito vectors of arboviruses from Saudi Arabia

10.21203/rs.3.rs-35782/v2 ◽

2020 ◽

Author(s):

Yuan Fang ◽

Tambo Ernest ◽

Jing-Bo Xue ◽

Yi Zhang ◽

Xiao-Nong Zhou ◽

...

Keyword(s):

Saudi Arabia ◽

Aedes Aegypti ◽

Insecticide Resistance ◽

Resistance Gene ◽

Culex Quinquefasciatus ◽

Direct Sequencing ◽

Mosquito Species ◽

Gene Mutations ◽

Mosquito Vector ◽

The Status

Abstract Background: Gene mutations on target sites can be a valuable reference to the status of insecticide resistance. Jeddah, a global commercial and major port-of-entry city, is bearing the most (>70%) dengue disease burden and the population of the dengue vector Aedes aegypti (Linnaeus, 1762) in Saudi Arabia. Culex quinquefasciatus Say, 1823 is a second example as one of the major arboviral vectors in the region. However, the status of insecticide resistance and targeted site mutations on the responsible genes are not fully characterized.Methods: We randomly sampled both mosquito species, Ae. aegypti (25 individuals) and Cx. quinquefasciatus (24 individuals) across Jeddah by daily mosquito surveillance in 2016 to detect the resistance-associated target site mutations on the voltage gated sodium channel (VGSC) and acetylcholinesterase 1 (ace-1) genes by PCR amplification and direct sequencing. Results: Our findings showed that Ae. aegypti resistance-associated VGSC gene mutations revealed polymorphic mutations on the 989, 1016, and 1534 sites. Additionally, we documented two types of introns between exons 20 and 21, however, the I1011M point mutation was undetected. Linkage disequilibrium associations were shown between V1016G with S989P, V1016G with F1534, and V1016G with type A intron. Furthermore, no mutation on ace-1 was identified in Ae. aegypti. In Cx. quinquefasciatus, homozygous L1014F/L1014F (95.23%) on the VGSC and heterozygous G119/G119S (100%) on ace-1 were widely distributed in the samples studied. Analysis of intron sequences obtained in our study and homologous sequences retrieved from GenBank showed that the intron type was significantly associated with 1016 (P = 0.000) allele type, and may reflect the history of insecticide treatment in different continents.Conclusions: To the best of our knowledge, this is the first record of the intron types between exons 20 and 21 on VGSC of Ae. aegypti populations. It can serve as a reference marker to evaluate the local insecticide resistance status. In addition, the first report of insecticide resistance gene mutation being present in field caught Cx. quinquefasciatus in Saudi Arabia. High prevalence of insecticide resistance gene mutations in local primary mosquito vector species alert the urgent need to carry out a comprehensive insecticide resistance surveillance.

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Molecular Analysis of Targeted Insecticide Resistance Gene Mutations in field Aedes Aegypti and Culex Quinquefasciatus Mosquito Vectors of Arboviruses from Saudi Arabia

10.21203/rs.3.rs-28170/v1 ◽

2020 ◽

Author(s):

Yuan Fang ◽

Tambo Ernest ◽

Jin-Bo Xue ◽

Yi Zhang ◽

Hany A. Kamal ◽

...

Keyword(s):

