Multiple Insecticide Resistance Target Sites in Adult Field Strains of An. Gambiae s.l. From Southeast Senegal.

Background: Malaria prevention strategies are based on the use of long-lasting insecticide-treated mosquito nets (LLINs), indoor residual spraying of insecticides (IRS) and seasonal malaria chemoprevention (SMC). The combination of these strategies with artemisinin-based combination therapy (ACTs) has led to a signicant reduction in malaria cases. However, malaria remains a major public health issue in most sub-Saharan African countries. Indeed, the resistance of vectors to most WHO-approved insecticides could jeopardize vector-control strategies. This study examines insecticide resistance and associated genetic mutations among malaria vectors in southeast Senegal. Methods: The study was conducted in October and November 2014 in two sites in southeast Senegal. An. gambiae s.l. populations were sampled from Kedougou (Kedougou district) and Wassadou-Badi (Tambacounda district) and were evaluated for insecticide resistance according to WHO susceptibility tests. Specimens were 3 to 5-day-old adults raised from collected larvae. Eleven insecticides belonging to the four known classes of insecticides were assessed. Mosquito species were identied and mutations associated with insecticide resistance (ace-1, rdl (A296S or A296G), Vgsc-1014F and Vgsc-1014S) were determined. Results: A total of 3,742 An. gambiae s.l. were exposed to insecticides (2,439 from Kedougou and 1,303 from Wassadou-Badi). In both sites, mosquitoes showed high levels of resistance to all the ve pyrethroids tested (mortality rates ranged from 42.8 to 81.4% in Kedougou and 52.4 to 86.4% in Wassadou-Badi) as well as to dieldrin (67.8 and 83%) and DDT (12.7 and 55%). The mosquitoes were susceptible to pirimiphos-methyl (mortality rate 100%) and malathion (mortality rates 100% and 99% in Kedougou and Wassadou-Badi respectively). An. gambiae s.l. populations from Kedougou were also resistant to bendiocarb. genotyped An. gambiae s.s. (71.6%) was the predominant species, followed by An. arabiensis (21.7%), An. coluzzii (6.3%) and hybrids (An. gambiae s.s./An. coluzzii; 0.4%). The Vgsc-1014F kdr mutation was widely distributed and is predominant in An. gambiae s.s. and An. coluzzii in comparison to An. arabiensis. Vgsc-1014S was present in An. gambiae s.l. populations in Wassadou but not in Kedougou. The ace-1 and rdl mutations were more frequent in An. gambiae s.s. compared to An. arabiensis but were not detected in all populations of An. coluzzii. Conclusion: The present study demonstrates the resistance of malaria vectors to pyrethroids and organo chlorines in southeast Senegal as well as the presence of genetic mutations associated with this resistance in An. gambiae s.l. There was no Vgsc-1014S mutation in An. gambiae s.s. population in Kedougou. These ndings are key for monitoring and managing the resistance of vectors to insecticides in this region. shmeal (Tetramin Baby®). Pupae collected daily and introduced into rearing cages. At emergence, mosquito adults fed using absorbent cotton soaked with 10% sucrose solution. from larvae. Eleven insecticides four insecticide ve pyrethroids (0.05% deltamethrin, 0.75% permethrin, 0.05% lambda cyhalothrin, 0.1% alpha cypermethrin and 0.15% cyuthrin), two organo chlorines (4% DDT and 4% dieldrin), three organophosphates (1% fenitrothion, 5% malathion and 1% pirimiphos-methyl) and one carbamate (0.1% bendiocarb).For the pyrethroids and DDT, the number of knock down individuals was recorded at 10, 15, 20, 30, 40, 50 and 60 minutes during the exposure period. Mortality rates were determined 24 hours post-exposure. The mortality rates in the tested groups were corrected when needed, using Abbot’s formula [20] to validate tests results according to mortality rate in controls. tested specimens were identied morphologically under a binocular microscope using a conventional key [21] and then individually stored in Eppendorf tubes containing silica-gel. All surviving specimens and ten for bendiocarb, twenty for organo chlorines, thirty for organophosphates and fty for pyrethroids randomly-selected dead specimens were individually stored for laboratory analysis. phenotypic in An. and An. coluzzii. the Vgsc-1014F in An. coluzzii. nding explained by introgression from An. gambiae s.s.to An. coluzzii [36, show an absence of the Vgsc-1014S mutation in An. coluzzii. to in [40], [41] [42].The (An. s.s. in mutation


