https://orcid.org/0009-0005-1469-0481
Figures
Abstract
Although malaria is endemic in coastal Côte d’Ivoire, updated data on the resistance profile of the main vector, Anopheles gambiae sensu lato (s.l.), are still lacking, thus compromising decision-making for an effective vector control intervention. This study investigated the complex members and the insecticide resistance in the Anopheles gambiae s.l. populations in coastal Côte d’Ivoire. Between 2018 and 2020, cross sectional survey bioassays were conducted on female An. gambiae s.l. mosquitoes in three coastal health districts (Aboisso, Jacqueville and San Pedro) of Côte d’Ivoire. Pyrethroids deltamethrin, permethrin and alphacypermethrin (1X, 5X and 10X), clothianidin and synergist piperonyl butoxide (PBO) combined with pyrethroid 1X were tested using WHO tube bioassays. Chlorfenapyr was evaluated using CDC bottle bioassays. An. gambiae complex members and kdr 995F, kdr 995S and Ace-1 280S mutations were identified using polymerase chain reaction (PCR) technique. Overall, An. gambiae s.l. populations were primarily composed of Anopheles coluzzii (88.24%, n = 312), followed by Anopheles gambiae sensu stricto (7.56%) and hybrids (4.17%). These populations displayed strong resistance to pyrethroids at standard diagnostic doses, with mortality remaining below 98% even at 10X doses, except for alphacypermethrin in Aboisso. Pre-exposure to PBO significantly increased mortality but did not induce susceptibility, except for alphacypermethrin in Jacqueville. Clothianidin induced full susceptibility in Jacqueville and San Pedro, while chlorfenapyr induced susceptibility in Aboisso at 100 μg ai/bottle and all three districts at 200 μg ai/bottle. kdr 995F mutation dominated, with frequencies varying from 71.2% to 79.3%. kdr 995S had low, rates with frequencies ranging from 2.3% to 5.7%. Ace-1 280S prevalence varied between 4.2% and 42.9%. Coastal Côte d’Ivoire’s An. gambiae s.l. populations were mainly composed of An. coluzzii and showed high resistance to pyrethroids. Clothianidin, chlorfenapyr, and PBO with pyrethroids increased mortality, indicating their potential use as an alternative for malaria vector control.
Citation: Kouamé JKI, Edi CVA, Zahouli JBZ, Kouamé RMA, Kacou YAK, Yokoly FN, et al. (2024) Assessing species composition and insecticide resistance of Anopheles gambiae complex members in three coastal health districts of Côte d’Ivoire. PLoS ONE 19(12): e0297604. https://doi.org/10.1371/journal.pone.0297604
Editor: Luca Nelli, University of Glasgow College of Medical Veterinary and Life Sciences, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND
Received: January 8, 2024; Accepted: September 27, 2024; Published: December 10, 2024
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors of this article formally declare the complete absence of any conflict of interest among them. Their commitment to integrity and transparency ensures an impartial contribution to the presented research. We declare that there is no conflict of interest regarding the publication of this paper. We certify that we have no financial or personal relationships with individuals or organizations that could inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the content of this paper.
Introduction
The World Health Organization (WHO) has recently reported that there were 249 million cases and 608,000 deaths from malaria in 2022 [1]. Malaria is most prevalent in sub-Saharan Africa, where the majority of cases and deaths occur. Within sub-Saharan Africa, countries such as Nigeria, the Democratic Republic of the Congo, and Mozambique have the highest malaria burdens. Malaria is one of the deadliest public health diseases among children less than 5 years old, particularly in sub-Saharan Africa. The WHO African region accounted for 95% of global malaria cases and 96% of deaths [2]. More than US$ 3.5 billion have been invested in 2021 for malaria, with a third of this investment (around US$ 1.1 billion) spent by the governments of malaria-endemic countries [2]. The WHO recommends malaria prevention strategies, including effective vector control tools that have major impacts in reducing the global burden of this disease. The WHO Global Technical Strategy (GTS) aims to reduce malaria incidence and mortality by at least 75% by 2025 and 90% by 2030, which seems to be challenging [2]. In 2021, 242 million Artemisinin-based Combination Therapies (ACTs) were distributed to the public health sector by National Malaria Control Programs (NMCPs) and about 590 million Insecticide Treated Nets (ITNs) were delivered to communities between2019 and2021 [2]. To achieve these goals, GTS has called for the development of new vector control tools that must incorporate new insecticide molecules (e.g., clothianidin and chlorfenapyr), synergists (e.g., pyperonyl butoxide: PBO) and/or insecticide mixtures containing at least two active ingredients with different modes of action to mitigate malaria vector resistance to insecticides [2–4]. Côte d’Ivoire’s population lives in high malaria risk areas [5]. In 2021, malaria in the country was estimated at 7,443,146 cases with 14,906 deaths [2]. The main objectives of the National Control Malaria Program (NMCP) are to reduce malaria morbidity by 40% and the malaria mortality in high-burden by 33% by 2026 compared to the 2015 baseline [6]. The vector control strategy implemented by the NMCP is mainly based on the mass deployment of long-lasting insecticidal nets (LLINs) every three years and recently by implementing indoor residual sprays (IRS) [7–9] in one district. In Côte d’Ivoire, the National Malaria Strategic Plan 2016–2020 has prioritized indoor residual spraying (IRS) as an additional vector control method to reduce malaria morbidity and mortality [10]. The transmission of malaria in Côte d’Ivoire is via An. gambiae s.l., An. funestus s.l. and An. nili [7, 11]. An. gambiae s.l., or the An. gambiae complex, comprises nine species, including An. gambiae sensu stricto (s.s.), An. coluzzii, An. arabiensis, An. melas, An. merus, An. bwambae, An. quadriannulatus, An. amharicus, and An. fontenillei, which cannot be morphologically distinguished [12]. The species An. gambiae s.s., An. coluzzii, and the hybrids resulting from their interbreeding, all part of the An. gambiae complex, have been identified throughout the country [7, 13–16]. However, Ivorian An. gambiae s.l., populations exhibit strong resistance to many traditional classes of insecticides (i.e., DDT, pyrethroid, carbamate, and organophosphate) [7, 14–18]. Local An. gambiae s.l. resistance to insecticides is a severe challenge to the NMCP efforts because all LLINs distributed in Côte d’Ivoire before 2021 were treated with pyrethroid only [7, 10]. Therefore, it is urgent to develop effective alternative strategies for the sustainable control of insecticide-resistant malaria vectors. Since 2018, the National Malaria Strategic Plan (NMSP) supported by the U.S. President’s Malaria Initiative (PMI) project has contributed to the generation of insecticide resistance data to conduct a stratification of vector control interventions across the country. From 2018 to 2022, the NMCP and PMI deployed IRS in the health districts of Nassian and Sakassou using clothianidin-based