Distribution of knock-down resistance mutations in Anopheles gambiae molecular forms in west and west-central Africa

Background Knock-down resistance (kdr) to DDT and pyrethroids in the major Afrotropical vector species, Anopheles gambiae sensu stricto, is associated with two alternative point mutations at amino acid position 1014 of the voltage-gated sodium channel gene, resulting in either a leucine-phenylalanine (L1014F), or a leucine-serine (L1014S) substitution. In An. gambiae S-form populations, the former mutation appears to be widespread in west Africa and has been recently reported from Uganda, while the latter, originally recorded in Kenya, has been recently found in Gabon, Cameroon and Equatorial Guinea. In M-form populations surveyed to date, only the L1014F mutation has been found, although less widespread and at lower frequencies than in sympatric S-form populations. Methods Anopheles gambiae M- and S-form specimens from 19 sites from 11 west and west-central African countries were identified to molecular form and genotyped at the kdr locus either by Hot Oligonucleotide Ligation Assay (HOLA) or allele-specific PCR (AS-PCR). Results The kdr genotype was determined for about 1,000 An. gambiae specimens. The L1014F allele was found at frequencies ranging from 6% to 100% in all S-form samples (N = 628), with the exception of two samples from Angola, where it was absent, and coexisted with the L1014S allele in samples from Cameroon, Gabon and north-western Angola. The L1014F allele was present in M-form samples (N = 354) from Benin, Nigeria, and Cameroon, where both M- and S-forms were sympatric. Conclusion The results represent the most comprehensive effort to analyse the overall distribution of the L1014F and L1014S mutations in An. gambiae molecular forms, and will serve as baseline data for resistance monitoring. The overall picture shows that the emergence and spread of kdr alleles in An. gambiae is a dynamic process and that there is marked intra- and inter-form heterogeneity in resistance allele frequencies. Further studies are needed to determine: i) the importance of selection pressure exerted by both agricultural and public health use of pyrethroid insecticides, ii) the phenotypic effects, particularly when the two mutations co-occur; and iii) the epidemiological importance of kdr for both pyrethroid- and DDT-based malaria control operations, particularly if/when the two insecticides are to be used in concert.

baseline data for resistance monitoring. The overall picture shows that the emergence and spread of kdr alleles in An. gambiae is a dynamic process and that there is marked intra-and inter-form heterogeneity in resistance allele frequencies. Further studies are needed to determine: i) the importance of selection pressure exerted by both agricultural and public health use of pyrethroid insecticides, ii) the phenotypic effects, particularly when the two mutations co-occur; and iii) the epidemiological importance of kdr for both pyrethroid-and DDT-based malaria control operations, particularly if/when the two insecticides are to be used in concert.

Background
DDT and pyrethroids share the same mode of action on the insect nervous system, targeting the neuronal voltagegated sodium ion channels. Molecular characterizations have revealed that various mutations in the S1-S6 transmembrane segments of domain II of the sodium ion channel gene give rise to resistance to these insecticides in a number of insect species [1][2][3]. In the major Afrotropical malaria vector, Anopheles gambiae sensu stricto (hereafter named simply An. gambiae), two point mutations at amino acid position 1014 of the voltage-gated sodium channel gene have been described, resulting in either a leucine-phenylalanine (L1014F) [2], or a leucine-serine (L1014S) [4] substitution. Both mutations have been shown to be linked with DDT and pyrethroid resistance phenotypes in field An. gambiae populations [2,[4][5][6][7][8][9]. A relatively small number of studies have been conducted to determine the epidemiological impact of kdr target site insensitivity on the efficacy of pyrethroid and DDT based vector control [9][10][11][12][13]. Although a consensus has not been reached yet, some groups suggest that it may be associated with decreased efficacy of ITNs and pyrethroid based IRS [10,11]. In the latter case, programme staff were able to utilize carbamate insecticides, which have a different mode of action, and thereby overcome the resistance problem. Therefore, studies for determining the epidemiological impact and the extent of distribution of kdr mutations in An. gambiae populations are considered relevant for the design of insecticide-based control programmes and for informing the debate about the re-introduction of DDT into the vector control. This manuscript details the first effort to analyse the distribution of the L1014F and L1014S mutations in An. gambiae populations from Sub-Saharan Africa west of the Rift Valley. It is envisaged that the data will serve as baseline for both researchers and, more importantly, for control staff who manage the deployment of insecticides, the efficacy of which could be compromised by the presence of kdr alleles.

Sample collections
Anopheles gambiae samples were collected from 19 sites in 11 west and west-central African countries, between 1998 and 2006. For Kedougou (Senegal), two samples were analysed, collected in 2001 and 2005, respectively. Table  1 shows the sampling sites (listed from west to east and north to south), their geographical coordinates, dates and methods of collection, the ecology of the sampling sites and the total number of M and S molecular forms analysed for kdr genotype.

Molecular analyses
DNA was extracted by a variety of standard methods (e.g. phenol/chloroform, DNAzol kit by Molecular Research Center Inc. and Easy-DNA Invitrogen kit) and specimens were identified to molecular form by PCR-RFLP [34]. Samples were genotyped at the kdr locus either using the Hot Oligonucleotide Ligation Assay (HOLA) developed by Lynd et al [35] or by the Allele-Specific PCR (AS-PCR) [2,4].
A sub-sample of the HOLA-analysed sample had been previously genotyped by PCR for the L1014F allele [2] and the results of the two approaches were compared. When the results of the two approaches were not consistent, sequencing of a fragment of the domain II of the voltagegated sodium channel gene containing the kdr locus was performed using standard primers Agd1 and Agd2 [2]. The PCR products were cleaned using a commercial kit (SureClean PCR Clean Up, BIOLINE) and bi-directionally sequenced.