Study area
The study was conducted in October and November 2014 in two sites of southeast Senegal: Kedougou (12 ° 33'11.3 "N and 12 ° 10'09.5" W in Kedougou district) and Wassadou-Badi (13°22′22.3″N and 13°22′53.5″W in Tambacounda district) (Fig. 1).The area is bordered by the Republics of Mali and Guinea. The climate is tropical with a sudano-Guinean climate. The rainy season generally lasts from May to October [14]. The average precipitation is between 1, 200 mm and 1,300 mm per year. Daily average temperatures vary from 21-25 ° C to 33-42 ° C. The temperature range is between 4 to 6 ° C. Agriculture is the main economic activity mainly sorghum, maize, fonio, rice and cotton. The area of Kedougou is also a gold-mining zone, and has a signi cant potential for mineral resources. With a malaria incidence greater than 25 per 1000 [17], the study area remains the most holo endemic area in Senegal. In 2014, 265,624 clinical cases were recorded; including 12,636 severe cases [18]. Malaria transmission is seasonal, and occurs during the rainy season and the beginning of the dry season. An. gambiae s.s., An. coluzzii and An. arabiensis are responsible for most malaria transmission, but in some speci c settings, An. funestus and An. nili are involved [15,16].

Anopheles immature stages collection and mosquito rearing
Larval collections were carried out in Kedougou, Wassadou and Badi. Wassadou and Badi belong to the same area and are just 1.5 km apart. The larval sites for An. gambiae s.l. consisted of temporary water collections, footprints and hollows associated with human activities. During the study period, immature stages were collected from positive larval sites located in or around villages. All larval collections from Kedougou were pooled to form a sample and those of Wassadou and Badi a sample. After collection, immature stages were transferred to a local insectary for rearing. Anopheles larvae were fed with shmeal (Tetramin Baby®). Pupae were collected daily and introduced into rearing cages. At emergence, mosquito adults were fed using absorbent cotton soaked with 10% sucrose solution.
WHO bioassay tests and morphological identi cation WHO susceptibility tests were performed according to the standardized protocol [19] with adults 3 to 5 days post emergence from eld collected larvae. Eleven insecticides belonging to four insecticide classes were tested: ve pyrethroids (0.05% deltamethrin, 0.75% permethrin, 0.05% lambda cyhalothrin, 0.1% alpha cypermethrin and 0.15% cy uthrin), two organo chlorines (4% DDT and 4% dieldrin), three organophosphates (1% fenitrothion, 5% malathion and 1% pirimiphos-methyl) and one carbamate (0.1% bendiocarb).For the pyrethroids and DDT, the number of knock down individuals was recorded at 10,15,20,30,40,50 and 60 minutes during the exposure period. Mortality rates were determined 24 hours post-exposure. The mortality rates in the tested groups were corrected when needed, using Abbot's formula [20] to validate tests results according to mortality rate in controls.
Finally, tested specimens were identi ed morphologically under a binocular microscope using a conventional key [21] and then individually stored in Eppendorf tubes containing silica-gel. All surviving specimens and ten for bendiocarb, twenty for organo chlorines, thirty for organophosphates and fty for pyrethroids randomly-selected dead specimens were individually stored for laboratory analysis.
DNA extraction, molecular identi cation of species and detection of kdr, ace-1 and rdl.
Genomic DNA extraction was carried out by the 2% CTAB method (Cetyl trimethyl ammonium bromide). Each sample was grounded in an Eppendorf tube containing 200 µl of CTAB and incubated at 65 ° C for one hour. Then 200 µl of chloroform was added and mixed by inversion. The mixture was centrifuged at 12,000 rpm for 5 min, after which the supernatant containing DNA was recovered in a new Eppendorf tube.DNA was then precipitated with isopropanol and the mixture was then centrifuged at 12,000 rpm for 15 min and washed with 70° ethanol after a centrifugation of 12.000 rpm for 5 min and then brought to speed-vac for drying. The DNA, thus obtained was suspended in molecular biology grade water: DNA/RNA free (Invitrogen, 10977 035). One tenth of dilution was carried out before PCR (identi cation of species of the An. gambiae complex and detection of target site mutations). Species were identi ed using IMP-PCR (intentional mismatch primer-PCR) as described by Wilkins et al. [22]. Kdr mutations (Vgsc-1014F and Vgsc-1014S), G119S (ace-1 R ) and rdl-296S (An. arabiensis) and rdl-296G (An. gambiae s.s. and An. coluzzii) were determined using the protocols described by Huynh et al. [23] and by Weill et al. [24] and Du et al [12] respectively.

