The study took place in Benin, a country in West Africa. It lies in the intertropical zone between the equator and the Tropic of Cancer, more precisely between parallels 6˚30' and 12˚30' north latitude and meridians 1˚ and 3˚40' East longitude [19]. It is bounded to the north by the River Niger, to the south by the Atlantic Ocean, to the west by Togo and Burkina Faso and to the east by Nigeria. It covers an area of 114,763 km² [19].

This study was conducted during the rainy season between 2021 and 2023 in 4 types of cropping area in Benin (Figure 1). These are cotton-growing areas, cereal-growing areas, rice-growing areas, lagoon areas or market garden areas.

Cereal-growing zones farming system is essentially composed of food crops such as rice, sorghum, maize, etc [20]. These are areas where insecticides and other pesticides are used sparingly. The lagoon areas are generally market-garden areas with a pseudo-development of food crops. These are areas of relative pesticide use, generally represented by fungicides and other acaricides [20]. The cotton-growing areas in northern and central Benin, are areas of high insecticide use and rice-growing areas that use little or no insecticides. We collected Anopheles larvae in N’dali and Natitingou (cotton-growing areas), Glazoué and Malanville (rice-growing areas), Boukoumbé, Dogbo and Toffo (cereal-growing areas) and Cotonou, Comè and Semè-Kpodji (lagoon and market-garden areas). These different zones constitute the agro-ecological zones of Benin and are subject to different insecticide pressures, mainly due to cultivation practices. For example, in the cotton zone, insecticide use is very high because of production, compared with other zones where cotton is not produced. It should also be noted that pyrethroid-impregnated mosquito nets are the main vector control tools in all the different crop-growing zones.

Figure 1. Geographical distribution of study sites for the period January 2021 to July 2023.

The sampling method used to obtain imagos was the dipping method [21]. This consisted of collecting Anopheles larvae from the breeding sites using ladles. The larvae collected were transported to the insectarium of the Centre de Recherche Entomologique de Cotonou (CREC) in jars containing water from the collection sites. They were then reared until the adults emerged. The resulting imagos were fed with sugar juice. In the insectarium, the temperature was maintained at 27˚C ± 2˚C with a relative humidity of 70% ± 10%.

Morphological identification of anopheles

The female anopheles used for the tests were identified morphologically as belonging to the Anopheles gambiae complex using the key of Gillies and De Meillon [22] and Gillies and Coetzee [23].

Insecticides tested

Female Anopheles gambiaes.l. were subjected to 4 insecticides belonging to three families:

● Organophosphates: pirimiphos-methyl 0.5%;

● Carbamates: bendiocarb 0.01%; and

● Pyrethroids: deltamethrin 0.05% and alphacypermethrin 0.05%.

Insecticide sensitivity tests

Susceptibility testing was performed on fasting An. gambiaes.l. females aged 2 to 5 days, according to the standard WHO protocol for adults using WHO kits [24][25]. The tests were performed at room temperature and relative humidity of 25˚C ± 2˚C and 70% ± 10% respectively. Nine tubes were used for each test. In each tube, females were introduced in batches of approximately 25 females. Each test was carried out in two stages: firstly, the batches of mosquitoes were observed for one hour in tubes fitted with unimpregnated paper (12 × 15 cm); secondly, the females were brought into contact with the insecticide in tubes fitted with impregnated paper (12 × 15 cm), while the control females were introduced into tubes fitted with unimpregnated paper. During the 60-minute exposure period, the knock-down effect was observed every 5 minutes. At the end of this period, the females were transferred to observation tubes. A cotton swab soaked in a 10% sugar solution was then placed on each observation tube to feed the mosquitoes, thus ruling out hunger as a cause of mortality. Mortality was then assessed 24 hours after exposure to the insecticide [24][25]. The impregnated papers used came from the reference center (Vector Control Research Unit, Sains Malaysia University, Penang, Malaysia). These papers were used during their period of validity. Repetition of the tests under the same conditions and the absence of mortality in the controls enabled us to guarantee the presence of the insecticide and the quality of the impregnation of the papers used. All this confirms the results we obtained during the various tests.