Statistical analyses
The maximum predicted frequency (y) for an allele to be present in a sample of a given size (x) but undetected in a sample was obtained from the upper 95% confidence limit of a binomial distribution, given by y = 1-0.05 1/x , following the example of Post and Millest [36].

Results
Eight-hundred and two An. gambiae were kdr genotyped using the HOLA approach and 193 specimens from Gabon and São Tomé and Príncipe Islands were analysed by AS-PCR. The kdr genotype was successfully determined for more that 98% of the specimens. Table 2

Discussion
This study shows that kdr mutations are widespread in An. Merging the above results with all the data available to date on the simultaneous occurrence of the two kdr mutations in S-form [23-28], three main geographic areas can be distinguished based on kdr allele distribution and frequencies ( Figure 1a): i) a western area, from Senegal to Nigeria, in which only the L1014F allele is present with  The data obtained do not allow to determine whether the geographical differences in kdr allele frequencies among S-form populations is a result of non-uniform selective pressures due to variation in insecticide use, as recently hypothesized by Pinto et al [28]. However, it is interesting to note that the data from Angola suggest an impact of human activities on kdr distribution. All samples from Angola were characterized by the wild-type 1014L/1014L genotype, with the only exception of an S-form sample collected in Cabinda, less than 100 km apart from the sampling site of Kikudo [37]. Although it is not possible to completely rule out the presence of kdr alleles in the relatively small Kikudo sample (N = 46), as there is a 95% probability that an allele with a frequency of 3% or below would not be detected in the analysed sample (equivalent to mis-scoring one heterozygote genotype), the difference in the kdr allele frequencies between these sites is significant (chi-square = 67.9, df = 2, P < 0.001). This likely reflects the fact that the Kikudo area was involved in the civil war that ravaged Angola for the last 30 years, whilst Cabinda is located in an Angolan enclave within the Democratic Republic of Congo, which has been only marginally involved in the war, thus allowing for a larger scale use of pyrethroids against agricultural pests and/or malaria vectors [38].
This is the first regional survey of M-form specimens with respect to both resistance mutations. The results show the absence of the L1014S allele in all samples analysed and the presence of the L1014F allele in samples from a relatively restricted geographic region in the central part of Gulf of Guinea (i.e. Benin, Nigeria and Cameroon) (Figure 1b). Interestingly, the first report of the presence of L1014F in M-form was from Benin in 1998 [39], where it was shown to have introgressed from sympatric S-form populations [21] and where it has recently reached frequencies around 80% [15]. Inter alia, it can be hypothesized that the mutation is spreading from this region eastwards (i.e. Nigeria and later in Cameroon, where the L1014F frequency is 19% and 6%, respectively) and westwards in Burkina Faso [7]. Moreover, the hypothesis of a further "possible subdivision within the M molecular form" [40] may further complicate the picture.
Distribution of 1014L, L1014F and L1014S alleles in Anopheles gambiae S-form (1a) and M-form (1b) populations Moreover, the different distribution of the two kdr mutations between sympatric M and S populations could also reflect different ecological/behavioural traits between M and S-forms, that might promote different exposure to insecticide selective pressures. For example, the M-form may be more adapted to urbanized, man-influenced ecological settings, whereas the S-form tends to prevail in rural settings [29,41], where a use of insecticides for agricultural purposes is expected to be greater.

Conclusion
Although the results obtained refer to relatively few An. gambiae samples from a very large geographical range, they represent a first effort to analyse the overall distribution of the L1014F and L1014S mutations in sub-Saharan Africa, particularly west of the Rift Valley, where both molecular forms of this species co-occur in most of their range of distribution.
The results on S-form populations, combined with previously published data (Figure 1a), show a non-uniform distribution of kdr mutations throughout the continent: the L1014F is the only allele present west of 10°W latitude, while the two mutations co-exist in a wide geographic area in western-central Africa (ranging approximately from 6°N to 6°S).
The L1014F allele is present also in the M-form in an area comprised approximately between 5°W and 15°E, showing higher frequencies (>75%) in Benin and in the island of Bioko [15,33], and lower frequencies (<20%) in Burkina Faso, Nigeria and Cameroon. The absence of the L1014S allele in the M-form samples analysed will serve as baseline data for further studies on the possible occurrence and dispersal of this mutation in this form ( Figure  1b).
The overall picture strongly suggests that the origin and spread of kdr alleles in An. gambiae is an ongoing process. There is evidence that both kdr alleles have originated in the S-form through multiple mutation events [28], whereas the L1014F allele may have arisen in the M-form either through introgression with the S-form or through independent mutation events [19,21,42]. Insect control activities, such as the large scale use of pyretroid insecticides for agricultural purposes, and possibly for domestic protection, may contribute largely to this process, as suggested by Pinto et al [28], who hypothesized a possible link between the recent expansion of cotton production in West Africa and an increased selective pressure due to pyrethroid insecticide use. This highlights the need to carefully monitor the evolution and spread of kdr target site insensitivity with a sensitive and precise method for the detection of both alleles and through more specifically designed spatial and temporal transects. Such surveys should be accompanied with the molecular analysis of non-target site resistance mechanisms for An. gambiae. This would facilitate an analysis of the relative contribution of kdr mutations into the resistance phenotype. Given the large geographic area where the two mutations co-occur in the S-form, it is also extremely important to undertake insecticide resistance surveys to correlate kdr genotypes to DDT and pyrethroid resistance phenotypes. This would allow to ascertain the phenotypic effect of both kdr mutations, particularly when co-occurring in the same individual, and to evaluate their possible effect on the efficacy of ITNs and pyrethroid based IRS, thus allowing control programme managers to make informed choices about the chemicals they use for vector control.