Insecticide Resistance in Aedes aegypti Mosquitoes: Possible Detection of kdr F1534C, S989P, and V1016G Triple Mutation in Benin, West Africa

Simple Summary The effectiveness of chemical control of Aedes (Ae.) aegypti is threatened by the increasing frequency of insecticide resistance. This study aimed to determine, in two cities of Benin, the insecticide resistance profiles of Ae. aegypti, the presence of detoxification enzymes, and the frequency of kdr mutations. Ae. aegypti eggs were collected in the study areas using gravid Aedes traps (GATs). Centers for Disease Control and Prevention (CDC) bottle bioassays were used to assess the susceptibility status of adult female Ae. aegypti, followed by kdr screening using allele-specific PCR. The activity levels of key detoxification enzymes were measured among individual, unexposed, and un-engorged adult female Ae. aegypti. In both study sites, Ae. aegypti was resistant to the pyrethroids deltamethrin and permethrin, but susceptible to the carbamate bendiocarb. Significant over-expression of glutathione-S-transferases and under-expression of α and β esterases were observed in these vector populations. Three kdr mutations (F1534C, S989P, and V1016G) were possibly present in resistant Ae. aegypti at high frequencies, including the simultaneous occurrence of all three mutations in individual mosquitoes. Study findings will be used to inform prospective vector control strategies in Benin. Abstract Epidemics of arboviruses in general, and dengue fever in particular, are an increasing threat in areas where Aedes (Ae.) aegypti is present. The effectiveness of chemical control of Ae. aegypti is jeopardized by the increasing frequency of insecticide resistance. The aim of this study was to determine the susceptibility status of Ae. aegypti to public health insecticides and assess the underlying mechanisms driving insecticide resistance. Ae. aegypti eggs were collected in two study sites in the vicinity of houses for two weeks using gravid Aedes traps (GATs). After rearing the mosquitoes to adulthood, female Ae. aegypti were exposed to diagnostic doses of permethrin, deltamethrin and bendiocarb, using Centers for Disease Control and Prevention (CDC) bottle bioassays. Unexposed, un-engorged female Ae. aegypti were tested individually for mixed-function oxidase (MFO), glutathione-S-transferase (GST) and α and β esterase activities. Finally, allele-specific PCR (AS-PCR) was used to detect possible kdr mutations (F1534C, S989P, and V1016G) in the voltage-gated sodium channel gene in insecticide-exposed Ae. aegypti. Most traps were oviposition positive; 93.2% and 97% of traps contained Ae. aegypti eggs in the 10ème arrondissement of Cotonou and in Godomey-Togoudo, respectively. Insecticide bioassays detected resistance to permethrin and deltamethrin in both study sites and complete susceptibility to bendiocarb. By comparison to the insecticide-susceptible Rockefeller strain, field Ae. aegypti populations had significantly higher levels of GSTs and significantly lower levels of α and β esterases; there was no significant difference between levels of MFOs. AS-PCR genotyping revealed the possible presence of 3 kdr mutations (F1534C, S989P, and V1016G) at high frequencies; 80.9% (228/282) of the Ae. aegypti tested had at least 1 mutation, while the simultaneous presence of all 3 kdr mutations was identified in 13 resistant individuals. Study findings demonstrated phenotypic pyrethroid resistance, the over-expression of key detoxification enzymes, and the possible presence of several kdr mutations in Ae. aegypti populations, emphasizing the urgent need to implement vector control strategies targeting arbovirus vector species in Benin.


