The study sites were the city of Brazzaville, the urban commune of Kintele and the village of Djoumouna as shown in Figure 1.

The city of Brazzaville (4˚15'S and 15˚16'E) is the political capital of the Republic of Congo. It is situated at an average altitude of 326 meters [17]. The climate is equatorial, with a dry season from June to September and a rainy season from October to May. The climatic parameters for the last 5 years (2015 to 2019) showed an average temperature of 26.08˚C and rainfall of 127.22 mm of water on average per month. The average maximum and minimum relative humidity were 89.57% and 55.63% respectively [18]. Brazzaville is irrigated by numerous rivers, including the Djoue, Djiri, Tsieme, Mfoa, Madoukou-tsekele, Mfilou and Kelekele.

The vegetation of the city and its surroundings consists of forest and savanna. With its 9 districts, the city is home to more than half of the Congolese population. Studies carried out in Brazzaville have shown that species of the Anophelesgambiae s.l. complex are mainly involved in malaria transmission [19]-[23]. Regarding the intensity of transmission, the study by Trape and Zoulani in 1989 showed that in the densely populated districts (Poto-Poto, Moungali and Ouenze), one person received an infectious bite every three years. It was also shown that in a recently urbanized district, such as Mfilou-Ngamaba in those years, one person received more than 100 infectious bites per year [21].

Regarding the sensitivity of Anopheles to insecticides, studies conducted in the 1950s and 1960s had shown that An.gambiae s.l. was resistant to organochlorines (DDT, HCH, dieldrin) and sensitive to malathion [20][23]. In 2014, Nianga Bikouta showed that vector populations were resistant to DDT and pyrethroids (deltamethrin, permethrin, and lambda-cyhalothrin), but susceptible to bendiocarb and malathion [22].

During the present study, larvae were collected from six (6) sites in Brazzaville: three (3) integrated with horticultural activities (The bled, Test garden and Mayanga) with breeding grounds consisting of wells dug in the ground and used as water reserves for watering vegetables, and one (1) considered as polluted (Make-lekele). Polluted and non-polluted breeding sites were distinguished by the presence of plastic and organic waste. Finally, three (3) consisted of puddles left over from the rainy season (Bacongo, Moungali and Madibou).

The commune of Kintele is located 25 km north of Brazzaville (4˚9'0"S, 15˚20'32"E) with an average altitude of 201 m [24]. The population is estimated at 11,105 inhabitants. The economic activity of the municipality of Kintele is flourishing. There are 265 small and medium-sized commercial enterprises, 64 small and medium-sized industries, mainly specialized in construction, and 6 small state markets. The houses are made of cement bricks with corrugated iron roofs. These dwellings generally have gaps that allow many arthropods, including mosquitoes, to pass through. The relief consists of valleys, plains, hills and plateaus. The climate is tropical and humid. With two seasons, a rainy season from October to May and a dry season from June to September, the average annual rainfall is 1370 mm and the average temperature is 25.5˚C [25]. Kintele is irrigated by two rivers, the Djiri and the Blue Chatelet. The vegetation is mainly wooded savannah, sometimes with scrub, where manioc and pineapple are grown. Vegetables are grown on the banks of the Congo River [26]. A study of malaria transmission in 1987 showed that the An.gambiae s.l. complex contained the main malaria vector species in Kintele [27].

Figure 1. Map of Congo including Brazzaville, Kintele and Djoumouna (CERGEC, 2020).

Djoumouna (4˚22'34"S, 15˚9'36"E) is a rural area located 25 km southeast of Brazzaville in the Pool department, with an average altitude of 217 m [28]. The population is estimated at 635 inhabitants. The main activities are agriculture, fish farming and cattle rearing. Djoumouna is located in an area of degraded secondary forest. The climate is similar to that of Brazzaville. The town is bordered by four rivers: the Lomba, the Kinkoue, the Loumbangala and the Djoumouna. These rivers supply water to a number of fish ponds. These ponds are permanent breeding grounds for the larvae proliferation of the Anopheles mosquito. In addition to these breeding sites, there are many other temporary breeding sites. Many studies were carried out in the village between the 1970s and 1990s [29]-[31]. These showed that species belonging to the An.gambiae s.l. complex were the main vector species of malaria and that the intensity of malaria transmission was expressed as more than 1000 ft/h/a [32]. In terms of insecticide resistance, An.gambiae s.l. was sensitive to deltamethrin [29].

Two sampling methods were used to obtain adults for susceptibility testing in the three study sites from May 2016 to December 2018.

In the first method, Anopheles larvae were collected by the dipping method according to the WHO protocol [33]. The larval sites consisted of unpolluted sites (river backwaters, water pools, ruts and ditches), a site polluted by the presence of numerous plastic and organic wastes (a decorative fountain) and sites in market gardens.

