# Insecticide Resistance in the Disease VectorCulex pipiens in Morocco: Intensity, Mechanisms, and Contribution in Insecticide Resistance Management

Authors: Souhail Aboulfadl; Fouad Mellouki; Btissam Ameur; Chafika Faraj
Journal: Psyche: A Journal of Entomology
 (2022)

---

## Abstract

West Nile (WN) virus has been detected in Morocco in 1996, 2003, and 2010. One WN human case and 94 equine cases with 42 deaths were reported in the first outbreak. The second and the last epizootics were in Kénitra and Mohammedia regions, respectively.Culex pipiens mosquitoes were strongly suspected as WN vectors during these epizootics in Morocco. To help propose the first insecticide resistance management strategy in Morocco, we provide baseline data on the resistance status of C. pipiens mosquitoes to insecticides most used in public health in Morocco. We investigated the resistance intensity of C. pipiens and the role of monooxygenases in pyrethroids resistance using the synergist piperonyl butoxide (PBO) in three prefectures of central Morocco: Mohammedia, Benslimane, and Skhirate. Bioassays were carried out using WHO (World Health Organization) diagnostic test kits for adult mosquitoes, with DDT 4%; deltamethrin 0.05%, 0.25%, and 0.5%; bendiocarb 0.1%, 0.5%, and 1%; and malathion 5%. The synergist PBO 4% was used to check for the involvement of detoxification enzymes in pyrethroids resistant populations. Mosquito populations tested displayed high resistance intensity to pyrethroids and carbamates in Mohammedia and moderate resistance intensity in Skhirate. In Benslimane, the resistance intensity of C. pipiens was high to carbamate and moderate to pyrethroids. The pre-exposure to PBO restored full or partial susceptibility to pyrethroids in the different regions. According to our finding, Mohammedia and Benslimane are at risk level 3 of control failure, while Skhirate is at risk level 2 of it. The employment of IRM is therefore imperative in all study sites.


---

## Body

## 1. Introduction

Culex pipiens mosquitoes are the most widely distributed and abundant species, and their nuisance have serious negative impacts on the standard of living in many Moroccan urban communities. Moreover, this species is the principal vector of WNV (West Nile virus) in Morocco [1]. C. pipiens is highly suspected in the WN virus transmission during epizootics that had affected Morocco in 1996, 2003, and 2010 [1–3]. Until now, there are no highly effective vaccines for all mosquito-borne infections other than yellow fever and Japanese encephalitis [4]. For this reason, considerable efforts in mosquito control have been performed since the 1980s to decrease the nuisance generated by C. pipiens and prevent the WNV transmission in Morocco [5].Chemical insecticides usage was the main strategy in the vector control national program using organophosphates and pyrethroids against both larvae and adult mosquitoes, respectively. According to Moroccan public health services, mazout diesel, chlorpyriphos, malathion, and then temephos were used in larval control through time. On the other side, DDT, permethrin, cypermethrin, tetrametrin, and deltamethrin were used in adult control. The state of susceptibility to insecticides of this mosquito is the object of regular surveillance by the Ministry of Health since 2003, but the published data remain scarce [2]. Most of the published studies, mainly conducted on larvae, showed significant resistance to insecticides used in the majority of urban areas: Rabat, Sale, Skhirate, and Mohammedia to organophosphates (OP) [6]; Khemisset to organochlorines (OC) [7]; Fes to OP [8]; Smir to OP [9]; Casablanca, Tanger, and Marrakech to pyrethroids (PYR) [10]; and Mohammedia, Benslimane, Skhirate, and Sale to OP [5]. However, the continuous chemical treatment of resistant mosquitoes can make the use of insecticides ineffective compromising the strategy of mosquito control.In order to better manageC. pipiens resistance in Morocco, it is essential to study the resistance intensity and the mechanisms involved in this species. In other words, the operational impact of a given resistance might not be achieved yet what makes the resistance intensity (RI) investigation so important [11]. The objective of our study was first to assess the current susceptibility status of C. pipiens adult mosquitoes to DDT, deltamethrin, bendiocarb, and malathion and second to know the resistance intensity as well as the mechanisms involved in those populations from Mohammedia, Skhirate, and Benslimane in Northwestern Morocco.The results of our study can be used, as a guide to the choice of mosquito control and resistance management strategies. However, it is important to collaborate with public hygiene services to ensure the transfer of our basic research to field operations in order to assess and optimize the vector strategy proposed. To our knowledge, this is the first study that gives basic data on resistance intensity and metabolic mechanism and has practical implications for resistance management in adultC. pipiens from Morocco.