insecticides that resulted in effective malaria vector control [6, 7]. Insecticide sensitivity tests have also been conducted so that these data can facilitate the development of mosquito nets incorporating pyrethroids and other active ingredients (synergists, pyrroles, etc.), as well as combinations of active ingredients, for the upcoming LLIN distribution campaign, based on entomological stratification data [6, 7]. Recently, pyrethroids in combination with either an insect growth regulator, a pyrrole, or a synergist that inhibits the primary metabolic mechanism of pyrethroid resistance within mosquitoes were used in LLINs [3, 4, 7]. In the current study, insecticide resistance status and complex members of An. gambiae s.l. were assessed in three coastal health districts of Côte d’Ivoire, namely Aboisso, Jacqueville and San Pedro. Although a low entomological inoculation rate (10.9 ib/p/y in Bardo) is recorded in the coastal zone of Côte d’Ivoire [11], this area has reported a high incidence of malaria. The health district of Jacqueville records one of the highest malaria incidences, three hundred sixty-seven per thousand (367‰), while a lower incidence is reported in the health district of San Pedro (127.1‰) [19, 20]. In Côte d’Ivoire, the coastal areas are characterized by a massive concentration of people [21]. These areas are the heart of the economic development of the country and have two main seaports, one in Abidjan and one in San-Pedro. This coastal region is home to the largest number of agro-industrial units, traditional agriculture and smallholdings [11, 15, 22]. The industrial and traditional crops (e.g., cocoa, rubber, oil palm, coffee, rice) [11, 13, 15, 16, 22] are intensively treated with pesticides and insecticides to protect crops [15]. The local Anopheles malaria vectors are constantly under strong pressure from insecticides and chemicals, which can lead to the development of their resistance and increased their survival. Thus there is an urgent need is to develop alternative insecticides and evaluate their effectiveness against malaria vectors in resistant areas, mainly on the coastline. The current study is part of a program designed to explore the landscape of malaria Anopheles vectors in coastal areas to inform disease control programs in Côte d’Ivoire.
Methodology
Study sites
The study was conducted in three health districts, Aboisso (latitude 5° 28’ 04” N and longitude 3° 12’ 25” W), Jacqueville (latitude 5° 12’ 02” N and longitude 4° 24’ 44” W) and San Pedro (latitude 4° 44’ 5” N and longitude 6° 38’ 10” W), located in the southern littoral area of Côte d’Ivoire (Fig 1). The local climate is characterized by two major seasons: the long rainy season from April to July, with the peak rainfall in June, and the long dry season from October to March. The annual rainfall average is 1848 mm with an annual temperature average of 27°C. In the three health districts, economic activities are dominated by agriculture with large agricultural areas for food crops and cash crops (rubber, oil palm, pineapple, cassava, yam, banana) [21, 23, 24]. Farmers use several types of pesticides (herbicides, insecticides, and fungicides) to protect crops and increase production [15, 22, 25].
The map was generated using QGIS software version 3.32.3-Lima (https://www.qgis.org/), with the basemap shapefile sourced from the Database of Global Administrative Areas (GADM, https://gadm.org/; license: https://gadm.org/license.html).
Larval sampling and rearing to adults
Surveys for potential mosquito larvae habitats were conducted in different locations such as water puddles, vegetable cultivation sites, rice fields, and other potential larval habitats within our study sites. Anopheles mosquito larvae and pupae were collected using the dipping method [26] and combined. All collection site within each health district were combined. The larvae and pupae were collected from larval habitats between January 2018 and December 2020. Larvae were transferred to distilled water and reared into adults using the mosquito mass-rearing method [27] under standard laboratory conditions (27 ± 2∘C temperature; 70 ± 10% relative humidity; 12:12 hour light: dark photoperiod). Emerged adults were provided with a cotton wool pad soaked in a 10% sugar solution. Species (An. gambiae s.l.) were morphologically identified using identification keys [28] before being utilized for various sensitivity tests and moleculars analyses.
WHO tube bioassay
The diagnostic dose tests of pyrethroids (alphacypermethrin, deltamethrin, and permethrin) were conducted in 2018, 2019, and 2020. The following tests, including intensity tests (1X, 5X, and 10X), synergist tests (PBO), and clothianidin tests, were exclusively conducted in 2020. Emerged adult females of An. gambiae s.l. were tested for insecticide susceptibility according to the WHO standard procedures [29]. Females of F0 generations aged 2–5 days were used in all tests. Four batches of 20–25 non-blood-fed females were introduced each in four tube tests garnished with insecticide-treated filter papers, while two batches were exposed to control tubes with untreated filter papers for one hour. Mosquitoes were exposed to the WHO discriminating dosages of deltamethrin (0.05%), permethrin (0.75%) and alpha-cypermethrin (0.05%) to determine their resistance status (WHO, 2018). Moreover, synergist effects were assessed by pre-exposing mosquitoes to 4% PBO for one hour before being exposed to deltamethrin 0.05%, permethrin 0.75% and alphacypermethrin 0.05% for an additional hour. To determine the pyrethroid-resistance intensity, mosquitoes were exposed to 1X, 5X and 10X diagnostic concentrations of alpha-cypermethrin (0.05%, 0.25% and 0.5%), permethrin (0.75%, 3.75% and 7.5%) and deltamethrin (0.05%, 0.25% and 0.5%). Mosquito mortality was recorded at 24 hours after exposure.
For clothianidin, impregnated papers were prepared in situ following the standard operating procedure (SOP) developed by the Vector Link entomology team. The diagnostic dose was set at 2% clothianidin. A solution of clothianidin was prepared by diluting 264 mg of the formulated product (Sumishield WG50) in 20 ml of distilled water. Four Whatman No.1 filter papers of 12 × 15 cm each were impregnated using a pipette to dispense 2 ml of solution on each filter paper, resulting in a concentration of 13.2 mg/ai clothianidin per paper. The untreated filter papers were treated using 2 ml of a solution of distilled water. Clothianidin was tested using WHO susceptibility tests, with the standard guidelines [30]. Mosquitoes were tested against clothianidin-treated papers as described above [31]. The mortality was recorded daily for up to seven days in order to capture any delayed mortality effects.
CDC bottle test
The Centers of Disease Control and Prevention (CDC) bottle tests were exclusively conducted in 2020. Chlorfenapyr susceptibility was determined using CDC bottle tests. The CDC bottle tests utilized 250 ml glass bottles coated with 100 μg ai/bottle and 200 μg ai/bottle. A set of 15 to 20 non-blood-fed female An. gambiae s.l. aged 2–5 days were exposed to two discriminating concentrations of chlorfenapyr following the CDC bottle test protocol [32]. Mortality was recorded daily for up to three days.