The mosquito larvae collected in the field were transferred to the insectarium of the National Institute for Research in Health Sciences (NIRHS), fed with fish food (Comipex^®) and reared to adult stage.

The second method was to collect resting females in the bedrooms. Susceptibility tests were carried out using first generation (F1) females from eggs collected from wild females.

The female Anopheles used in the tests were identified morphologically identified as belonging to the An.gambiae complex according to the key of Gillies and De Meillon 1968).

Susceptibility testing was performed using susceptibility test kits and the WHO standard protocol for adults [16][34]. Testing was performed at an average room temperature and relative humidity of 25˚C ± 2˚C and 70% ± 10% RH, respectively.

For each test series, 4 insecticide exposure tubes and 2 control tubes were monitored in two phases. In the first phase, 4 and 6 batches of 25 mosquitoes each were placed in non-insecticide-impregnated paper tubes and observed for one hour. Insecticide-impregnated papers were obtained through WHO/AFRO from its reference center (Vector Control Research Unit, Sains Malaysia University, Penang, Malaysia).

Four insecticides belonging to two families were tested with pyrethroids including lambda-cyhalothrin (0.05%), deltamethrin (0.05% and 0.5%), permethrin (0.75%) and an organochlorine DDT (4%). The use of the 0.5% (10×) dose of deltamethrin made it possible to assess the intensity of resistance according to WHO criteria. These results were interpreted as an indicator of the selective force exerted on local populations, in comparison with the standard 0.05% test.

In the second phase, mosquitoes were exposed to the insecticide in tubes with impregnated paper, while control mosquitoes were placed in tubes containing unimpregnated paper. During the 60 minutes exposure period, the knock-down effect was observed every 5 minutes from the 5th minute of exposure, and the number of knocked-out mosquitoes was recorded. At the end of the exposure period, females were transferred to observation tubes. Mortality was assessed 24 hours after exposure to the insecticide [16].

In order to assess the involvement of detoxifying enzymes in resistance to pyrethroids (deltamethrin), tests were conducted with 4% PBO (piperonyl butoxide), a monooxygenase inhibitor [16].

The PBO tests were conducted according to WHO protocols with four batches of 25 female An.gambiaes.l. aged 2 to 5 days. Two batches were pre-exposed for 1 hour to papers impregnated with 4% PBO, then immediately transferred to 0.05% deltamethrin papers for 1 hour of exposure. One batch was exposed only to 0.05% deltamethrin and a control batch was kept on untreated paper. Mortality was recorded 24 hours after exposure. Finally, 1 to 10 exposed Anopheles mosquitoes (survivors and dead) were stored individually in 1.5 ml Eppendorf tubes containing silica gel and cotton wool at −20˚C in order to identify the different species and the kdr mutation.

These tests were limited to the Makelekelé site, chosen because of its high level of domestic pollution, which favors the selection of metabolic resistance mechanisms. This choice was justified by the desire to document the potential role of detoxifying enzymes in a context where resistance is particularly pronounced.

The data collected were analyzed according to WHO criteria. The parameters measured were the knockdown times or the time required to knock down 50% (KD50) and 95% (KD95) of the specimens tested. The knockdown effect of the insecticide was assessed during the 1 h exposure period and mortality was assessed 24 h after the exposure period. Mortality was interpreted according to WHO criteria for determining susceptibility status [16].

The susceptibility, probable resistance, and resistance status of the tested mosquitoes were evaluated according to the following criteria: a mortality rate ≥ 98% indicates that the tested population is susceptible; a mortality rate between 90% and 97% indicates the presence of probable resistance; and a mortality rate < 90% corresponds to a population resistant to the insecticide used.

For PBO tests to be valid, there must be no mortality in batches exposed to PBO alone. Second, the mortality rates of the PBO and then deltamethrin tests must be greater than the mortality rates of deltamethrin alone, according the criteria are the same as those for the tests with papers impregnated with insecticides by comparing the tests with PBO-insecticide and the tests with insecticides alone [16].

DNA was extracted from mosquitoes preserved with silica gel according to the protocol described by Morlais etal., (2004) [35]. The method involved grinding the legs and wings in 200 μl of 2% CTAB (Cetyl Trimethylammonium Bromide) in an Eppendorf tube. The tubes were then incubated for at least 5 minutes at 65˚C in a dry bath and then placed on ice for 3 minutes. To each tube, 200 μl of chloroform was then added. After manual agitation, the tubes were centrifuged at 12,000 rpm for 5 minutes. The supernatant from each tube was then pipetted into a new tube to which 200 μl of isopropanol was added. The new tubes were again centrifuged at 12,000 rpm for 15 minutes, then emptied and drained. The resulting pellet was washed with 200 μl of cold 70˚ ethanol and then the tubes centrifuged at 12,000 rpm for 5 minutes. The drained was then removed, drained and placed in a Speed-vac to dry the DNA pellet for 5 minutes. The resulting DNA was solubilized in 20 μl of bi-distilled water and stored at −20˚C until PCR testing.