## 2. Materials and Methods

### 2.1. Study Area

This study was conducted in three urban districts: Ouled Hamimoun (33.673748, –7.445547), Lahjar (33.849538, –7.018181), and El-Jazeera (33.780420, –7.235220) from three coastal cities, respectively: Mohammedia, Skhirate, and Benslimane, Northwestern Morocco (Figure1). We chose Mohammedia for this study because it was the subject of a West Nile virus epizootic in 2010 transmitted by C. pipiens. Benslimane and Skhirate, as for them, are selected because they have recently experienced a very strong proliferation of C. pipiens due to the growth of urbanization in these regions. Ouled Hamimoun site from the Mohammedia region is an open drain very close to settlements and industrial companies. However, the Lahjar site from Skhirate and the El-Jazeera site from Benslimane are swamps too close to settlements and food companies, respectively. All sites are receiving a daily charge of waste that forms breeding sites very rich in organic matters and therefore suitable for C. pipiens proliferation. Although the three breeding sites are located in coastal cities, the geography of the regions affects the risk of a possible epidemic or epizootic. For example, the Mohammedia site exists in a semiurban wetland complex that represents a refuge for migratory birds, the main reservoir of the West Nile virus. The risk of a vector-borne disease in this region remains very high. On the other hand, Benslimane and Skhirate sites are inside an urban complex. The absence of migratory birds in these regions decreases the risk above, but it stills a nuisance for the inhabitants.Figure 1
Map of Mohammedia, Benslimane, and Skhirate cities showing the study sites.


### 2.2. Sampling

C. pipiens larvae and pupae were collected by dipping and netting methods using a ladle and a vacuum landing net with a mesh size of 0.1 mm and kept in the water sampled from the larval habitat at 27 ± 2°C until adult stages [12]. We morphologically identified the tested mosquitoes using the Culicidae of Mediterranean Africa Identification Software (CulAfMe) [13].


### 2.3. WHO Susceptibility Assays

We tested adult susceptibility using WHO discriminating dosages (DD) of four insecticides belonging to four chemical classes: organochlorine (DDT4%), pyrethroid (deltamethrin 0.05%), carbamate (bendiocarb 0.1%), and organophosphate (malathion 5%). We determined the resistance intensity by using 5x and 10x DD of deltamethrin (0.25% and 0.5%) and bendiocarb (0.5% and 1%). All bioassays were conducted following WHO protocols [11]. Four batches of 25 C. pipiens unfed females, aged 2–5 days, were exposed for 4 h to DDT insecticide-treated papers, 2 h to bendiocarb, and 1 h to deltamethrin and malathion. We recorded the knocked down number during the exposure time for DDT every 30 min and for deltamethrin every 10 min. After the exposure period, we transferred all mosquitoes to observation tubes with a 10% sugar solution supplement, and the mortality rate was recorded after 24 h. Batches exposed to oil-treated papers were run in parallel and used as control. Knockdown time (KdT50 and KdT90 indicating 50% and 90% of knocked down tested populations, respectively) were calculated using WinDL32 software [14].


### 2.4. Synergist-Insecticide Bioassay

In order to assess the involvement of detoxifying enzymes, mainly monooxygenases in producing resistant phenotypes to pyrethroids, the synergist-insecticide bioassay was carried out using PBO 4%. Unfed females, aged 2–5 days, were pre-exposed to 4% PBO-impregnated papers for 1 h before they were exposed to insecticide-impregnated papers (deltamethrin 0.05%). Control mosquitoes were exposed to oil-impregnated papers for 1 h. Mortality was recorded after 24 h. Three replicates were performed for each bioassay.