Data entry and statistical analysis
Data was recorded in a Microsoft Excel 2010 spreadsheet. Homogeneity tests of percentages and averages were performed using the standard chi-square tests with a 5% signi cance level threshold. Susceptibility tests were validated by considering mortality rates of control mosquitoes. If the control mortality was less than 5%, no correction of test results was necessary whereas mortality of ≥ 5% required Abbott's correction [19].KDT 50 and KDT 95 times with 95% con dence intervals were determined using a log-probit regression model. The mortality rates, the genotypes and allelic frequencies were estimated for each studied population. All statistical analyses and graphs were made using R software version 3.0.3 [43].

Susceptibility tests
A total of 3,742 specimens of An. gambiae complex (between 109 to 240 per insecticide per site) were exposed to the WHO recommended diagnostic doses (2,439 from Kedougou and 1,303 fromWassadou-Badi). In both sites, a high number of mosquitoes were resistant to all ve tested pyrethroids (mortality ranged from 42.8 to 86.4%) as well as to the organo chlorines (mortality rates ranged from 67.8 and 83% for dieldrin and 12.8 and 55.8% for DDT in Kedougou and Wassadou-Badi respectively) ( Fig. 2 and Table 1).In the group of organophosphates, the populations of An. gambiae s.l. tested in both areas were susceptible to 5% malathion and 1% pirimiphos-methyl. Fenitrothion resistance (89% mortality rate, 95% CI 85-95) was detected in Kedougou, where An. gambiae s.l. populations were also resistant to bendiocarb 0.1% (Fig. 2).  The wild-type allele was the most frequent allele for the ace-1 R for all species of the An. gambiae complex in both sites. The frequency of the ace-1 R (G119S) mutation was low in both sites and heterozygotes genotypes (GS) were predominant for carriers of an 119S allele.
In Wassadou-Badi, a relatively higher allelic frequency was noted in An. gambiae s.s., the only species in which all SS homozygotes were found ( Table 2). As with ace-1 R , the predominant allele for rdl gene was the wild type allele. The mean allelic frequencies of A296S or A296G were signi cantly different among species of the An. gambiae complex in Kedougou (Fisher's exact test: P = 0.0147), but not in Wassadou-Badi (Fisher's exact test: P = 0.1236). However, only An. gambiae s.s. population has homozygous (GG) for A296G rdl allele ( Table 2).
Allelic frequencies at the Vgsc-1014F, Vgsc-1014S, ace-1 R (G119S) and rdl-A296S or rdl-A296G locus according to the phenotype after insecticide exposure Table 3 shows the allelic frequencies of the Vgsc-1014F, Vgsc-1014S, G119S and rdl-A296G or rdl-A296S mutations in the selected specimens that survived or died after exposure to insecticides.  In An. gambiae s.s., the frequencies of resistant allele in surviving versus dead specimens were comparable for the 1014F allele (Fisher's exact test: P ≥ 0.058), and signi cantly different for the ace-l R (G119S) allele (Fisher's exact test: P ≤ 0.001) in both sites (Table 3).