Molecular analyses of An. gambiae s.l.complex members and resistance genes
The An. gambiae complex members and kdr and Ace-1 mutations are stored individually in Eppendorf tubes with silica gel, and maintained at −20°C for subsequent identification.
DNA extraction
The genomic DNA of individual mosquito was extracted according to the method described in Collins et al. [33]. Each mosquito was individually processed in a 1.5 ml Eppendorf tube, where it was soaked and crushed in 200 μl of 2% Cetyl Trimethyl Ammonium Bromide (CTAB), followed by incubation at 65°C for 5 minutes. Afterwards, 200 μl of chloroform was added, and the resultant mixture was centrifuged at 12,000 rounds per minute (rpm) for 5 minutes. The supernatant was carefully transferred to a new 1.5 ml tube. Subsequently, 200 μl of isopropanol was added to the supernatant, thoroughly mixed by pipetting, and then centrifuged at 12,000 rpm for 15 minutes to precipitate the DNA. The DNA pellet formed at the bottom of the tubes and the supernatant was discarded. The DNA of each sample was purified with 200 μl of 70% Ethanol and centrifuged at 12,000 rpm for 5 minutes. After removing the ethanol, the pellet was air-dried overnight on the bench. Finally, the extracted DNA was reconstituted in 30 μl of DNase-free water (Sigma-Aldrich, United Kingdom), incubated at 65°C for 15 minutes, and stored in the fridge at -20°C.
Complex member identification
The molecular identification of An. gambiae complex members was performed according to the SINE-PCR method [34], with minor modifications of reaction mixture. The PCR assay was performed using two primers, 6.1a (5’-TCGCCTTAGACCTTGCGTTA-3’) and 6.1b (5’-CGCTTCAAGAATTCGAGATAC-3’). Each reaction mixture was done in a final volume of 25 μl containing 12.5 μl One taq Quick-load 2X Master Mix, 5.5 μl free water, 0.5 μl Dimethyl sulphoxide (DMSO), 2 μl Bovine Serum Albumin (BSA), 1 μl of each primer, 0.5 μl Magnesium chloride (MgCl2) and 2 μl DNA template. The incubation took place in a thermocycler of LongGene® type (A200 Gradient Thermal cycler; LongGene Scientific Instruments Co., Ltd Hangzhou, P.R. China) according to the following program: 94 ∘C for 5 min, 94 ∘C for 25 sec, and 54 ∘C for 30 sec; 72 ∘C for 1 min repeated 35 times; and a final step at 72 ∘C for 10 min to terminate the reaction. After amplification, PCR products were run on either a 1.5% agarose gel in Tris/borate/EDTA (TBE) and stained with ethidium bromide solution for UV visualization. PCR products were loaded on the gel and allowed to migrate under a voltage of 140 V for an hour. The result was visualized with a UV illuminator (BioDoc- It Imaging System; Upland, CA, USA).
Identification of target site mutation
The presence of insecticide resistance genes including kdr-L995F (previously known as kdr-L1014F (kdr-West)), kdr-L995S (previously known as kdr-L1014S (kdr-East)), and Ace-1-G280S (previously known as Ace-1-G119S) [35] was investigated using real-time PCR. The primers Kdr-Forward (5’-CATTTTTCTTGGCCACTGTAGTGAT-3’) and Kdr-Reverse (5’-CGATCTTGGTCCATGTTAATTTGCA-3’) were standard oligonucleotides without any modifications. The probe WT (5’-CTTACGACTAAATTTC-3’), labeled with HEX at the 5’ end, was used for detecting the wildtype allele. The probes kdr W (5’-ACGACAAAATTTC-3’) and kdr E (5’-ACGACTGAATTTC-3’) were labeled with FAM for detecting the kdr-W and kdr-E alleles, respectively. Similarly, the primers Ace-1-Forward (5’-GGCCGTCATGCTGTGGAT-3’) and Ace-1-Reverse (5’-GCGGTGCCGGAGTAGA-3’) were also standard oligonucleotides. The probe WT (5’-TTCGGCGGCGGCT-3’) was labeled with HEX, while the probe (5’-TTCGGCGGCAGCT-3’) was labeled with FAM. The TaqMan assays as detailed by Bass [36] were used to screen for the 995F, 995S kdr and 280S mutations. The reaction was carried out in an Agilent Stratagene MX3005 qPCR thermocycler (Agilent Technologies, Santa Clara, CA, USA). Each 1 μl of gene DNA was combined with a total volume of 9 μl of master mix, comprising 3.875 μl of DNase-free water, 5 μl of SensiMix, and 0.125 μl of specific primer/probe for either kdr 995F, 995S, or Ace-1-280S. KdrAce-1 The specific probe contains FAM and HEX fluorochromes. FAM was used to detect the mutant allele, while HEX detected the wild-type susceptible allele. PCR conditions used were 10 min at 95 ∘C (1 cycle), followed by 40 cycles of 10 sec at 95 ∘C, and 45 sec at 60 ∘C.
Data analysis
All data were entered in Excel. R software version 4.2.1 (2022-06-23 ucrt) was used for the various statistical analyses. The resistance status of each mosquito population was determined according to the WHO criteria with mortality after 24-hour, 72-hour and 7-day post-exposure for pyrethroids, chlorfenapyr and clothianidin, respectively [37]. Mortality was corrected using Abbott’s formula (), when the mortality of the control tubes was above 5% and less than 20% [38]. There was a confirmed resistance if the mortality percentage was <90%, possible resistance if the mortality rate was between 90 and 98%, and susceptibility if the mortality rate was ≥98%. The package ‘lsmeans’ version 2.30–0 was used for various analyses. The mortality recorded each year was compared between each insecticide (pyrethroid diagnostic concentration) using the pairwise tests of the generalized linear model (GLM). For the resistance intensity, if corrected mortality was 98–100% at 5X the diagnostic dose indicated low resistance intensity. However, if mortality was less than 98% at 5X diagnostic dose implied testing the 10X diagnostic dose.At 10 times the diagnostic dose, a corrected mortality rate of 98–100% confirms a moderate resistance intensity.; A corrected mortality rate of less than 98% at 10 X the diagnostic dose indicates a high level of resistance. For the synergist assays, the mortality of mosquitoes exposed to PBO with pyrethroid was compared with that of the insecticides without PBO pre-exposure. Comparison was made between mortality rates with and without PBO pre-exposure using the prop.test with software R version 4.2.1. The frequency of resistance mutations (kdr-995F, kdr-995S and Ace-1 280S) was determined using the formula: F = [(2AA+ Aa)] / [2(AA+Aa+aa)] [39] with aa, homozygous susceptible genotype; Aa, heterozygous genotype; AA, homozygous resistant genotype.