### 2.5. Data Analysis

The WHO criteria were adopted for distinguishing between resistance/susceptibility status and resistance intensity of the tested mosquito populations [11]. When more than 98% mortality at DD was observed, the population was considered “susceptible”; when less than 90% was observed, the population was considered “resistant.” Mortality of 98–100% at the 5x DD indicates low resistance intensity. When mortality <98% but ≥98% at the 10x DD, resistance intensity is moderate, and when mortality <98% at the 10x DD, resistance intensity is high. In regard to the population pre-treated by PBO 4%, when mean mortality was ≥98% in the “PBO followed by insecticide” samples, this implies that a monooxygenase-based resistance mechanism fully accounts for the expression of the resistant phenotype in the tested population. When mean mortality in the “PBO followed by insecticide” samples is superior to mean mortality in the “insecticide only” samples but <98%, the phenotype resistance observed is partially due to a monooxygenase-based resistance mechanism, and other resistance mechanisms are likely to be present in the tested population. When mean mortality in the “PBO followed by insecticide” samples is ≤= mean mortality in the “insecticide only” samples, the resistance phenotype detected is not based on monooxgenase-mediated detoxification.


## 2.1. Study Area

This study was conducted in three urban districts: Ouled Hamimoun (33.673748, –7.445547), Lahjar (33.849538, –7.018181), and El-Jazeera (33.780420, –7.235220) from three coastal cities, respectively: Mohammedia, Skhirate, and Benslimane, Northwestern Morocco (Figure1). We chose Mohammedia for this study because it was the subject of a West Nile virus epizootic in 2010 transmitted by C. pipiens. Benslimane and Skhirate, as for them, are selected because they have recently experienced a very strong proliferation of C. pipiens due to the growth of urbanization in these regions. Ouled Hamimoun site from the Mohammedia region is an open drain very close to settlements and industrial companies. However, the Lahjar site from Skhirate and the El-Jazeera site from Benslimane are swamps too close to settlements and food companies, respectively. All sites are receiving a daily charge of waste that forms breeding sites very rich in organic matters and therefore suitable for C. pipiens proliferation. Although the three breeding sites are located in coastal cities, the geography of the regions affects the risk of a possible epidemic or epizootic. For example, the Mohammedia site exists in a semiurban wetland complex that represents a refuge for migratory birds, the main reservoir of the West Nile virus. The risk of a vector-borne disease in this region remains very high. On the other hand, Benslimane and Skhirate sites are inside an urban complex. The absence of migratory birds in these regions decreases the risk above, but it stills a nuisance for the inhabitants.Figure 1
Map of Mohammedia, Benslimane, and Skhirate cities showing the study sites.


## 2.2. Sampling

C. pipiens larvae and pupae were collected by dipping and netting methods using a ladle and a vacuum landing net with a mesh size of 0.1 mm and kept in the water sampled from the larval habitat at 27 ± 2°C until adult stages [12]. We morphologically identified the tested mosquitoes using the Culicidae of Mediterranean Africa Identification Software (CulAfMe) [13].


## 2.3. WHO Susceptibility Assays

We tested adult susceptibility using WHO discriminating dosages (DD) of four insecticides belonging to four chemical classes: organochlorine (DDT4%), pyrethroid (deltamethrin 0.05%), carbamate (bendiocarb 0.1%), and organophosphate (malathion 5%). We determined the resistance intensity by using 5x and 10x DD of deltamethrin (0.25% and 0.5%) and bendiocarb (0.5% and 1%). All bioassays were conducted following WHO protocols [11]. Four batches of 25 C. pipiens unfed females, aged 2–5 days, were exposed for 4 h to DDT insecticide-treated papers, 2 h to bendiocarb, and 1 h to deltamethrin and malathion. We recorded the knocked down number during the exposure time for DDT every 30 min and for deltamethrin every 10 min. After the exposure period, we transferred all mosquitoes to observation tubes with a 10% sugar solution supplement, and the mortality rate was recorded after 24 h. Batches exposed to oil-treated papers were run in parallel and used as control. Knockdown time (KdT50 and KdT90 indicating 50% and 90% of knocked down tested populations, respectively) were calculated using WinDL32 software [14].