Introduction
Aedes (Ae.) aegypti mosquitoes are the main vector species of dengue viruses worldwide.The number of dengue fever cases reported to the World Health Organization (WHO) has increased dramatically by more than eightfold over the previous twenty years, from 505,430 cases in 2000 to over 2.4 million cases in 2010 and 5.2 million cases in 2019 [1].Ae. aegypti is present in Benin and proliferating globally due to the development of trade, rapid urbanization, and high frequency of international travel [2].Between 2010 and 2019, dengue fever cases have been diagnosed in Benin, resulting in at least one death [2].
In the absence of effective vaccines and available treatments, vector control remains the main strategy for dengue virus prevention.Vector control relies on the use of insecticides, such as pyrethroids, for house spraying and personal protection [3].As a result of the strong selection pressures exerted using insecticides in agricultural practices and malaria control, including pyrethroid-only long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) with pyrethroids, insecticide resistance among Ae.aegypti populations is common and widespread worldwide.According to the WHO, insecticide resistance is defined as "the ability of mosquitoes to survive exposure to a standard dose of insecticide; this ability may result from physiological or behavioral adaptation" [4].Two of the main mechanisms underlying insecticide resistance in mosquitoes are alterations in the insecticide target site, including knock-down resistance (kdr) mutations in the voltage-gated sodium channel (vgsc) gene and increased metabolic activity [5,6].Metabolic resistance to pyrethroids may be mediated by glutathione-S-transferases (GSTs), esterases, and mixed-function oxidases (MFOs) [7][8][9].
The emergence and re-emergence of dengue fever epidemics requires effective vector control responses, including monitoring of vector population insecticide susceptibility.To date, there is a considerable paucity of insecticide resistance information for Ae.aegypti populations in Benin.

Study Sites and Mosquito Sampling
Adult Ae. aegypti mosquitoes were reared from eggs collected over a 2-week period in the 10 ème arrondissement of Cotonou and in Godomey-Togoudo, Abomey-Calavi from 31 December 2021 (Figure 1).This collection period coincided with the dry season, which Insects 2024, 15, 295 3 of 11 is a period characterized by a scarcity of Aedes breeding sites.During this sampling period, a total of 73 gravid Aedes traps were set in the 10 ème arrondissement of Cotonou and 74 in Godomey-Togoudo.The traps used are black plastic pots that can contain half a liter of water, in which wooden egg-laying supports have been immersed.The traps were hung on the box or wall with a nail and a wire.The egg carriers were removed from each trap after 7 days, eggs were counted and then hatched according to standard insectary rearing procedures for Aedes species.A dipper was used to visually count the number of larvae per bowl.Larval hatching rate was measured by dividing the number of larvae by the total number of immersed eggs ×100.Emergent adults were counted by aspiration.Adult emergence rate was measured by dividing the number of emerged adults by the total number of larvae ×100.

Study Sites and Mosquito Sampling
Adult Ae. aegypti mosquitoes were reared from eggs collected over a 2-week period in the 10 ème arrondissement of Cotonou and in Godomey-Togoudo, Abomey-Calavi from 31 December 2021 (Figure 1).This collection period coincided with the dry season, which is a period characterized by a scarcity of Aedes breeding sites.During this sampling period, a total of 73 gravid Aedes traps were set in the 10 ème arrondissement of Cotonou and 74 in Godomey-Togoudo.The traps used are black plastic pots that can contain half a liter of water, in which wooden egg-laying supports have been immersed.The traps were hung on the box or wall with a nail and a wire.The eggs carriers were removed from each trap after 7 days, eggs were counted and then hatched according to standard insectary rearing procedures for Aedes species.A dipper was used to visually count the number of larvae per bowl.Larval hatching rate was measured by dividing the number of larvae by the total number of immersed eggs ×100.Emergent adults were counted by aspiration.Adult emergence rate was measured by dividing the number of emerged adults by the total number of larvae ×100.

Aedes Morphological Identification
Adult Ae. aegypti for insecticide resistance bioassays were identified using Fontenille's taxonomic keys [18].Ae. aegypti and others aedes were distinguished by characteristic white stripes on their legs.Then, the thorax was used to differentiate the two species;

Aedes Morphological Identification
Adult Ae. aegypti for insecticide resistance bioassays were identified using Fontenille's taxonomic keys [18].Ae. aegypti and other Aedes were distinguished by characteristic white stripes on their legs.Then, the thorax was used to differentiate the two species; Ae. aegypti has two thin white median lines with a lyre pattern, whereas Ae. albopictus has only one distinct white central line.