Results
Species composition of the Anopheles gambiae complex
A total of 672 An. gambiae s.l. were selected from the three sites for species identification. Overall, An. gambiae complex was mainly composed of An. coluzzii (88.2%, n = 593), followed by An. gambiae s.s. (7.6%, n = 51) and hybrids (An. coluzzii/An. gambiae s.s.) (4.2%, n = 27). In Jacqueville and San Pedro, all mosquitoes were identified as An. coluzzii. The two species were found in sympatry in Aboisso. However, An. coluzzii dominated in Aboisso (66.7%, n = 158), followed by An. gambiae s.s. (21.5%, n = 51) and hybrids (11.8%, n = 28) (Table 1).
Insecticide susceptibility in An. gambiae s.l.
Table 2 presents the mortality rates of An. gambiae s.l. populations from the surveyed sites (Aboisso, Jacqueville, and San Pedro) when exposed to pyrethroids (alphacypermethrin 0.05%, permethrin 0.75%, and deltamethrin 0.05%) over the years 2018, 2019, and 2020. Across all sites, the mortality rates for all three pyrethroids remained below 30%, confirming significant resistance of An. gambiae s.l. to these insecticides.
Table 3 summarizes the comparison of insecticide-induced mortality rates by site and year. In 2020, a statistically significant difference was observed in Aboisso between mortality rates due to deltamethrin and alpha-cypermethrin (estimate: 2.45; Z-ratio: 3.25; p-value = 0.0033). Similarly, in 2020, permethrin also showed a statistically significant difference compared to alpha-cypermethrin in Aboisso (estimate: 3.42; Z-ratio: 3.32; p-value = 0.0026). In Jacqueville, the 2020 comparison between permethrin and alpha-cypermethrin revealed a statistically significant difference (estimate: 2.08; Z ratio: 2.73; p-value = 0.0174).
Intensity of resistance
The results of the pyrethroid intensity test are summarized in Fig 2. These results confirmed the strong resistance of An. gambiae s.l. against alpha-cypermethrin, permethrin and deltamethrin. In the resistance pyrethroid intensity tests, conducted on samples from Aboisso, Jacqueville, and San Pedro, mortalities remained below the 98% threshold when exposed to 1X and 5X concentrations of all insecticides(Fig 2). However, at a concentration of 10X, a high intensity of resistance was still observed across all three mosquito populations for the insecticides, except for a case of moderate intensity resistance recorded with alphacypermethrin 0.5% in the Aboisso populations, resulting in 100% mortality.
The red dashed line represents the susceptibility threshold.
Effect of piperonyl butoxide (PBO)
Table 4 shows the mortality rates in the Aboisso, Jacqueville and San Pedro An. gambiae s.l. populations exposed to alpha-cypermethrin, permethrin and deltamethrin without and with pre-exposure to PBO. The results showed that synergist effect of PBO was strongest in all mosquito populations. Mortality increased significantly when mosquitoes were pre-exposed to PBO (df = 1, p <0.0001) (Table 1). Although a significant increase was observed in mortality after pre-exposure to PBO, susceptibility was not fully restored. The mortality rates after PBO pre-exposure were still under the 98% threshold in all three study sites, except for the Jacqueville populations in which alphacypermethrin mortality after pre-exposure to PBO was 98.5% (Table 4).
Effect of clothianidin and chlorfenapyr
After seven days of observation, An. gambiae s.l. populations from Jacqueville and San Pedro were found to be completely susceptible to clothianidin (Fig 3). The San Pedro population achieved a 100% mortality rate by the sixth day (Fig 3). In Jacqueville, the sensitivity threshold was reached on the seventh day, with a mortality rate of 98.8%. However, in Aboisso, susceptibility was not fully restored after seven days of post-exposure observation, with the mortality rate only reaching 55.7%.
The red dashed line represents the susceptibility treshold.
For chlorfenapyr at 100 μg/bottle, the Aboisso population was fully susceptible, with a 72-hour mortality of 100%. However, chlorfenapyr did not fully induce susceptibility in Jacqueville and San Pedro populations with 100 μg/bottle. With 100 μg/bottle, the 72-hour mortality was 88.7% for Jacqueville and 97.1% for San Pedro populations (Fig 4). With 200 μg/bottle, the mortality rate was 100% for the San Pedro population after 24-hours post-exposure. Except for the Jacqueville population where 100% mortality was recorded after 48-hours post-exposure (Fig 4). The population of Aboisso was not tested at 200 μg/bottle, because susceptibility was achieved at 100 μg/bottle.
The red dashed line represents the susceptibility treshold.
Resistance mutation
Table 5 shows the kdr (L995F and L995S) mutation allelic frequencies in the population from Aboisso, Jacqueville and San Pedro (Table 2). The kdr L995F mutation allelic frequencies was from 79.3% for An. coluzzii, 76.0% for hybrids, and 72.1% for An. gambiae s.s. in the Aboisso population. In the population from Jacqueville and San Pedro, the kdr L995F (kdr west) frequencies were respectively 38.1% and 71.2%. For the kdr L995S (kdr East), the mutation allelic frequencies were low. The mutation allelic frequencies were 2.3% for An. gambiae s.s. in Aboisso population and 5.7% in San Pedro population. For the population of Aboisso, and San Pedro mutation was not detected. The mutation allelic frequencies of G280S were summarized in Table 5. The mutation allelic frequencies of G280S were 27.4% for An. coluzzii, and 42.9% for An. gambiae s.s. in the population of Aboisso. The allelic frequencies were 31.1% and 4.2% respectively for the population of Jacqueville and San Pedro.
Discussion
This study investigated the An. gambiae complex members resistance levels and mechanisms to pyrethroids (i.e., alpha-cypermethrin permethrin and deltamethrin) with and without PBO, clothianidin and chlorfenapyr in three coastal health districts, Aboisso, Jacqueville and San Pedro in southern Côte d’Ivoire. To our knowledge, this is one of the rare studies investigating both, the resistance status and complex members of malaria Anopheles mosquitoes in Costal Côte d’Ivoire. The current study showed that An. gambiae s.l. was composed of two sibling species, namely An. coluzzii, An. gambiae s.s, and the hybrids. Among the two sibling species An. coluzzii was the predominant (88.24%) species. The samples from Jacqueville and San Pedro were composed only of An. coluzzii. Both sibling species were represented in the Aboisso An. gambiae s.l. population. Among the sibling species, An. coluzzii (66.95%) was most abundant, followed by An. gambiae s.s. and hybrids. Moreover, An. gambiae s.l. showed high phenotypic and genotypic resistance to pyrethroids, with increased mortality while pre-exposed to PBO. Overall, An. gambiae s.l. susceptibility was fully reestablished by clothianidin and chlorfenapyr throughout, except for the Aboisso population with clothianidin. Kdr 995F allele frequency was very high (0.72–0.80%) in all An. gambiae s.l. populations. kdr 995S was very low (0–0.06%) in all An. gambiae s.l. populations. For Ace-1 280S, the high frequency (0.57%) was recorded in San Pedro and the low (0) from the Aboisso hybrid population.