## 2.4. Synergist-Insecticide Bioassay

In order to assess the involvement of detoxifying enzymes, mainly monooxygenases in producing resistant phenotypes to pyrethroids, the synergist-insecticide bioassay was carried out using PBO 4%. Unfed females, aged 2–5 days, were pre-exposed to 4% PBO-impregnated papers for 1 h before they were exposed to insecticide-impregnated papers (deltamethrin 0.05%). Control mosquitoes were exposed to oil-impregnated papers for 1 h. Mortality was recorded after 24 h. Three replicates were performed for each bioassay.


## 2.5. Data Analysis

The WHO criteria were adopted for distinguishing between resistance/susceptibility status and resistance intensity of the tested mosquito populations [11]. When more than 98% mortality at DD was observed, the population was considered “susceptible”; when less than 90% was observed, the population was considered “resistant.” Mortality of 98–100% at the 5x DD indicates low resistance intensity. When mortality <98% but ≥98% at the 10x DD, resistance intensity is moderate, and when mortality <98% at the 10x DD, resistance intensity is high. In regard to the population pre-treated by PBO 4%, when mean mortality was ≥98% in the “PBO followed by insecticide” samples, this implies that a monooxygenase-based resistance mechanism fully accounts for the expression of the resistant phenotype in the tested population. When mean mortality in the “PBO followed by insecticide” samples is superior to mean mortality in the “insecticide only” samples but <98%, the phenotype resistance observed is partially due to a monooxygenase-based resistance mechanism, and other resistance mechanisms are likely to be present in the tested population. When mean mortality in the “PBO followed by insecticide” samples is ≤= mean mortality in the “insecticide only” samples, the resistance phenotype detected is not based on monooxgenase-mediated detoxification.