Insecticide Resistance Bioassays
Insecticide resistance profiles for Ae.aegypti field populations were measured using Centers for Disease Control and Prevention (CDC) bottle bioassays [19].Two-to-five-day-old female Ae.aegypti mosquitoes were exposed to diagnostic doses of deltamethrin (10 µg/bottle), permethrin (15 µg/bottle), and bendiocarb (12.5 µg/bottle) in Wheaton 250 mL bottles, alongside an acetone-treated control bottle.In each bioassay, 10-25 unfed mosquitoes were introduced into each bottle and knock-down was scored every 15 min until all were dead, or up to two hours had elapsed; data were reported for the diagnostic time of 30 min for all insecticides.Approximately 100 mosquitoes (in 3-4 bottle bioassays) were tested per insecticide dose.

Measurement of Detoxification Enzyme Activity
Biochemical tests were performed to quantify the activity of families of detoxification enzymes, including non-specific esterases (α and β esterases), mixed-function oxidases (MFOs), glutathione S-transferases (GSTs) and total proteins in 3-5-day old female Ae.aegypti mosquitoes; a total of 80 non-insecticide exposed mosquitoes were tested per study site, in parallel with the Rockfeller susceptible strain of Ae. aegypti as a control [20].
Individual mosquitoes were ground in 200 µL of distilled water using sterile pestles, after which the lysate was centrifuged at 14,000 rpm for two minutes.For non-specific esterases, we used 2 substrates: α-naphthyl acetate and β-naphthyl acetate.Each plate was calibrated with the products of the esterase-substrate reaction (α-naphthyl acetate or β-naphthyl acetate).The reaction consisted of hydrolysis of α-naphthyl (or β-naphthyl) acetate by esterase with the formation of α-(or β)-naphthol with Fast Garnett Salt.This assay involved determining the quantity of α-(or β)-naphthol formed as a function of time.For each esterase plate, 10 µL of mosquito lysate was added in duplicate.Then, the standard curves of α or β naphthol were generated with Gen5 software (version 2.0) to determine each well concentration.To each well, 10 µL of supernatant or standards and 90 µL of Phosphate Saline Buffer (PBS) pH 6.5 + 1% triton ×100 was added.The plate was then incubated at 25 • C for 10 min, after which 100 µL of working solution (500 µL α-naphthyl acetate or β-naphthyl (0.03 M) + 2.5 mL PBS buffer (pH mL 6.5) + 7 mL H 2 O), was added to each well and the plate was again incubated at 25 • C for 30 min.Finally, 100 µL of 3 mM of a solution of Fast Garnett Salt dissolved in 10 mL distilled water was added to each well and the plate was incubated at 25 • C for 10 min.Plate absorbance values were read on an ELx808 spectrophotometer at 550 nm.The standard curve fitted a straight line: DO = ax + b where x is the quantity of αor β-naphthol in the well.Using this formula with the optical density (OD) of the sample we calculated the amount of αor β-naphthol produced by 10 µL of mosquito supernatant for 30 min.Esterase activity of each mosquito was expressed as µmol αor β-naphthol produced/min/mg protein and calculated according to the following: αor β-naphthol in µmol per mL/amount of protein in mg per mL/30.
For MFOs, 20 µL of mosquito lysate was added to 2 wells (technical duplicate) of a 96-well ELISA plate.Then, the standards curve of cytochrome C was generated with Gen5 software to determine the each well concentration.The first 2 columns are reserved for the H 2 O control and the standard range.For each duplicate of 20 µL of supernatant, 80 µL of 0.0625M Potassium Phosphate Buffer (KHPO 4 ; pH 7.2) was added; then, 200 µL of working solution (1.6 mM of 3,3,5,5-tetramethylbenzidine dihydrochloride was dissolved in 5 mL ethanol and 15 mL of 0.25 M sodium acetate buffer pH 5.0).After adding 25 µL of 3% hydrogen peroxide, the plate was incubated for 30 min, and plate absorbance values were read on an ELx808 spectrophotometer at 630 nm.The standard curve was fitted to a second-degree curve: OD = ax2 +bx + c, where x is the amount of P450 equivalent units in the homogenates.Using this formula with the ODs of the samples, we calculated the quantity of "P450 equivalent units" (in nmol) produced by 20 µL of supernatant.This transformation was usually performed automatically by the software supplied with the spectrophotometer.The oxidase activity of each mosquito in nmol P450 equivalent units/mg protein is as follows: (nmol P450 equivalent units for 20 µL supernatant/2 mg protein in 10 µL supernatant).For GSTs, to each duplicate of 10 µL supernatant we added 200 µL of working solution: (10 mM GSH prepared in 0.1 M phosphate buffer, pH 6.5-, and 3-mM Di chloronitrobenzene dissolve in methanol).For each microtiter plate, 2 blanks with 10 µL of distilled water + 200 µL of working solution was added and the absorbance was measured at 340 nm for 5 min.The GST activity for individual mosquitos was calculated as µmol GSH conjugated/min/mg protein using known extinction coefficients: (Milli DO × 0.21)/(5.76× 1000)/(amount of protein in mg in 10 µL of supernatant. For total protein, 10 µL of supernatant was added to 2 wells (technical duplicate) of 96-well ELISA plate.Then, the standards curve of Bovine Serum Albumin (BSA) was generated with Gen5 software to determine the each well concentration.We added 200 µL of working solution (19 mL bicinchoninic Acid Solution + 380 mL Copper (II) Sulfate Solution) as described by the manufacturer.The plate was incubated for 30 min at room temperature, after which the OD was read as an end point at 595 nm.