Saudi Arabia ◽

Aedes Aegypti ◽

Insecticide Resistance ◽

Resistance Gene ◽

Allele Frequency ◽

Culex Quinquefasciatus ◽

Rift Valley ◽

Gene Mutations ◽

Mosquito Vectors ◽

The Status

Abstract Background In absence of effective and safe vaccines or drugs against mosquito-borne arboviral diseases (such as dengue, Zika and Rift Valley and West Nile), there have been an increasing pattern of insecticide use against the mosquito vectors of these diseases in integrated vector management (IVM) programs in Saudi Arabia. However, the efficacy of IVM programmes is threatened by the development of insecticide resistance in vectors. Gene mutations on target sites can be a valuable reference to the status of insecticide resistance. Jeddah, a global commercial and major port-of-entry city, is bearing the most (> 70%) dengue disease burden and the population of the dengue vector Aedes aegypti in Saudi Arabia. Culex quinquefasciatus is a second example as one of the major arboviral vectors in the region and a proven vector of Rift Valley fever and wide-spread in Jeddah and the rest of the western region of Saudi Arabia. However, the status of insecticide resistance and targeted site mutations on the responsible genes are not fully characterized.Methods We randomly sampled both mosquito species, Ae. aegypti and Cx. quinquefasciatus across Jeddah by daily mosquito surveillance in 2016 to detect the resistance-associated target site mutations on the voltage gated sodium channel (VGSC) and acetylcholinesterase 1 (ace-1) genes.Results Our findings showed that Ae. aegypti resistance-associated VGSC gene mutations revealed polymorphic mutations on the 989 (allele frequency = 65.91%), 1016 (allele frequency = 65.91%), and 1534 (allele frequency = 52.27%) sites. Additionally, we documented two types of introns between exons 20 and 21, however, the I1011M point mutation was undetected. Linkage disequilibrium associations were shown between V1016G with S989P, V1016G with F1534, and V1016G with type A intron. Furthermore, no mutation on ace-1 was identified in Ae. aegypti. In Cx. quinquefasciatus, homozygous L1014F/L1014F (95.23%) on the VGSC and heterozygous G119/G119S (100%) on ace-1 were widely distributed in the samples studied.Conclusions To the best of our knowledge, this is the first record of the intron types between exons 20 and 21 on VGSC of Ae. aegypti populations, and the first report of insecticide resistance gene mutation being present in field caught Cx. quinquefasciatus in Jeddah, Saudi Arabia. High prevalence of insecticide resistance gene mutations in local primary mosquito vector species, implies that the local Ae. aegypti mosquitoes are resistant to both pyrethroid and DDT, while the resistance to all four insecticide groups may possessed in Cx. quinquefasciatus populations. Our findings alert the urgent need to carry out a comprehensive insecticide resistance gene mutation surveillance and monitoring to guide sustained and effective IVM planning and innovative guidelines in local predominate mosquito populations and mosquito-borne disease control and elimination in Saudi Arabia.

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Molecular Analysis of Targeted Insecticide Resistance Gene Mutations in Field-Caught Mosquitos of Medical Importance From Saudi Arabia

Journal of Medical Entomology ◽

10.1093/jme/tjab048 ◽

2021 ◽

Author(s):

Yuan Fang ◽

Ernest Tambo ◽

Jing-Bo Xue ◽

Yi Zhang ◽

Xiao-Nong Zhou ◽

...

Keyword(s):

Saudi Arabia ◽

Insecticide Resistance ◽

Resistance Gene ◽

Phylogenetic Analyses ◽

Gene Mutations ◽

Mosquito Vector ◽

Dengue Disease ◽

The Status ◽

History Of ◽

Exon 20

Abstract Gene mutations on target sites can be a valuable indicator of the status of insecticide resistance. Jeddah, a global commercial and major port-of-entry city, is bearing the brunt of dengue disease burden in Saudi Arabia. In the current study, six genotypes of three codon combinations (989, 1016, and 1534) were observed on voltage-gated sodium channel (VGSC) gene in Jeddah’s Aedes aegypti population, with PGF/PGC as the dominant one. Two types of introns between exon 20 and 21 on VGSC have been identified for the first time in Ae. aegypti in Saudi Arabia. Statistical and phylogenetic analyses showed that the intron type was significantly associated with the 1016 allele and may reflect the history of insecticide treatment in different continents. In addition, fixation of the L1014F allele on VGSC and G119S on acetylcholinesterase 1 gene was detected in local Culex quinquefasciatus populations, with frequencies of 95.24 and 100%, respectively. To the best of our knowledge, this is the first report of resistant-associated mutations in field-caught Cx. quinquefasciatus in Saudi Arabia. The high prevalence of insecticide resistance gene mutations in local primary mosquito vector species highlights the urgent need to carry out comprehensive insecticide resistance surveillance in Saudi Arabia.

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Voltage-sensitive sodium channel (Vssc) mutations associated with pyrethroid insecticide resistance in Aedes aegypti (L.) from Jeddah, Kingdom of Saudi Arabia - baseline information for a Wolbachia release program

10.1101/2021.01.26.428347 ◽

2021 ◽

Author(s):

Nancy M Endersby-Harshman ◽

AboElgasim Ali ◽

Basim Alhumrani ◽

Mohammed Abdullah Alkuriji ◽

Mohammed B. Al- Fageeh ◽

...