Molecular identification of species belonging to the An.gambiae s.l. complex was performed according to the method developed by Wilkins etal., (2006) [36]. PCR was performed using Thermus aquaticus polymerase (Sigma, USA). The reaction volume was 12.5 μl with the following composition: 6.4 μl of water; 1.25 μl of 10X buffer; 1.25 μl of 2mM dNTP; 0.5 μl of each primer (UN, AR, ML, M1, and S1); 0.1 μl of Taq, 1 μl of DNA template.

The reaction mixture was initially incubated at 94˚C for 5 minutes followed by 30 cycles of amplification at 94˚C for 30 seconds for denaturation to 58˚C for 30 seconds for hybridization and 72˚C for 30 seconds for extension, followed by final extension at 72˚C for 5 minutes.

The size of the amplified DNA sequences was assessed after electrophoresis on a 2% agarose gel. The agarose gel was prepared by dissolving 2 g of agarose in 100 ml of 0.5X TBE buffer to which 5 μl of ethidium bromide (BET) was added. The gel was then migrated for 20 minutes, in a tank connected to 170 volts. Visualization was performed under UV light. The size of the bands obtained was estimated using a 1 kb scale; 221 bp for An.gambiae, 333 bp for An.coluzzii and 387 bp for An.arabiensis.

Detection of the Kdr mutation (L1014F) was performed according to the method developed by Huynh etal., (2007) [37]. The L1014F (Kdr-w) mutation was detected using IMP PCR primers. PCR was performed using Taq DNA polymerase. An 11.5 μl reaction mixture was prepared with the following composition: 3.95 μl of water; 2.5 μl buffer without MgCl2; 0.7 μl of 25 mM MgCl₂; 1.25 μl of 2 mM dNTP; 0.5 μl of each primer (IPCF, ALTRev, WT and West); 0.1 μl of Go Taq; and 1 μl of DNA template. The reaction mixture was initially incubated at 95˚C for 5 minutes, followed by 35 cycles of amplification (95˚C for 30 seconds, 59˚C for 30 seconds, and 72˚C for 30 seconds), and a final extension at 72˚C for 5 minutes.

The size of the amplified DNA sequences was assessed after electrophoresis on a 2% agarose gel. The agarose gel was prepared by dissolving 2 g of agarose in 100 ml of 0.5X TBE buffer to which 5 μl of ethidium bromide (BET) was added. Gel migration was performed for 20 min in a 170-volt connected vessel. The bands were visualized under UV light. The size of the bands obtained was estimated using a 1 kb scale; 156 bp for the resistant band, 214 bp for the susceptible band and 314 bp for the common band.

Data for the present study were entered into a Microsoft Office Excel 365 file and analyzed using R software (version 8.4.1). Mortality rates were calculated for each location and study site, but also for anopheline species and insecticide type. For pyrethroids, the 50% and 95% knockdown times with their confidence intervals were determined using the log-time probit model of the Dose Effect Function package on XLSTAT 2020 software. Mortality rates were compared using Pearson’s or Fisher’s Chi2 tests or non-parametric Kruskal-Wallis and Mann-Whitney tests for numbers less than 5. The significance level of the tests was set at 0.05 (p value < 0.05).

3.1.1. Knock-Down Effects

The KD50 and KD95 times for DDT of An.gambiae s.l. in 5 sites from 2016 to 2018 were not determined.

KD50 knockdown times ranged from 10 to 60 min and KD95 ranged from 15 to 60 min in the 3 locations (Tables 1-5), mainly for deltamethrin. In Brazzaville, the KD50 knockdown time was between 30 and 60 min for deltamethrin 0.05% (Table 2). For DDT, lambda-cyhalothrin and permethrin, the knock-down times were indeterminate because they exceeded 60 min. In Djoumouna, the KD50 and KD95 knockdown times were calculated only for deltamethrin, which were 25 min and 45.5 min respectively. For deltamethrin 0.5% (10× diagnostic dose) the KD50 was 10 and 30 min and the KD95 was 10 and 40 min.

The highest KD50 and KD95 knockdown times were recorded at the market garden site The bled, where they were 30 and 40 min respectively. At Kintele, these times were the lowest observed, being 10 min each. At Djoumouna, the KD50 and KD95 were 10 and 15 minutes respectively. For DDT, permethrin and lambda-cyhalothrin, the KD50 and KD95 knockdown times were not determined.

Regarding the tests with the synergist PBO, the KD50 and KD95 knockdown times for deltamethrin alone were 33.3 and 51 min respectively. For the PBO + deltamethrin test, the KD50 and KD95 knockdown times were 23.3 and 33.3 min, respectively (Table 4).