Ae. aegypti kdr Genotyping
After grinding each mosquito in 200 µL of 2% CTAB, samples were then incubated at 65 • C for 5 min.Thereafter, 200 µL of chloroform was added and each sample was centrifuged at 12,000 rpm for 5 min at room temperature after mixing by inversion at least 10 times.The supernatant was mixed with 200 µL of isopropanol and centrifuged at 12,000 rpm at room temperature for 10 min.The DNA pellet was washed in 200 µL of 70% ethanol by centrifugation for 5 min at 12,000 rpm.The DNA pellet was dried at room temperature and re-suspended in 140 µL of sterile H 2 O.

Data Analysis
Global Positioning System (GPS) coordinates of the gravid Aedes traps were recorded using the OSM Tracker for Android application.Insecticide susceptibility test results were recorded and analyzed using Microsoft Excel 2016.All biochemical data were saved after reading on a computer connected to the plate reader.Transformations from the various right-hand equations to determine the quantity of the final product were performed automatically by the software (GeneS.1)supplied with the spectrophotometer.Statistical analyses were also performed using GraphPad-Prism 5 software (version 5.00, San Diego, CA, USA).The Mann-Whitney test test was chosen for comparison between Rockefeller (susceptible strain), and field mosquitoes.Statistical significance was determined if p < 0.05.Statistical analyses were conducted in Stata/SE 17.0, including Pearson's Chi-squared test to investigate deviations from Hardy-Weinberg equilibrium.

Mosquito Sampling
A total of 147 gravid Aedes traps were used in the 2 study areas, of which 142 were positive (i.e., female Aedes oviposited in them); yielding an attractiveness rate of 97%.The total number of eggs obtained was 15,844; 8846 eggs in the 10 ème arrondissement of Cotonou, and 6998 eggs in Godomey-Togoudo.

Insecticide Resistance Profiles
Resistance to permethrin and deltamethrin was evident in both populations of Ae. aegypti.Mosquito mortality was 85.7% and 82.7%, following exposure to the diagnostic dose of deltamethrin or permethrin after 30 min, respectively, in the 10 ème arrondissement of Cotonou (Figure 2).Similarly, in Godomey-Togoudo, mortality was 87.9% and 88.8% with deltamethrin and permethrin, respectively.By comparison, complete susceptibility to bendiocarb was observed in both vector populations (100% mortality) (Figure 2).
reading on a computer connected to the plate reader.Transformations from the various right-hand equations to determine the quantity of the final product were performed automatically by the software (GeneS.1)supplied with the spectrophotometer.Statistical analyses were also performed using GraphPad-Prism 5 software (version 5.00, San Diego, CA, USA).Mann-Whitney test was chosen for comparison between Rockefeller (susceptible strain), and field mosquitoes.Statistical significance was determined if p < 0.05.Statistical analyses were conducted in Stata/SE 17.0, including Pearson's Chi-squared test to investigate deviations from Hardy-Weinberg equilibrium.