Keyword(s):

Saudi Arabia ◽

Aedes Aegypti ◽

Insecticide Resistance ◽

Sodium Channel ◽

Dominant Role ◽

Public Health Problem ◽

Type I ◽

Mosquito Vector ◽

Genotype Frequencies ◽

The Impact

Background: Dengue suppression often relies on control of the mosquito vector, Aedes aegypti, through applications of insecticides of which the pyrethroid group has played a dominant role. Insecticide resistance is prevalent in Ae. aegypti around the world and the resulting reduction of insecticide efficacy is likely to exacerbate the impact of dengue. Dengue has been a public health problem in Saudi Arabia, particularly in Jeddah, since its discovery there in the 1990s and insecticide use for vector control is widespread throughout the city. An alternative approach to insecticide use, based on blocking dengue transmission in mosquitoes by the endosymbiont Wolbachia, is being trialled in Jeddah following the success of this approach in Australia and Malaysia. Knowledge of insecticide resistance status of mosquito populations in Jeddah is a prerequisite for establishing a Wolbachia-based dengue control program as releases of Wolbachia mosquitoes succeed when resistance status of the release population is similar to that of the wild population. Methods: WHO resistance bioassays of mosquitoes with deltamethrin, permethrin and DDT were used in conjunction with TaqMan® SNP Genotyping Assays to characterise mutation profiles of Ae. aegypti from Jeddah. Results: Screening of the voltage sensitive sodium channel (Vssc), the pyrethroid target-site, revealed mutations at codons 989, 1016 and 1534 in Ae. aegypti from two districts of Jeddah. The triple mutant homozygote (1016G/1534C/989P) was confirmed from Al Safa and Al Rawabi. Bioassays with pyrethroids (Type I and II) and DDT showed that mosquitoes were resistant to each of these compounds based on WHO definitions. An association between Vssc mutations and resistance was established for the Type II pyrethroid, deltamethrin, with one genotype (989P/1016G/1534F) conferring a survival advantage over two others (989S/1016V/1534C and the triple heterozygote). An indication of synergism of Type I pyrethroid activity with piperonyl butoxide suggests that detoxification by cytochrome P450s accounts for some of the pyrethroid resistance response in Ae. aegypti populations from Jeddah. Conclusions: The results provide a baseline for monitoring and management of resistance as well as knowledge of Vssc genotype frequencies required in Wolbachia release populations to ensure homogeneity with the target field population.

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Voltage-sensitive sodium channel (Vssc) mutations associated with pyrethroid insecticide resistance in Aedes aegypti (L.) from two districts of Jeddah, Kingdom of Saudi Arabia: baseline information for a Wolbachia release program

Parasites & Vectors ◽

10.1186/s13071-021-04867-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Nancy M. Endersby-Harshman ◽

AboElgasim Ali ◽

Basim Alhumrani ◽

Mohammed Abdullah Alkuriji ◽

Mohammed B. Al-Fageeh ◽

...

Keyword(s):

Saudi Arabia ◽

Aedes Aegypti ◽

Insecticide Resistance ◽

Sodium Channel ◽

Public Health Problem ◽

Population History ◽

Type I ◽

Mosquito Vector ◽

Triple Mutant ◽

The Impact

Abstract Background Dengue suppression often relies on control of the mosquito vector, Aedes aegypti, through applications of insecticides of which the pyrethroid group has played a dominant role. Insecticide resistance is prevalent in Ae. aegypti around the world, and the resulting reduction of insecticide efficacy is likely to exacerbate the impact of dengue. Dengue has been a public health problem in Saudi Arabia, particularly in Jeddah, since its discovery there in the 1990s, and insecticide use for vector control is widespread throughout the city. An alternative approach to insecticide use, based on blocking dengue transmission in mosquitoes by the endosymbiont Wolbachia, is being trialed in Jeddah following the success of this approach in Australia and Malaysia. Knowledge of insecticide resistance status of mosquito populations in Jeddah is a prerequisite for establishing a Wolbachia-based dengue control program as releases of Wolbachia mosquitoes succeed when resistance status of the release population is similar to that of the wild population. Methods WHO resistance bioassays of mosquitoes with deltamethrin, permethrin and DDT were used in conjunction with TaqMan® SNP Genotyping Assays to characterize mutation profiles of Ae. aegypti. Results Screening of the voltage-sensitive sodium channel (Vssc), the pyrethroid target site, revealed mutations at codons 989, 1016 and 1534 in Ae. aegypti from two districts of Jeddah. The triple mutant homozygote (1016G/1534C/989P) was confirmed from Al Safa and Al Rawabi. Bioassays with pyrethroids (Type I and II) and DDT showed that mosquitoes were resistant to each of these compounds based on WHO definitions. An association between Vssc mutations and resistance was established for the Type II pyrethroid, deltamethrin, with one genotype (989P/1016G/1534F) conferring a survival advantage over two others (989S/1016V/1534C and the triple heterozygote). An indication of synergism of Type I pyrethroid activity with piperonyl butoxide suggests that detoxification by cytochrome P450s accounts for some of the pyrethroid resistance response in Ae. aegypti populations from Jeddah. Conclusions The results provide a baseline for monitoring and management of resistance as well as knowledge of Vssc genotype frequencies required in Wolbachia release populations to ensure homogeneity with the target field population. Vssc mutation haplotypes observed show some similarity with those from Ae. aegypti in southeast Asia and the Indo-Pacific, but the presence of the triple mutant haplotype in three genotypes indicates that the species in this region may have a unique population history. Graphical Abstract