Synergistic bioassays

In order to assess the involvement of detoxification enzymes in resistance to deltamethrin and alphacypermethrin (insecticides used in the impregnation of mosquito nets in Benin), tests with synergists were carried out with pre-exposure to 4% PBO (piperonyl butoxide), an inhibitor mainly of mono-oxygenases. This test was carried out in three stages using three batches of 20 to 25 non-gorged An. gambiaes.l. females aged between 2 and 5 days. The three batches were divided as follows: 1 batch to be tested with insecticide (deltamethrin 0.05% or alphacypermethrin 0.05%) alone, 1 batch to be tested with synergist followed by insecticide and 1 batch serving as a control exposed to unimpregnated paper. Initially, the batches of females were observed for one hour in tubes fitted with unimpregnated paper (12 × 15 cm). Secondly, 2 batches of 20 to 25 females were each introduced into a tube lined with paper impregnated with synergist for 1 hour. Finally, the females were transferred to the exposure tubes. The transfer was carried out as follows: the batch to be tested with synergist and then with insecticide was transferred to the tube containing insecticide, the batch to be tested with insecticide was transferred to a tube containing paper impregnated with insecticide and the control batch was transferred to a tube containing unimpregnated paper. After 1 hour exposure, the three batches were transferred back into their respective observation tubes supplied with a 10% sugar water solution and maintained for 24 hours before recording mortality. It should be noted that these tests were carried out at a temperature of 25˚C and a humidity of 70%.

Sensitivity test validity criteria

The susceptibility of Anophelesgambaes.l. populations was interpreted according to WHO criteria [25]. The knock-down effect of the insecticide was assessed during the exposure period, which lasted 1 hour, and its lethal effect was assessed 24 hours after the exposure period. The interpretation criteria used were as follows:

- Mortality rate between 98% - 100%: population sensitive to the insecticide tested

- Mortality rate between 90% - 97%: decrease in sensitivity within the population; suspected resistance

- Mortality rate below 90%: population resistant to the insecticide tested

For tests with synergists, the validity criteria were applied in accordance with previous recommendations [25]-[27]. For the validation of the results, the mortality of the controls must be less than 5%. When it is between 5% and 20%, Abbot's formula is used to correct the mortality of exposed mosquitoes. However, if the mortality is greater than 20%, the test is cancelled.

2.3.1. Determination of the L1014F Mutation in the Kdr Gene

The L1014F resistant allele of the kdr gene was determined using the protocol of Martinez-Torres et al. [28]. This diagnostic PCR test uses four oliogonucleotides or primers (Agd1, Agd2, Agd3, Agd4) and a Taq polymerase to amplify resistant or susceptible alleles on a DNA fragment coding for the voltage-gated sodium channel in each mosquito tested. The Agd1/Agd2 primer pair identifies the kdr gene by amplifying a 293-bp product as a control. The Agd3/Agd1 primer pair associates only with the resistance part of the kdr gene to amplify a 195 bp fragment. The Agd4/Agd2 pair associates only with the sensitive part of the gene, amplifying a 137 bp fragment. The nucleotide sequences of these primers are as follows: Agd1: 5'-ATAGATTCCGACCATG-3'; Agd2: 5'-ACAAGGATGATGAACC3'; Agd3: 5'-AATTTGCATTACTTACGACA-3'; Agd4: 5'-CTGTAGTGATAGGAAATTTA-3'

2.3.2. Determination of the G119S Mutation of the Ace1 Gene

For the Ace1 marker, PCR diagnosis of the G119S mutation was carried out according to the protocol of Weill et al. [29] using two primers with the following nucleotide sequences: Ex3AGdir: 5'-GATCGTGGACACCGTGTTCG-3' Ex3AGrev: 5'-AGGAT GGCCCGCTGGAACAG-3'

In this study, data were entered into Excel 2010 and analysed using R software (version 3.3.3) [30]. The Chi2 test was used to compare mortality rates. A probability value of p less than or equal to 0.05 was considered significant. The frequencies of the L1014F and G119S allele of the Kdr and Ace1 gene were determined for each growing zone. Frequency confidence intervals were calculated using the binomial formula. The proportion test [30] in R software, version 3.3.3 was used to compare the distribution of the different species and the frequency of L1014F alleles within these species in the different agro-ecosystems.