Mosquito Sampling
A total of 147 gravid Aedes traps were used in the 2 study areas, of which 142 were positive (i.e., female Aedes oviposited in them); yielding an attractiveness rate of 97%.The total number of eggs obtained was 15,844; 8846 eggs in the 10 ème arrondissement of Cotonou, and 6998 eggs in Godomey-Togoudo.

Insecticide Resistance Profiles
Resistance to permethrin and deltamethrin was evident in both populations of Ae. aegypti.Mosquito mortality was 85.7% and 82.7%, following exposure to the diagnostic dose of deltamethrin or permethrin after 30 min, respectively, in Godomey-Togoudo (Figure 2).Similarly, in the 10 ème arrondissement of Cotonou, mortality was 88% with both deltamethrin and permethrin.By comparison, complete susceptibility to bendiocarb was observed in both vector populations (100% mortality) (Figure 2).

Expression of Detoxification Enzymes
Field insecticide-resistant Ae. aegypti populations had significantly higher median levels of GSTs compared to the insecticide-susceptible Rockefeller reference strain in Godomey-Togoudo (p = 0.0003) and in the 10 ème arrondissement of Cotonou (p = 0.0046) (Figure 3A).By comparison, field Ae. aegypti did not differ in their expression levels of MFOs, compared to the susceptible strain (Figure 3B) and had significantly lower median levels of non-specific esterases (α and β esterases) (Figure 3C,D).

Expression of Detoxification Enzymes
Field insecticide-resistant Ae. aegypti populations had significantly higher median levels of GSTs compared to the insecticide-susceptible Rockefeller reference strain in Godomey-Togoudo (p = 0.0003) and in the 10 ème arrondissement of Cotonou (p = 0.0046) (Figure 3A).By comparison, field Ae. aegypti did not differ in their expression levels of MFOs, compared to the susceptible strain (Figure 3B) and had significantly lower median levels of non-specific esterases (α and β esterases) (Figure 3C,D).