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A Simple PCR Assay for Discrimination of Dengue Vectors from Nsukka LGA, Nigeria

International Annals of Science ◽

10.21467/ias.10.1.67-77 ◽

2020 ◽

Vol 10 (1) ◽

pp. 67-77

Author(s):

Amos Watentena ◽

Ikem Chris Okoye ◽

Ikechukwu Eugene Onah ◽

Onwude Cosmas Ogbonnaya ◽

Emmanuel Ogudu

Keyword(s):

Aedes Aegypti ◽

Aedes Albopictus ◽

Mosquito Species ◽

Public Health Problem ◽

Major Public Health Problem ◽

Mosquito Vector ◽

Aedes Mosquitoes ◽

Allele Specific ◽

Arboviral Diseases ◽

Dengue Vectors

Mosquitoes of Aedes species are vectors of several arboviral diseases which continue to be a major public health problem in Nigeria. This study among other things, morphologically identified Aedes mosquitoes collected from Nsukka LGA and used an allele specific PCR amplification for discrimination of dengue vectors. Larval sampling, BG-sentinel traps and modified human landing catches were used for mosquito sampling in two selected autonomous communities of Nsukka LGA (Nsukka and Obimo). A total of 124 Aedes mosquitoes consisting of five (5) different species were collected from April to June, 2019 in a cross-sectional study that covered 126 households, under 76 distinct geographical coordinates. Larvae was mainly collected from plastic containers 73% (n=224), metallic containers 14% (n=43), earthen pots 9% (n=29) and used car tyres 3% (n=9), reared to adult stage 69.35% (n=86), and all mosquitoes were identified using standard morphological keys. Five (5) Aedes mosquito species were captured; Aedes aegypti 83(66.94%), Aedes albopictus 33(26.61%), Aedes simpsoni (4.48%), Aedes luteocephalus (≤1%) and Aedes vittatus (≤1%). Nsukka autonomous community had higher species diversity than Obimo. Allele specific amplification confirmed dengue vectors, Aedes aegypti and Aedes albopictus species on a 2% agarose gel. Since the most recent re-emergence of arboviral diseases is closely associated with Aedes species, findings of this study, therefore, give further evidence about the presence of potential arboviral vectors in Nigeria and describe the role of a simple PCR in discriminating some. Further entomological studies should integrate PCR assays in mosquito vector surveillance.

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Behavioural response of mosquito vectors Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus to synthetic pyrethroid and organophosphorus-based slow-release insecticidal paint

Parasites & Vectors ◽

10.1186/s13071-021-04746-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Sunil Dhiman ◽

Kavita Yadav ◽

B. N. Acharya ◽

Raj Kumar Ahirwar ◽

D. Sukumaran

Keyword(s):