3.1.2. Mortality Rates

Figures 2-7 show mortality rates (%) of An.gambiae s.l. after 24 h exposure to insecticides.

Table 1. KD50 and KD95 times for deltamethrin with and without PBO in Makelekele in 2018.

Table 2. KD50 and KD95 times for DDT 4%, deltamethrin 0.05%, deltamethrin 0.5%, lambdacyhalothrin 0.05% and permethrin 0.75% for An.gambiaes.l. in the 5 sites from 2016 to 2018.

KD50: time required to knock out 50% of mosquitoes; KD95: time required to knock out 95% of mosquitoes; min: minute. Susceptibility testing conducted in Bacongo and Madibou in 2016 showed that, resistance is not homogeneous within a locality.

Table 3. Mortality for DDT 4%, deltamethrin 0.05%, deltamethrin 0.5%, lambdacyhalothrin 0.05% and permethrin 0.75% for An.gambiaes.l. in the 5 sites from 2016 to 2018.

Table 4. Frequencies of the kdr L1014F allele in survivors, deaths and controls of An.gambiae and An.coluzzii between 2016-2018.

N: number tested; SS: sensitive; RS: resistant heterozygous; RR: homozygous resistant; F [R]: frequency of the allele.

Table 5. Frequencies of the kdr L1014F allele from An.gambiae and An.coluzzii in the 8 sites between 2016-2018.

N: number sampled; SS: sensitive; RS: resistant heterozygous; RR: homozygous resistant; F [R]: frequency of the allele; P (HW): P. value provided by the Hardy-Weinberg test.

Figure 2. Mortality to DDT 4% 24 hours after exposure of An. gambiae s.l. in 4 sites in Brazzaville and in Djoumouna between 2016 and 2018.

Figure 3. Mortality to 0.05% deltamethrin 24 hours after exposure of An. gambiae s.l. in 6 sites in Brazzaville and Djoumouna between 2016 and 2018.

Figure 4. Mortality to 0.5% deltamethrin 24 hours after exposure of An. gambiae s.l. in 4 sites in Brazzaville, in Kintele and in Djoumouna in 2017 and 2018.

Figure 5. Mortality of An. gambiae s.l. with deltamethrin 0.05% with or without PBO in Makelekele in 2018.

Figure 6. Mortality to lambdacyhalothrin 0.05% 24 hours after exposure of An. gambiae s.l. at the Test garden and Djoumouna 2018.

Figure 7. Mortality to permethrin 0.75% 24 hours after exposure of An. gambiae s.l. in 4 sites in Brazzaville between 2017 and 2018.

Mortality rates for DDT were less than 50% and ranged from 48% to 98.6%. For deltamethrin 0.05%, the highest mortality rates were recorded in the Bacongo and Madibou districts in 2016.

For deltamethrin 0.5%, the highest mortality rates (100%) were observed in the The bled neighborhood in Kintele and in Djoumouna. The lowest mortality rates (84% and 85%) were observed in the districts of Test garden and Mayanga. For permethrin 0.75%, mortality rates ranged from 1% to 85.3%, with the lowest (1%) was recorded in the Test Garden market garden area and the highest (85.3%) in Madibou. For lambda-cyhalothrin 0.05%, the lowest mortality rate (29%) was recorded in Djoumouna and the highest (49%) in Test garden. Statistical tests showed that there was a relationship between sensitivity to these insecticides and the collection site (p < 0.001).

Regarding the tests with PBO, there was a recovery of sensitivity to deltamethrin after the use of PBO. Statistical tests indicated a relationship between sensitivity to deltamethrin and the use of PBO at p = 0.0054.

Of the females tested for insecticide susceptibility, 244 females were sampled for species and kdr mutation identification. Two species were identified at the study sites, 223 An.gambiae and 21 An.coluzzii.

An.gambiae was the main species identified in the three sites, with frequencies of 94.8%, 80% and 67.9% in Brazzaville, Kintele and Djoumouna, respectively.

Of the 244 tested females tested, 239 carried the kdr L1014F mutation. Among the 223 An.gambiae females, 218 carried the mutation and 5 were susceptible. In addition, all 21 An.coluzzii carried the kdr L1014F mutation. The frequency of the kdr mutation varied between 0.5 and 1 in the three sites. In the Brazzaville sites, the frequency ranged from 0.6 to 1, in Kintele from 0.5 to 0.6 and in Djoumouna from 0.6 to 0.7 (Table 4).

Comparison of kdr frequencies between surviving and dead An.gambiae carrying the kdr mutation showed no difference between homozygous and heterozygous individuals. However, for An.coluzzii, there was a statistically significant difference between surviving and dead individuals carrying the kdr mutation in the homozygous and heterozygous states (Table 5).