The resistance of vectors to insecticides was monitored in the different growing zones (cereal-growing zones, rice-growing zones, lagoon zones and cotton-growing zones) using the WHO tube test (Table 1). Mortality rates were below 40% for both insecticides in all four crop zones, indicating high resistance to both pyrethroids. There was no significant difference in the susceptibility level of Anopheles gambiaes.l. populations in three of the four growing zones (cotton, cereals and rice). However, in the lagoon zone, the level of susceptibility of Anopheles gambiaes.l. populations to deltamethrin was significantly higher than to alphacypermethrin (Figure 2).

Table 1. Results of sensitivity tests on Anopheles gambiaes.l. to pyrethroids.

Figure 2. Mortality rate observed in Anopheles gambiaes.l. after exposure to pyrethroids (Deltamethrin and alphacypermethrin) in different growing zones.

Pre-exposure of An.gambiaes.l. to PBO induced a significant increase in mortality in the different populations tested with alphacypermethrin and deltamethrin alone, indicating resistance management due to the action of the PBO synergist. This means that MFOs are partially involved in the phenotypic resistance of An.gambiaes.l. to pyrethroids. No significant differences were observed between the mortality rates of An.gambiaes.l. populations in the different growing zones (Table 2 and Figure 3).

Table 2. Results of sensitivity tests on Anopheles gambiaes.l. to pyrethroids + PBO.

Figure 3. Mortality rate observed in Anopheles gambiaes.l. after exposure to pyrethroids + PBO (Deltamethrin + PBO and alphacypermethrin + PBO) in different growing zones.

Table 3 shows the mortality rates observed in Anopheles gambiaes.l. in the different growing zones. The results show phenotypic resistance to bendiocarb in the lagoon zone and suspected resistance to this insecticide in the cereal, cotton and rice-growing zones. As for pirimiphos methyl, there was sensitivity in the cotton-growing zone, suspected resistance in the rice-growing zone and resistance in the cereal-growing and lagoon-growing areas of the different populations of Anopheles gambiaes.l. (Figure 4).

Table 3. Results of sensitivity tests on Anopheles gambiaes.l. to carbamates and Organophosphates.

Figure 4. Mortality rate observed in Anopheles gambiaes.l. after exposure to carbamates (bendiocarb) and organophosphates (pirimiphos methyl) in different growing zones.

Table 4. Frequency of the L1014F allele of the Kdr gene in An. gambiaes.l. per zone.

Frequencies with the same letter in the columns do not differ significantly from one locality to another. N: Number of genotypes; (F %): frequency; homozygous resistant genotype 1014F/1014F; heterozygous genotype 1014F/1014L; susceptible homozygous genotype 1014L/1014L.

Table 5. Frequency of the G119S allele of the Ace1 gene in An. gambiaes.l. per zone.

Frequencies with the same letter in the columns do not differ significantly from one locality to another. (F %): frequency; homozygous resistant genotype 119S/119S; heterozygous genotype 119S/119G; susceptible homozygous genotype 119G/119G.

Table 4 and Table5 show the allelic frequencies of the Kdr L1014F gene and the Ace1 G119S gene in Anopheles gambiaes.l. in different cropping zones in Benin. Kdr L1014F gene frequency values are high (above 76%) in all cropping zones: the highest value is observed in the cereal-growing areas (84.84%) and the lowest value in the cotton-zone (76.58%). A comparison of these different frequencies reveals a significant difference between the allelic frequencies observed (Table 4). As for the frequency of the Ace 1 gene, the values observed in the different cropping zones were generally low (below 5%). The highest value was observed in the rice-growing zone with a frequency of 4.86% and the lowest value was observed in the cotton-zone with a frequency of 3.52%. No significant difference was observed between the allele frequencies of the Ace1 gene in the different cropping zones (Table 5).

CA, GGP and MCA designed the study. SA, AF, ZCK, AS, EO, OO Performed the experiment. SA, ZCK, AF and MCA analysed the data. SA wrote the manuscript. ZCK, AF and MCA reviewed the manuscript critically. All authors read and approved the final manuscript.

We thank the anonymous reviewers for the relevant corrections. We would also like to thank the Cotonou Entomological Research Center (CREC) for its technical and financial support in carrying out this study.