## 3. Results

Results showed the presence of resistance from the threeC. pipiens populations to the four chemical classes: organochlorates (DDT), pyrethroids (deltamethrin), carbamates (bendiocarb), and organophosphates (malathion; Table 1). The highest knockdown time (KdT) was observed in the population exposed to DDT from Benslimane with KdT50 = 1,771, and the lowest KdT was observed in the population exposed to deltamethrin from Mohammedia with KdT50 = 12. These values agree with the resistance state obtained in both populations. Mortality, as for it, is very high in the Skhirate population exposed to deltamethrin with %Mr = 85 and is very low in the Benslimane population exposed to DDT with %Mr = 2.Table 1
Susceptibility ofCulex pipiens to DDT, deltamethrin, bendiocarb, and malathion in Mohammedia, Benslimane, and Skhirate.
InsecticideDDT 4%Deltamethrin 0.05%Bendiocarb 0.1%Malathion 5%Study sitesNKdT50 [CI]KdT90 [CI]%Mr [SE]SNKdT50 [CI]KdT90 [CI]%Mr [SE]SN%Mr [SE]SN%Mr [SE]SMohammedia91166 [45–262]816 [424–1,700]74 [0.9]R10012 [0.9–19]30 [19–183]75 [0.5]R10074 [1.1]R8982 [0.9]RBenslimane1001,771 [1,200–1,886]6,978 [5,661–10,523]2 [0.2]R10050 [24–65]122 [80–211]44 [0.7]R9432 [0.5]R10370 [2]RSkhirate101112 [98–123]266 [238–311]84 [0.7]R9923 [21–25]49 [44–55]85 [0.7]R10172 [0.5]R9883 [1.2]RN: number of mosquitoes, KdT50: time of which 50% of the tested population suffers a knockdown, KdT90: time of which 90% of the tested population suffers a knockdown, %Mr: mortality percentage, S: status, and R: resistant.The mortality obtained of adultC. pipiens from Mohammedia, Benslimane, and Skhirate after being exposed to deltamethrin ×10 was 97%, 99%, and 99%, respectively, and to bendiocarb ×10 was 94%, 92%, and 98%, respectively. On the other side, KdT50 decreased in all three populations after being exposed first to deltamethrin ×5 and then to deltamethrin ×10. The resistance intensity of C. pipiens from Mohammedia was high to both deltamethrin and bendiocarb, whereas, in Benslimane, it was high to bendiocarb and moderate to deltamethrin. In Skhirate, it was moderate to both deltamethrin and bendiocarb (Table 2).Table 2
Resistance intensity of adultCulex pipiens to deltamethrin and bendiocarb in Mohammedia, Benslimane, and Skhirate.
InsecticideDeltamethrin 0.25%Deltamethrin 0.5%Bendiocarb 0.5%Bendiocarb 1%Study sitesNKdT50 [CI]KdT90 [CI]%Mr [SE]NKdT50 [CI]KdT90 [CI]%Mr [SE]RIN%Mr [SE]N%Mr [SE]RIMohammedia10013 [11–14]23 [21–26]93 [0.7]1035 [1–11]69 [42–138]97 [0.7]HR9496 [0.9]10294 [0.6]HRBenslimane9825 [23–27]44 [41–49]89 [0.9]10125 [4–169]86 [42–172]99 [0.3]MR9987 [1.3]10292 [1]HRSkhirate1009 [7–10]21 [19–24]84 [0.6]1016 [4–7.5]21 [10–42]99 [0.3]MR10296 [0.2]10798 [1.1]MRN: number of mosquitoes, KdT50: time of which 50% of the tested population suffers a knockdown, KdT90: time of which 90% of the tested population suffers a knockdown, %Mr: mortality percentage, RI: resistance intensity, HR: high resistance, and MR: moderate resistance.The pre-exposure to piperonyl butoxide (PBO) completely eliminated the phenotypic resistance to deltamethrin observed in adultC. pipiens from Mohammedia and Skhirate. The populations pre-exposed to the synergist showed mortality of 99 and 100, respectively. However, the resistance to deltamethrin in the population of Benslimane has been partially removed after a pre-exposure to PBO with a mortality of 87% (Table 3).Table 3
Mortality rates ofCulex pipiens with deltamethrin alone and deltamethrin + PBO in Mohammedia, Benslimane, and Skhirate, 24 hours after exposure.
InsecticidePBO onlyPBO + deltamethrin 0.05%Deltamethrin 0.05% onlySolvent controlStudy sitesNKdT50KdT90%MrNKdT50 [CI]KdT90 [CI]%Mr [SE]NKdT50 [CI]KdT90 [CI]%Mr [SE]NKdT50KdT90%MrMohammedia75——07518 [16–19]27 [25–29]99 [0.2]7623 [7.5–30]37 [28–104]83 [0.8]75——0Benslimane75——07111 [0.02–24]25 [1.1–39]87 [1.3]6919 [13–24]43 [33–68]59 [1.3]76——0Skhirate77——07811 [2–21]24 [3–34]100 [0]7718 [6–26]46 [34–55]88 [0.7]75——0N: number of mosquitoes, KdT50: time of which 50% of the tested population suffers a knockdown, KdT90: time of which 90% of the tested population suffers a knockdown, and %Mr: mortality percentage.