Discussion
To deploy appropriate control strategies targeting arbovirus vectors, it is crucial to understand the distribution of key mosquito species, their bionomics and insecticide resistance profiles.Study findings report Ae.aegypti in the 10 ème arrondissement of Cotonou and Godomey-Togoudo during the dry season, which might be explained by climate change, transport development, and increasing urbanization.Both populations of Ae. aegypti were characterized by pyrethroid resistance (to deltamethrin and permethrin), but complete susceptibility to bendiocarb.Pyrethroid resistance in these populations is not expected, given that insecticide treatment specifically targeting Ae. aegypti in Benin is rare; rather, it may have been driven by several alternate factors.It is possible that insecticidal interventions used to control other vector species, e.g., LLINs, IRS, or household mosquito repellents targeting Anopheles vectors of malaria, may have exerted indirect selection pressure on Ae. aegypti for the evolution of pyrethroid resistance.Furthermore, water contamination caused by pesticides used for agricultural practices may have also played a role in the development of resistance.
Regarding insecticide resistance mechanisms, Ae. aegypti populations were characterized by over-expression of GSTs and a slight, but non-significant, increase in the activity of MFOs.It is likely that over-expression of both types of metabolic enzymes could confer pyrethroid resistance in these populations.Further investigation is warranted of molecular mechanisms involving point mutations in the GSTe2 gene, which may be contributing to pyrethroid resistance in Ae. aegypti in Benin.By comparison, under expression of non-specific esterases (α and β esterases) observed in these same populations may explain the strong sensitivity to bendiocarb; therefore, this insecticide has the potential to be used in both areas to suppress Ae. aegypti population densities.
Several mutations at nine different loci in Ae. aegypti have been identified, which are implicated in reduced insecticide susceptibility.Of these, F1534C, S989P, and V1016I are widely reported kdr mutations and have been associated with DDT and pyrethroid resistance [13][14][15][16].In this study, AS-PCR genotyping revealed the presence of S989P, F1534C, and V1016G mutations in both populations of Ae. aegypti in Benin; 82.9% of genotyped mosquitoes carried at least one mutation.The frequencies of S989P in both study sites (63% and 78%) were much higher compared to that observed in Nigeria (7%) [16].Paradoxically the high mutation frequencies identified in this study were accompanied by only a moderate amount of phenotypic pyrethroid resistance.It is possible that the high fitness cost associated with these mutations may be adversely impacting mosquito survival [21].To our knowledge, this is the first report of V1016G in Africa, and this is the first time all three kdr mutations have been detected in Ae. aegypti in Benin.The presence of the V1016G gene mutation in West Africa represents a further expansion of the geographic range of this mutation and is an important finding.However, it would be necessary to confirm this mutation in positive samples using Sanger sequencing; this is a limitation of this study.
We identified 13 insecticide-resistant Ae. aegypti with the simultaneous presence of all three kdr mutations.The co-occurrence of two or three kdr mutations has been previously reported in China, Nigeria and Malaysia, which resulted in highly intense pyrethroid resistance [12,16,22,23].Other kdr mutations, T1520I and F1534L, have also been found in Ae. aegypti; however, the S989P/V1016G/F1534C mutation is the most widespread, followed by S989P/V1016V and V1016G/F1534C.A quadruple mutation, S989P/V1016G/T1520I/F1534C, was also identified in 2022 in Myanmar in Ae. aegypti [24].High prevalence of the F1534C mutation has been reported previously, but intense phenotypic resistance to pyrethroids is only found when accompanied by the V1016I mutation or V1016G, S989P, and/or V410L [24,25].Recent work has also highlighted specific profiles of metabolic gene overexpression associated with resistance to pyrethroids and organophosphates in Ae. aegypti popula-tions from two localities in Puerto Rico [26].In this study, the detection of multiple kdr mutations and overexpressed detoxification enzymes in the major dengue virus vector Ae.aegypti is concerning and cautions against the use of pyrethroids in arbovirus control programs in Benin.In this part of West Africa, additional surveillance activities are needed to further investigate other co-occurring molecular and metabolic insecticide resistance mechanisms, as well as to assess the susceptibility of these Ae.aegypti populations to alternate pyrethroids (e.g., alpha-cypermethrin, cyfluthrin, or lambda-cyhalothrin), with or without synergists (e.g., piperonyl butoxide), or other classes of insecticides (e.g., organophosphates, neonicotinoids, pyrroles etc.) to identify efficacious insecticidal control measures.In addition to these vector control tools, information education communication and behavior change communication (IEC/BCC) initiatives are needed to encourage local community members to properly dispose of stagnant water sources, which act as potential Aedes breeding sites, and to promote the use personal protective measures (including the use of topical repellents and wearing of long clothing) to prevent mosquito bites.In the event of a dengue fever epidemic, the Ministry of Health in Benin is cautioned against relying exclusively on pyrethroids for vector control; combination and/or rotation of insecticides with different active ingredients may be required to suppress populations of pyrethroid-resistant Ae. aegypti.

Conclusions
This study reports resistance to deltamethrin and permethrin in Ae. aegypti populations, collected from the 10 ème arrondissement of Cotonou and Godomey-Togoudo in Abomey-Calavi.Molecular and metabolic mechanisms associated with pyrethroid resistance included the kdr mutations F1534C, S989P, and V1016G, and significant overexpression of certain detoxification enzymes.To our knowledge, this is the first report of V1016G in Africa, and this is the first time all three kdr mutations have been detected in Ae. aegypti in Benin, suggesting alternative vector tools may be required for arbovirus control in this part of West Africa.Study results highlight the importance of strengthening and scaling-up surveillance activities to respond to the control of vector-borne diseases.

Figure 1 .
Figure 1.Map of trapping sites in Cotonou and Godomey-Togoudo, Benin.The dots correspond to the different places where the traps were set.

Figure 1 .
Figure 1.Map of trapping sites in Cotonou and Godomey-Togoudo, Benin.The dots correspond to different traps sites.

Figure 2 .
Figure 2. Susceptibility of Ae. aegypti to diagnostic doses of three insecticides in Godomey-Togoudo and the 10 ème arrondissement of Cotonou.

Figure 2 .
Figure 2. Susceptibility of Ae. aegypti to diagnostic doses of three insecticides in Godomey-Togoudo and the 10 ème arrondissement of Cotonou.