Species identification of An. gambiae s.l. showed that An. coluzzii constituted the major species among the An. gambiae complex. In San Pedro and Jacqueville, only the species An. coluzzii was identified. This selective distribution of vectors is of considerable importance for understanding malaria transmission in these regions. Anopheles coluzzii’s preference for hosts and specific breeding habitats, especially its attraction to the littoral region, may account for its dominance in these zones [40]. Furthermore, the geographical distribution of various An. gambiae s.l. species may result from local ecological variations and adaptations to specific microclimates at each site. In Aboisso, the dominance of An. coluzzii over An. gambiae s.s. and crossbreeds carries profound implications for malaria transmission and vector control [41]. Anopheles coluzzii prevalence underscores its role as the primary vector, given its affinity for human hosts and urban breeding sites, which enhances the efficiency of malaria transmission [42]. The lower prevalence of An. gambiae s.s. may be attributed to ecological factors or competition with An. coluzzii [13]. Hybrid mosquitoes, comprising 11.8% of the complex, introduce variables such as insecticide resistance and altered behavior [43, 44]. Customizing vector control strategies, including the use of insecticide-treated bed nets and indoor spraying, to the dominant species, particularly An. coluzzii, is crucial [45]. The dominance of An. coluzzii species among An. gambiae complex is commonly reported in southern Côte d’Ivoire [7, 11, 13, 15, 18, 22]. Human activities such as urban Agriculture (vegetable gardens, irrigated rice fields) could have contributed to the development of the breeding sites of An. gambiae s.l. [25, 46]. The presence of sibling species, namely An. coluzzii, An. gambiae s.s. and hybrids, which is consistent with previous studies in the same area [13, 18]. Along with the presence of both An. coluzzii and An. gambiae s.s., and hybridsin the same area found here may imply that the mating appears to be occurring between the two species.
In the bioassays conducted in this study with pyrethroid, the highest mortalities were obtained with deltamethrin. Indeed, deltamethrin and alphacypermethrin are type II pyrethroids contrary to permethrin which is type I. Type II pyrethroids are distinguished from type I pyrethroids by the presence of a cyano group at the carbon of the esterified alcohol [47]. The effect of the cyano group on insecticidal activity increases mortality [48]. In such settings, use of deltamethrin or alphacypermethrin would probably be a good choice to treat nets. Several studies have shown the performance of type II pyrethroids in comparison to Type I [49]. However, the mortality varied between both insecticides. Several reasons would explain deferred mortalities induced by the same insecticide in different years. Pyrethroid resistance was detected more than 30 years ago in malaria vectors (An. gambiae s.l.) in Côte d’Ivoire for the first time in vectors from the Bouaké population [50]. In this study, An. gambiae s.l. from Aboisso, Jacqueville and San Pedro showed very strong phenotypic resistance to pyrethroids resulting in a very low mortality at standard diagnostic doses. The result is consistent with the general trends of An. gambiae s.l. resistance reported by previous studies in Côte d’Ivoire [16, 18] and reinforces the view that Côte d’Ivoire is a hotspot of resistance in West Africa [14, 16, 18]. Phenotypic resistance to pyrethroids may indicate the presence of genetic resistance resulting from modifications to the target sites of the insecticides. This could be linked to the extensive use of the same insecticides in both agricultural and public health contexts. Additionally, other factors such as the inherent genetic variability of insect populations and agricultural practices may also contribute to the emergence and spread of resistance [22–24]. The coastal zones in southern Côte d’Ivoire are devoted to large agro-industrial units (cocoa, rubber and oil palm plantations [21, 23, 51]) which are treated with chemicals, including insecticides, to protect the crops [14, 33–35, 52]. Indeed, the massive and incorrect use of pesticides by untrained farmers can contribute to the development of multiple resistance mutations in An. gambiae s.l. against insecticides used in public health [53].
In this study, very high allelic frequencies of the L995F kdr gene (>38%) were found in An. gambiae s.l. from the coastal health districts of Aboisso, Jacqueville, and San Pedro. These elevated allele frequencies indicate the widespread presence of the L995F and kdr-West mutations within the local An. gambiae s.l. population, as observed in several regions of Côte d’Ivoires.l. [12, 14, 15]. The low allelic frequencies of the L995S kdr gene (<6%) were detected in local An. gambiae s.l. The 995S allele was only found in An. gambiae s.s. from Aboisso and An. coluzzii from Jacqueville. Nonetheless, the presence of this allele can deliver further evidence for a pan-African propagation of the kdr resistance. This spread of the kdr 995F and 995S gene in An. gambiae s.l. populations in littoral areas could compromise the effectiveness of the vector control tools that are currently in use. The kdr genes are known to confer resistance to pyrethroid insecticides, which are commonly used in long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). If the kdr 995F and 995S mutations spread, the effectiveness of these malaria vector control tools (LLINs and IRS) would be compromised [54]. The high frequencies of the G119S mutation in Aboisso and Jacqueville suggest its widespread presence in the coastal areas of Côte d’Ivoire. Ace-1 G119S allele confers resistance to organophosphates and carbamates that are commonly used in Côte d’Ivoire for crop protection [14, 35].
The present study demonstrates that increased insecticide concentrations and PBO pre-exposure significantly heightened mortality in all three coastal populations of An. gambiae s.l. against all pyrethroids. However, this approach did not fully achieve susceptibility. Indeed, the current study showed that high intensity resistance is present in all three An. gambiae s.l. populations, with overall mortality being under 98% at 1X, 5X and 10X for all pyrethroid insecticides as reported by Kouassi [18] who carried out similar tests in Southern and central districts of Cote d’Ivoire. Reduced insecticidal activity can result in lower personal protection by allowing more insects to survive [55]. This increases the likelihood of bites and the spread of insect-borne diseases, thereby threatening the effectiveness of LLINs [32]. According to WHO, vector control operational failure is likely to happen if resistance is confirmed at the 5X and especially at the 10X concentrations [32]. The various anthropogenic or natural xenobiotics present in local mosquito breeding sites and the biotic interaction could lead to the development of very high levels of resistance observed in the study [56]. The high intensity resistance recorded using only pyrethroid could be explained by high kdr mutation frequency, mixed function oxidases (MFO) activities, and an increase in esterase activity. However, moderate resistance intensity was recorded with a 10X concentration of alphacypermethrin in the Aboisso populations, and additional investigations are required for better understanding. Although insecticide-induced mortality increased, full sensitivity with PBO was only observed in the Jacqueville strain with deltamethrin. However, full susceptibility was not recovered using PBO, except with the strain of Jacqueville with deltamethrin. Indeed, mortalities in PBO-pre-exposed mosquitoes tested against pyrethroids were still lower than the WHO susceptibility cut-off, as observed in other areas in Côte d’Ivoire [18, 57]. Significant increase in mortality after exposure to PBO may indicate the presence of metabolic resistance (MFO, P450s, and esterases) [57], and this can compromise the effectiveness of non-PBO LLINs (e.g., Panda.Net, MagNet, PermaNet 2.0). In addition, non-restoration of full susceptibility with PBO might reduce the efficacy of PBO-treated LLINs (e.g., PermaNet 3.0) [58].