Aedes Aegypti ◽

Culex Quinquefasciatus ◽

Anopheles Stephensi ◽

Slow Release ◽

Disease Risk ◽

Mosquito Species ◽

Escape Time ◽

Behavioural Avoidance ◽

Vector Mosquito ◽

Contact Exposure

Abstract Background The direct toxicological impact of insecticides on vector mosquitoes has been well emphasized; however, behavioural responses such as excito-repellency and physical avoidance as a result of insecticide exposure have not been much studied. We have demonstrated the excito-repellency and behavioural avoidance in certain vector mosquito species on exposure to a slow-release insecticidal paint (SRIP) formulation in addition to direct toxicity. Methods A SRIP formulation developed by the Defence Research and Development Establishment, Gwalior, contains chlorpyriphos, deltamethrin and pyriproxyfen as active insecticides. Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti mosquitoes were used to study the excito-repellency response of the formulation. The experiments were performed in a specially designed dual-choice exposure and escape chamber made of transparent polymethyl methacrylate. For the experiments, the SRIP formulation was applied undiluted at a rate of 8 m2 per kg on 15 cm2 metallic surfaces. Mosquitoes were introduced into the exposure chamber, and observations of the movement of mosquitoes into the escape chamber through the exit portal were taken at 1-min intervals for up to 30 min. Results The evaluated formulation displayed strong excito-repellency against all three tested vector mosquito species. Results showed that the ET50 (escape time 50%) for Ae. aegypti, An. stephensi and Cx. quinquefasciatus was 20.9 min, 14.5 min and 17.9 min for contact exposure (CE) respectively. Altogether in CE, the escape rates were stronger in An. stephensi mosquitoes at different time intervals compared to Ae. aegypti and Cx. quinquefasciatus mosquitoes. The probit analysis revealed that the determined ET did not deviate from linearity for both non-contact exposure (NCE) and placebo exposure (PE) (χ2 ≤ 7.9; p = 1.0) for Ae. aegypti mosquitoes and for NCE (χ2 = 8.3; p = 1.0) and PE (χ2 = 1.7; p = 1.0) treatments in Cx. quinquefasciatus. Mortality (24 h) was found to be statistically higher (F = 6.4; p = 0.02) in An. stephensi for CE but did not vary for NCE (p ≥ 0.3) and PE (p = 0.6) treatments among the tested mosquito species. Survival probability response suggested that all the three tested species displayed similar survival responses for similar exposures (χ2 ≤ 2.3; p ≥ 0.1). Conclusion The study demonstrates the toxicity and strong behavioural avoidance in known vector mosquito species on exposure to an insecticide-based paint formulation. The combination of insecticides in the present formulation will broaden the overall impact spectrum for protecting users from mosquito bites. The efficacy data generated in the study provide crucial information on the effectiveness of the tested formulation and could be useful in reducing the transmission intensity and disease risk in endemic countries.

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Contrasted transmission efficiency of Zika virus strains by mosquito species Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Reunion Island

Parasites & Vectors ◽

10.1186/s13071-020-04267-z ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Yann Gomard ◽

Cyrille Lebon ◽

Patrick Mavingui ◽

Célestine M. Atyame

Keyword(s):

Aedes Aegypti ◽

Aedes Albopictus ◽

Culex Quinquefasciatus ◽

Zika Virus ◽

Mosquito Species ◽

Transmission Efficiency ◽

Reunion Island ◽

Réunion Island ◽

Virus Strains

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Co-occurrence of kdr Mutations V1016I and F1534C and Its Association With Phenotypic Resistance to Pyrethroids in Aedes aegypti (Diptera: Culicidae) Populations From Costa Rica

Journal of Medical Entomology ◽

10.1093/jme/tjz241 ◽

2019 ◽

Vol 57 (3) ◽

pp. 830-836

Author(s):

Aryana Zardkoohi ◽

David Castañeda ◽

Juan C Lol ◽

Carmen Castillo ◽

Francisco Lopez ◽

...

Keyword(s):

Costa Rica ◽

Aedes Aegypti ◽

Insecticide Resistance ◽

Vector Control ◽

Strong Association ◽

Resistance Management ◽

Management Plan ◽

Costa Rican ◽

Mosquito Vector ◽

Phenotypic Resistance

Abstract Aedes aegypti (Linnaeus, 1762) is considered the most important mosquito vector species for several arboviruses (e.g., dengue, chikungunya, Zika) in Costa Rica. The primary strategy for the control and prevention of Aedes-borne diseases relies on insecticide-based vector control. However, the emergence of insecticide resistance in the mosquito populations presents a significant threat to these prevention actions. The characterization of the mechanisms driving the insecticide resistance in Ae. aegypti is vital for decision making in vector control programs. Therefore, we analyzed the voltage-gated sodium channel (VGSC) gene for the presence of the V1016I and F1534C kdr mutations in Ae. aegypti populations from Puntarenas and Limon provinces, Costa Rica. The CDC bottle bioassays showed that both Costa Rican Ae. aegypti populations were resistant to permethrin and deltamethrin. In the case of kdr genotyping, results revealed the co-occurrence of V1016I and F1534C mutations in permethrin and deltamethrin-resistant populations, as well as the fixation of the 1534C allele. A strong association between these mutations and permethrin and deltamethrin resistance was found in Puntarenas. Limon did not show this association; however, our results indicate that the Limon population analyzed is not under the same selective pressure as Puntarenas for the VGSC gene. Therefore, our findings make an urgent call to expand the knowledge about the insecticide resistance status and mechanisms in the Costa Rican populations of Ae. aegypti, which must be a priority to develop an effective resistance management plan.

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