Discussion
This study aimed to update data relating to insecticide susceptibility and to determine the frequencies of mutations of kdr (Vgsc-1014F and Vgsc-1014S), ace-1 R and rdl alleles associated with the resistance of An. gambiae s.l. populations to insecticides in southeastern Senegal.
The results of WHO susceptibility tests showed vector resistance to pyrethroids organo chlorines (DDT and dieldrin) and carbamates insecticides that are recommended WHOPES for vector control. These insecticides are the only ones currently approved for LLIN treatment [25,26], and are offered by nongovernmental organizations such as the United States President's Malaria Initiative (PMI) and Senegal River Basin Development Organization (OMVS).
The use of LLINs over several years could have led to the increase of resistance genes in vectors of An. gambiae species complex, through selection pressure [27,35]. The resistance of An. gambiae s.l. to pyrethroids has been shown to be strongly associated with their excessive use in agriculture especially in cotton growing areas [28].
Moreover, An. gambiae s.l. populations in the area were also resistant to organo chlorines (DDT and dieldrin). Since the rst malaria eradication attempt, DDT and dieldrin resistance phenotypes have been reported in many African countries by Hamon [29]. Despite several decades of non-use, DDT may persist in the environment due to lack of microbial degradation system [49].
Previous studies have reported resistance only to DDT and pyrethroids in southeastern and central Senegal where LLIN use is high, [30,31]. However, unlike previous studies conducted in Senegal, this study shows that vectors are resistant to almost all pyrethroids and bendiocarb.
Bioassays likewise showed resistance to bendiocarb in Kedougou. This resistance could come from selection pressure in larval from insecticide residues (bendiocarb) used on cotton crops by the cotton industry [47].This phenotypic resistance to bendiocarb should be closely monitored as there is often crossresistance to carbamates and organophosphates.
The search for mutations involved in the phenotypic resistance of An. gambiae s.l. population to insecticides showed the presence of Vgsc-1014F, Vgsc-1014S, ace-1 (G119S) and rdl-A296S or rdl-A296G mutations. The Vgsc-1014S mutation was not found in An. gambiae s.l. from Kedougou, where the Vgsc-1014F was at 0.99 in An. gambiae s.s.
Although not yet xed in Wassadou-Badi, the allelic frequency of Vgsc-1014F mutation was more than 0.50.
The frequency of the Vgsc-1014F mutation was higher both in the surviving and dead phenotypes in the An. gambiae s.s. populations from Kedougou. The frequency of the Vgsc-1014S mutation in An. arabiensis populations from Wassadou-Badi was higher in the surviving than the dead specimens whereas no correlations were detected between the Vgsc-1014F mutation and the resistance phenotype in An. gambiae s.s. and An. coluzzii species. It is therefore likely that mechanisms other than Vgsc-1014F mutation are involved in the insecticide-resistance of these species. This hypothesis should be investigated in the future. These results are in line with those of Thiaw et al. [31] and Ahoua et al. [32], who found no correlation between the kdr mutation and the phenotypic alive or dead phenotypic respectively in An. arabiensis and An. coluzzii. Only the Vgsc-1014F mutation was noted in An. coluzzii. This nding could be explained by introgression from An. gambiae s.s.to An. coluzzii [36,37]. Furthermore, our results show an absence of the Vgsc-1014S mutation in An. coluzzii. This nding is similar to results obtained in Benin [40], but not those obtained in Cameroon [41] and in Equatorial Guinea Republic [42].The occurrence of the Vgsc-1014F mutation was detected in 2 hybrids (An. gambiae s.s./An. coluzzii), of which two were homozygote resistant genotype (FF). This is the rst report of this mutation in hybrids from An. gambiae s.s. and An. coluzzii in Senegal. Other mutations could be involved in resistance of An. gambiae s.l. to insecticides, including the Vgsc-1575Y mutation [48] that was not investigated in this study.
With a signi cantly higher frequency in surviving specimens after exposure, the study shows that the ace-1 R mutation was implicated in phenotypic resistance of An. gambiae s.s. to bendiocarb. The involvement of the ace-1 R mutation in the phenotypic resistance to bendiocarb has been reported in An. gambiae s.s. populations from Côte Ivoire [32] and Ghana [33]. However, it was not present in surviving An. arabienesis.
The presence of heterozygotes in surviving specimens may explain the resistance of An. gambiae s.l. population to carbamates (bendiocarb) from Kedougou and Wassadou-Badi and organophosphates (fenitrothion) from Kedougou area.
Associated with rdl mutation (rdl-A246S or rdl-A296G), the phenotypic resistance to dieldrin was found in An. gambiae s.l. population in both localities. A similar result was obtained in Benin [39]. The allelic frequencies obtained in our study are quite similar to those described by Corbel [39]. The phenotypic resistance to dieldrin could be explained by the long use of dieldrinin the past of other insecticides belonging to different families (such as pronil or lindane) with the same mode of action as dieldrin on one hand and by the presence of rdl-A296G mutation, which is associated with a 2La chromosomal polymorphic on the other hand [37]. This is a very stable polymorphic inversion that limits crossover and would help preserve this mutation in a given population. The occurrence of multiple-resistance locus in An. gambiae s.s., the main malaria vector in the study area, is indicative of the genes involved in resistance to the insecticides used in this area.

Conclusion
The study demonstrates phenotypic resistance in An. gambiae s.l. population to DDT, pyrethroids, benbiocarb and fenitrothion in southeastern Senegal. The relatively higher frequency in specimens surviving insecticide exposure demonstrates the role of target site modi cations, including Vgsc-1014F and Vgsc-