In contrast to PBO, chlorfenapyr and clothianidin reestablished fully the susceptibility in all populations of An. gambiae s.l. from Aboisso, Jacqueville and San Pedro, except for clothianidin in Aboisso population in the current study. The chlorfenapyr (pyrrole) and clothianidin (neonicotinoid) have new modes of action and are good candidates for malaria vector control in areas comprised of mutation and/or metabolic resistance to pyrethroids [59]. Indeed, after exposure to chlorfenapyr, full susceptibility was recovered in one of three districts at 100μg ai/bottle. However, when the samples were tested with higher dosages (200μg ai/bottle), the mortality increased, and susceptibility was achieved in all districts. Chlorfenapyr requires activation, via cytochrome P450 monooxygenases [60]. Chlorfenapyr-treated LLINs (e.g., Interceptor G2 and PermaNet Dual) could be effective against An. gambiae s.l. in coastal Côte d’Ivoire [3, 4, 59]. Moreover, An. gambiae s.l. susceptibility was achieved with clothianidin (100% mortality) in Jacqueville and San Pedro. As the National Malaria Strategic Plan of the National Malaria Control Programme of Côte d’Ivoire prioritizes indoor residual spraying (IRS), using clothianidin-based IRS (e.g., Fludora® Fusion) could be effective against the coastal An. gambiae s.l. populations in coastal Côte d’Ivoire.
Conclusion
The present study showed that An. gambiae s.l. was predominate by An. coluzzii, followed by An. gambiae s.s and hybrids in the three studied sites of costal Côte d’Ivoire. Local An. gambiae s.l. populations were highly resistant to pyrethroids and susceptible to clothianidin and chlorfenapyr. Pre-exposure to PBO increased the mortality, but susceptibility was not fully recovered. The coastal An. gambiae s.l. populations had high frequencies of target site mutation genes (kdr-West and kdr-East) and metabolic genes (Ace-1). An. gambiae s.l. proved fully susceptible to clothianidin and chlorfenapyr. This suggests that vector control tools, such as insecticide-treated nets and sprays, treated with these new insecticides could be highly effective in reducing malaria transmission in coastal Côte d’Ivoire. Their use could significantly improve malaria control and reduce the disease burden in the region.
Supporting information
S1 Table. Data base assessing species composition and insecticide resistance of Anopheles gambiae complex members in three coastal health districts of Côte d’Ivoire.
https://doi.org/10.1371/journal.pone.0297604.s001
(XLSX)
Acknowledgments
We gratefully acknowledge the contribution of technicians Assamoi Jean Baptiste, Bamogo Kassoum and Dobri Didier of the Centre Suisse de Recherches Scientifiques en Côte d’Ivoire for help and assistance during laboratory analysis. We also thank the local health district staff and a special thanks to the Programme National de Lutte contre le Paludisme Côte d’Ivoire for facilitating and participating in field data collection.
References
- 1.
WHO. World malaria report 2023. Geneva: World Health Organization; 2023. Available: https://reliefweb.int/attachments/f03b4f2c-af9a-4161-9ee2-f2cec1eb3dc8/World%20malaria%20report%202023.pdf
- 2.
WHO. World malaria report 2022. Geneva: World Health Organization; 2022. Available: https://www.who.int/publications/i/item/9789240064898
- 3. Oxborough RM, Seyoum A, Yihdego Y, Chabi J, Wat’senga F, Agossa FR, et al. Determination of the discriminating concentration of chlorfenapyr (pyrrole) and Anopheles gambiae sensu lato susceptibility testing in preparation for distribution of Interceptor® G2 insecticide-treated nets. Malar J. 2021;20: 316. pmid:34261475
- 4. Oxborough RM, N’Guessan R, Jones R, Kitau J, Ngufor C, Malone D, et al. The activity of the pyrrole insecticide chlorfenapyr in mosquito bioassay: towards a more rational testing and screening of non-neurotoxic insecticides for malaria vector control. Malar J. 2015;14: 124. pmid:25879231
- 5. Institut National de la Statistique (INS). Enquête de prévalence parasitaire du paludisme et de l’anémie 2016. 2016; 61.
- 6. PRESIDENT’S MALARIA INITIATIVE. U.S. President’s Malaria Initiative Côte d’Ivoire Malaria Operational Plan FY 2023. 2023; 56.
- 7. Kouassi BL, Edi C, Ouattara AF, Ekra AK, Bellai LG, Gouaméné J, et al. Entomological monitoring data driving decision-making for appropriate and sustainable malaria vector control in Côte d’Ivoire. Malar J. 2023;22: 14. pmid:36635720
- 8. Ouattara AF, Dagnogo M, Constant EA, Koné M, Raso G, Tanner M, et al. Transmission of malaria in relation to distribution and coverage of long-lasting insecticidal nets in central Côte d’Ivoire. Malar J. 2014;13: 109. pmid:24645751
- 9. N’Guessan GKD, Coulibaly FH, Barreaux AMG, Yapo RJ, Adou KA, Tia E, et al. Qualitative study on the use and maintenance of long-lasting insecticidal nets (LLINs) in Bouaké (Côte d’Ivoire), 17 months after the last mass distribution campaign. Malar J. 2022;21: 228. pmid:35906600
- 10. The PMI VectorLink Côte d’Ivoire 2021 Annual Entomological Report, January–December 2021.: 65.