## 4. Discussion

In our work, we give basic data on the resistance status ofC. pipiens adult populations from three regions in central Morocco. Our results showed that culicidian populations of adult C. pipiens from Mohammedia, Benslimane, and Skhirate have a resistant phenotype towards DDT 4% (OC), deltamethrin 0.05% (PYR), bendiocarb 0.1% (CX), and malathion 5% (OP) with dissimilar intensities. The resistance intensity was high in Mohammedia and moderate in Skhirate to both deltamethrin and bendiocarb, while, for Benslimane, it was moderate to deltamethrin and high to bendiocarb. The current insecticides used by public health services in all study sites are temephos and deltamethrin to control mosquito larvae and adults, respectively. However, the insecticide quantities and the treatment frequencies made in vector control might affect the resistance as well as its intensity. The average quantity of an organophosphate, for example, “AbateR” (temephos), over the past five years in prefectures of Mohammedia and Skhirate was 120 L on 100 ha of mosquito breeding sites and 47 L on 13 ha, respectively [5]. Despite the rate of 3.6 of temephos quantities used in Skhirate being 2.4 higher than the rate of 1.2 of temephos quantities used in Mohammedia, the resistance intensity of adult C. pipiens to CX and PYR in Mohammedia is still higher than Skhirate. This could be explained by the treatment frequency of each mosquito habitat. The Ouled Hamimoun (Mohammedia) breeding site is a permanent open drain located close to human habitats and is therefore regularly treated [5], contrary to the Lahjar (Skhirate) breeding site that is a temporary swamp and only treated depending on the presence of the water. El-Jazeera (Benslimane) breeding site is a permanent swamp located outside the city and is regularly treated by only temephos-based products. Larval and adult control is maintained in both Ouled Hamimoun and Lahjar breeding sites, whereas, at El-Jazeera, only larvae are treated. The different intensities of adult resistance to the three neurotoxic insecticides indicate that the chemicals present in the environment could not only induce direct adult resistance but also interact with other molecules from the environment and thus develop cross-resistance processes [15]. Our results agree with those obtained by Sinegre et al. [16] who have established an observable correlation between the level of resistance and the frequency of treatments.Moreover, we demonstrated that the pyrethroids resistance within mosquitoes from Mohammedia and Skhirate is completely metabolic and contributed to full involvement of P450 monooxygenases in the resistant populations. However, the pre-exposure to PBO inC. pipiens from Benslimane revealed that the pyrethroids resistance is partly metabolic and caused by limited involvement of P450 monooxygenases. The action of other resistance mechanisms in this population could also be in play.Our finding investigates for the first time the resistance intensity and the mechanism involved in the disease vectorC. pipiens from Mohammedia, Benslimane, and Skhirate. According to our results, we prove the necessity of the implementation of insecticide resistance management (IRM) in all study sites in order to maintain the effectiveness of chemical insecticides used in vector control in these regions. In other words, Mohammedia and Benslimane regions are at level 3 of control failure, and IRM should be implemented immediately with unrelated insecticides in these areas following the flow chart to support the decision-making of IRM strategy by Dusfour et al. [17]. Contrariwise, the Skhirate region is at level 2 of control failure, and IRM should be implemented without the need for unrelated insecticides at this level. Environment management, biological control, use of biocides, insecticide rotation, mosaic, and mixture or combination are all strategies for IRM. However, the use of these strategies must be appropriate to the Culicidean population resistance mechanism. Rotating insecticides with different modes of action, for example, will be unnecessary and will not reduce the resistant mosquitoes’ adaptive value in a case of metabolic cross-resistance [17]. The susceptibility state and resistance intensity of the same mosquito species could vary from one breeding site to another within the same prefecture and from one city to another within the same country. In this context, it is important for implementing an effective IRM to have precise, detailed, and deep knowledge of the Culicidean populations (susceptibility state, resistance intensity, mechanisms involved, etc.), their habitats (geographical data of the breeding sites, sites type, areas type, etc.), and their vector control strategies. According to our results, the best IRM strategy that could be applied in Mohammedia and Skhirate is the use of a combination of pyrethroids and the synergist PBO in adult control because of their resistance mechanism that is completely metabolic due to cytochromes P450 monooxygenases, whereas, in Benslimane, the combination of PYR and PBO synergist could be a good strategy but not sufficient for IRM. This is due to the limited involvement of P450 monooxygenases in metabolic resistance. For this reason, other complementary strategies must be implemented in this region as rotating insecticides with different target sites, biological control, or environment management.Definitely, the biosphere, which largely overlaps with what is now called the “environment,” is profoundly transformed (growing demography and/or population densification, the evolution of farming and breeding practices, draining of wetlands, hydro-agricultural developments, deforestation, reforestation, and climate change) making it very difficult to define constants [18]. These environmental factors have a crucial impact on the vector system and possibly on mosquito resistance, so our understanding of the interaction environment x mosquitoes could optimize IRM. The urbanization degree, for example, has a significant impact on mosquito resistance. A study showed that the occurrence of insecticide resistance is strongly related to increasing human urbanization [19]. Indeed, the resistant populations are highly resistant in most urbanized areas and low resistant in natural areas [20]. Moreover, Grossman et al. [21] highlighted the importance of gene × environment interaction in re-establishing mosquito susceptibility. They found that the Aedes aegypti resistant population was no longer phenotypically resistant at high density (93% were knocked down when exposed to permethrin with a decrease in C1534 kdr allele frequency from 0.98 ± 0.04 to 0.69 ± 0.04 in only one generation of selection). According to the authors, this gain of susceptibility is due to the selective force induced by density-dependent intraspecific competition. This environment influence leads us to take into account the local dimension, which is that of populations and not of species [18].Furthermore, mosquito resistance is mainly due to two mechanisms: metabolic resistance and target site modification. In metabolic resistance, several classes of enzymes are involved in the detoxification of chemical insecticides, the major ones being cytochrome P450 oxidases (monooxygenases), esterases, and glutathione-S-transferases. Cyt P450 oxidases play the main role in resistance to pyrethroids, while esterases are largely responsible for resistance to organophosphates and carbamates [22]. Monooxygenases are also responsible for activating organophosphorus molecules, that is, the OP that are able to quickly penetrate the insect’s cuticle, are applied in the form of thionates, and monooxygenases convert the thionate analogues to highly toxic oxon analogues inside the mosquito [23]. Metabolic resistance to pyrethroids is due to the overexpression of one or more P450s or also to mutations that transform the P450 protein [24]. However, there are more than 100 P450s in mosquitoes’ genomes, the thing that makes a determination of the specific P450(s) liable for the resistance a real challenge [25]. Molecular tools can help choose the best alternative insecticide, by determining which resistant alleles are responsible for certain cross-resistance mechanisms [26]. However, other resistance mechanisms such as metabolic resistance have not been sufficiently studied, and therefore, alleles that induce this type of mechanisms are rarely explored. Diagnostic tests for such resistance mechanisms are currently failing, which is mainly due to the lack of validated DNA markers [17].Our finding shows that metabolic resistance is involved completely in the resistant population from Mohammedia and Skhirate and partly in the resistant population from Benslimane. Although these results help in the decision-making of the insecticide resistance management, complementary studies are needed to know which cytochrome P450 genes are responsible for the observed resistance. Thus, understanding what kind of insecticide and the precise P450 behind its metabolism is important to delineate the potential strengths and liabilities of insecticide compounds [27].


## 5. Conclusion

The results of this study showed the presence of resistance with variable intensities in the threeC. pipiens populations to chemical insecticides used for mosquito control in three coastal cities: Mohammedia, Benslimane, and Skhirate. According to our finding, the implementation of insecticide resistance management in all study sites is imperative in order to avoid a critical resistance state in C. pipiens populations. In Mohammedia and Skhirate where resistance is proved completely metabolic, the IRM strategies such as insecticide rotation, mosaic, and mixture or combination will be unnecessary and will not reduce the proportion of resistant mosquitoes against the tested insecticides. However, the possible strategies that could be implemented in these areas are biological control, physical treatment, or combining PBO with the insecticide, whereas, in Benslimane, the resistance mechanism is partly metabolic and needs further studies to know which IRM strategy is ideal to implement. Insecticide resistance aspects in mosquitoes could be affected by several factors, but still decreasing insecticide pressure is the best strategy to apply in IRM. Thus, it is important to collaborate with public hygiene services to ensure the transfer of our basic research to field operations in order to assess and optimize the vector strategy proposed.



---

*Source: 5576317-2022-04-28.xml*