- 11. Assouho KF, Adja AM, Guindo-Coulibaly N, Tia E, Kouadio AMN, Zoh DD, et al. Vectorial Transmission of Malaria in Major Districts of Côte d’Ivoire. Norris D, editor. Journal of Medical Entomology. 2020;57: 908–914. pmid:31785095
- 12. Mbama Ntabi JD, Malda Bali ED, Lissom A, Akoton R, Djontu JC, Missontsa G, et al. Contribution of Anopheles gambiae sensu lato mosquitoes to malaria transmission during the dry season in Djoumouna and Ntoula villages in the Republic of the Congo. Parasites Vectors. 2024;17: 104. pmid:38431686
- 13. Yokoly FN, Zahouli JBZ, Small G, Ouattara AF, Opoku M, de Souza DK, et al. Assessing Anopheles vector species diversity and transmission of malaria in four health districts along the borders of Côte d’Ivoire. Malar J. 2021;20: 409. pmid:34663359
- 14. Kouamé RMA, Lynd A, Kouamé JKI, Vavassori L, Abo K, Donnelly MJ, et al. Widespread occurrence of copy number variants and fixation of pyrethroid target site resistance in Anopheles gambiae (s.l.) from southern Côte d’Ivoire. Current Research in Parasitology & Vector-Borne Diseases. 2023;3: 100117. pmid:36970448
- 15. Fodjo BK, Koudou BG, Tia E, Saric J, N’dri PB, Zoh MG, et al. Insecticides Resistance Status of An. gambiae in Areas of Varying Agrochemical Use in Côte D’Ivoire. BioMed Research International. 2018;2018: 1–9. pmid:30402467
- 16. Camara S, Koffi AA, Ahoua Alou LP, Koffi K, Kabran J-PK, Koné A, et al. Mapping insecticide resistance in Anopheles gambiae (s.l.) from Côte d’Ivoire. Parasites Vectors. 2018;11: 19. pmid:29310704
- 17. Edi CA, Koudou BG, Bellai L, Adja AM, Chouaibou M, Bonfoh B, et al. Long-term trends in Anopheles gambiae insecticide resistance in Côte d’Ivoire. 2014; 10.
- 18. Kouassi BL, Edi C, Tia E, Konan LY, Akré MA, Koffi AA, et al. Susceptibility of Anopheles gambiae from Côte d’Ivoire to insecticides used on insecticide-treated nets: evaluating the additional entomological impact of piperonyl butoxide and chlorfenapyr. Malar J. 2020;19: 454. pmid:33298071
- 19. PRESIDENT’S MALARIA INITIATIVE. U.S. President’s Malaria Initiative Côte d’Ivoire Malaria Operational Plan FY 2018 and FY 2019. 2018; 57.
- 20. MSHPCMU. Rapport annuel sur la situation sanitaire (RASS). Côte d’Ivoire; 2017. 2017; 414.
- 21. Anoh P, Pottier P. Géographie du littoral de Côte-d’Ivoire: éléments de réflexion pour une politique de gestion inté. com. 2010;63: 485–489.
- 22. Sadia-Kacou CMA, Alou LPA, Edi AVC, Yobo CM, Adja MA, Ouattara AF, et al. Presence of susceptible wild strains of Anopheles gambiae in a large industrial palm farm located in Aboisso, South-Eastern of Côte d’Ivoire. Malar J. 2017;16: 157. pmid:28427450
- 23. Gohourou F, Ahua ÉA, Gnanbe DR, Desse M. Fonctionnement, dynamique et structuration de l’espace littoral en Côte d’Ivoire.: 16.
- 24. Malan DF, Neuba DFR, Kouakou KL. Medicinal plants and traditional healing practices in ehotile people, around the aby lagoon (eastern littoral of Côte d’Ivoire). J Ethnobiology Ethnomedicine. 2015;11: 21. pmid:25888765
- 25. Chouaïbou MS, Fodjo BK, Fokou G, Allassane OF, Koudou BG, David J-P, et al. Influence of the agrochemicals used for rice and vegetable cultivation on insecticide resistance in malaria vectors in southern Côte d’Ivoire. Malar J. 2016;15: 426. pmid:27553959
- 26. Sattler MA, Mtasiwa D, Kiama M, Premji Z, Tanner M, Killeen GF, et al. Habitat characterization and spatial distribution of Anopheles sp. mosquito larvae in Dar es Salaam (Tanzania) during an extended dry period. Malar J. 2005;4: 4. pmid:15649333
- 27. Soma DD, Maïga H, Mamai W, Bimbile-Somda NS, Venter N, Ali AB, et al. Does mosquito mass-rearing produce an inferior mosquito? Malar J. 2017;16: 357. pmid:28882146
- 28.
Gillies MT, Meillon B de. The Anophelinae of Africa south of the Sahara. Suppl: Afrotropical region. Johannesburg; 1987.
- 29.
WHO. World malaria report: 2013. Geneva: World Health Organization; 2013. Available: https://apps.who.int/iris/handle/10665/97008
- 30. WHO. Malaria control improves for vulnerable in Africa, but global progress off-track = La lutte contre le paludisme s’améliore pour les personnes vulnérables en Afrique, mais les progrès stagnent à l’échelle mondiale. Is part of a journal: Weekly Epidemiological Record = Relevé épidémiologique hebdomadaire. 2016;91: 622–624.
- 31. Dagg K, Irish S, Wiegand RE, Shililu J, Yewhalaw D, Messenger LA. Evaluation of toxicity of clothianidin (neonicotinoid) and chlorfenapyr (pyrrole) insecticides and cross-resistance to other public health insecticides in Anopheles arabiensis from Ethiopia. Malar J. 2019;18: 49. pmid:30795768
- 32.
Brogdon WG, Chan A. Guideline for Evaluating Insecticide Resistance in Vectors Using the CDC Bottle Bioassay. Centers for Disease Control and Prevention, Atlanta, GA. 2012; 28.
- 33. Collins FH, Besansky NJ, Mendez MA, Rasmussen MO, Finnerty V, Mehaffey PC. A Ribosomal RNA Gene Probe Differentiates Member Species of the Anopheles gambiae Complex. The American Journal of Tropical Medicine and Hygiene. 1987;37: 37–41. pmid:2886070
- 34. Santolamazza F, Calzetta M, Etang J, Barrese E, Dia I, Caccone A, et al. Distribution of knock-down resistance mutations in Anopheles gambiae molecular forms in west and west-central Africa. Malar J. 2008;7: 74. pmid:18445265
- 35. Lucas ER, Rockett KA, Lynd A, Essandoh J, Grisales N, Kemei B, et al. A high throughput multi-locus insecticide resistance marker panel for tracking resistance emergence and spread in Anopheles gambiae. Sci Rep. 2019;9: 13335. pmid:31527637
- 36. Bass C, Nikou D, Donnelly MJ, Williamson MS, Ranson H, Ball A, et al. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods. Malar J. 2007;6: 111. pmid:17697325
- 37.
WHO. Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. 2nd ed. Geneva: World Health Organization; 2016. Available: https://iris.who.int/handle/10665/250677
- 38. Abbott WS. A Method of Computing the Effectiveness of an Insecticide. Journal of Economic Entomology. 1925;18: 265–267.
- 39. Hardy GH. Mendelian Proportions in a Mixed Population. Science. 1908;28: 49–50. pmid:17779291
- 40. Simard F, Ayala D, Kamdem G, Pombi M, Etouna J, Ose K, et al. Ecological niche partitioning between Anopheles gambiae molecular forms in Cameroon: the ecological side of speciation. BMC Ecol. 2009;9: 17. pmid:19460146
- 41. Smith DL, Battle KE, Hay SI, Barker CM, Scott TW, McKenzie FE. Ross, Macdonald, and a Theory for the Dynamics and Control of Mosquito-Transmitted Pathogens. Chitnis CE, editor. PLoS Pathog. 2012;8: e1002588. pmid:22496640
- 42. Gimonneau G, Pombi M, Choisy M, Morand S, Dabiré RK, Simard F. Larval habitat segregation between the molecular forms of the mosquito Anopheles gambiae in a rice field area of Burkina Faso, West Africa. Medical and Veterinary Entomology. 2012;26: 9–17. pmid:21501199
- 43. Norris LC, Main BJ, Lee Y, Collier TC, Fofana A, Cornel AJ, et al. Adaptive introgression in an African malaria mosquito coincident with the increased usage of insecticide-treated bed nets. Proc Natl Acad Sci USA. 2015;112: 815–820. pmid:25561525
- 44. Caputo B, Tondossoma N, Virgillito C, Pichler V, Serini P, Calzetta M, et al. Is Côte D’Ivoire a new high hybridization zone for the two major malaria vectors, Anopheles coluzzii and An. gambiae (Diptera, Culicidae)? Infection, Genetics and Evolution. 2022;98: 105215. pmid:35063691
- 45. Moyes CL, Vontas J, Martins AJ, Ng LC, Koou SY, Dusfour I, et al. Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting humans. Sinnis P, editor. PLoS Negl Trop Dis. 2017;11: e0005625. pmid:28727779
- 46. Chabi J, Eziefule MC, Pwalia R, Joannides J, Obuobi D, Amlalo G, et al. Impact of Urban Agriculture on the Species Distribution and Insecticide Resistance Profile of <i>Anopheles gambiae s.s. </i> and <i>Anopheles coluzzii</i> in Accra Metropolis, Ghana. AE. 2018;06: 198–211.
- 47. Godin SJ, Crow JA, Scollon EJ, Hughes MF, DeVito MJ, Ross MK. Identification of Rat and Human Cytochrome P450 Isoforms and a Rat Serum Esterase That Metabolize the Pyrethroid Insecticides Deltamethrin and Esfenvalerate. Drug Metab Dispos. 2007;35: 1664–1671. pmid:17576809
- 48. Kobayashi T, Nishimura K, Fujita T. Effects of the α-cyano group in the benzyl alcohol moiety on insecticidal and neurophysiological activities of pyrethroid esters. Pesticide Biochemistry and Physiology. 1989;35: 231–243.
- 49. Dadzie SK, Chabi J, Asafu-Adjaye A, Owusu-Akrofi O, Baffoe-Wilmot A, Malm K, et al. Evaluation of piperonyl butoxide in enhancing the efficacy of pyrethroid insecticides against resistant Anopheles gambiae s.l. in Ghana. Malar J. 2017;16: 342. pmid:28818077
- 50. Elissa N, Mouchet J, Riviere F, Meunier JY, Yao K. Resistance of Anopheles gambiaes.s. to pyrethroids in Côte d’Ivoire. Ann Soc Belg Med Trop. 1993;73: 291–294.
- 51. Pottier P. GÉOGRAPHIE DU LITTORAL DE CÔTE D IVOIRE Éléments de réflexion pour une politique de gestion intégrée. 2008 [cited 16 May 2022]. Available: https://docplayer.fr/56603270-Geographie-du-littoral-de-cote-d-ivoire-elements-de-reflexion-pour-une-politique-de-gestion-integree.html
- 52.
MINADER. Programme de Transformation de l’Agriculture en Afrique de l’Ouest (PTAAO) Plan de Gestion des Pestes (PGP). Abidjan; 2018 Apr p. 93. Available: http://www.waapp-ppaao.org/sites/default/files/ptaao-cote_divoire-document_de_suavegarde-plan_de_gestion_des_pestes.pdf
- 53.
WHO, FAO. International code of conduct on pesticide management: guidance on management of household pesticides. Geneva: World Health Organization; 2020. Available: https://apps.who.int/iris/handle/10665/337126
- 54. Mouhamadou CS, N’Dri PB, Fodjo BK, Sadia CG, Affoue F-PK, Koudou BG. Rapid spread of double East- and West-African kdr mutations in wild Anopheles coluzzi from Côte d’Ivoire. Wellcome Open Res. 2019;4: 31. pmid:31020049
- 55. Dabiré RK, Diabaté A, Baldet T, Paré-Toé L, Guiguemdé RT, Ouédraogo J-B, et al. Personal protection of long lasting insecticide-treated nets in areas of Anopheles gambiae s.s. resistance to pyrethroids. Malar J. 2006;5: 12. pmid:16472385
- 56. Poupardin R, Riaz MA, Vontas J, David JP, Reynaud S. Transcription profiling of eleven cytochrome P450s potentially involved in xenobiotic metabolism in the mosquito Aedes aegypti. Insect Molecular Biology. 2010;19: 185–193. pmid:20041961
- 57. Bingham G, Strode C, Tran L, Khoa PT, Jamet HP. Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid-resistant Aedes aegypti?: Can piperonyl butoxide enhance the efficacy of pyrethroids? Tropical Medicine & International Health. 2011;16: 492–500. pmid:21324051
- 58. Gleave K, Lissenden N, Chaplin M, Choi L, Ranson H. Piperonyl butoxide (PBO) combined with pyrethroids in insecticide-treated nets to prevent malaria in Africa. Cochrane Infectious Diseases Group, editor. Cochrane Database of Systematic Reviews. 2021;2021. pmid:34027998
- 59. N’Guessan R, Odjo A, Ngufor C, Malone D, Rowland M. A Chlorfenapyr Mixture Net Interceptor® G2 Shows High Efficacy and Wash Durability against Resistant Mosquitoes in West Africa. Vontas J, editor. PLoS ONE. 2016;11: e0165925. pmid:27851828
- 60. Paul A, Harrington LC, Scott JG. Evaluation of Novel Insecticides for Control of Dengue Vector Aedes aegypti (Diptera: Culicidae). me. 2006;43: 55–60.