Comparative analyses reveal discrepancies among results of commonly used methods for Anopheles gambiaemolecular form identification

Abstract

Background: Anopheles gambiae M and S molecular forms, the major malaria vectors in the Afro-tropical region, are ongoing a process of ecological diversification and adaptive lineage splitting, which is affecting malaria transmission and vector control strategies in West Africa. These two incipient species are defined on the basis of single nucleotide differences in the IGS and ITS regions of multicopy rDNA located on the X-chromosome. A number of PCR and PCR-RFLP approaches based on form-specific SNPs in the IGS region are used for M and S identification. Moreover, a PCR-method to detect the M-specific insertion of a short interspersed transposable element (SINE200) has recently been introduced as an alternative identification approach. However, a large-scale comparative analysis of four widely used PCR or PCR-RFLP genotyping methods for M and S identification was never carried out to evaluate whether they could be used interchangeably, as commonly assumed.

Results: The genotyping of more than 400 A. gambiae specimens from nine African countries, and the sequencing of the IGS-amplicon of 115 of them, highlighted discrepancies among results obtained by the different approaches due to different kinds of biases, which may result in an overestimation of MS putative hybrids, as follows: i) incorrect match of M and S specific primers used in the allele specific-PCR approach; ii) presence of polymorphisms in the recognition sequence of restriction enzymes used in the PCR-RFLP approaches; iii) incomplete cleavage during the restriction reactions; iv) presence of different copy numbers of M and S-specific IGS-arrays in single individuals in areas of secondary contact between the two forms.

Conclusions: The results reveal that the PCR and PCR-RFLP approaches most commonly utilized to identify A. gambiae M and S forms are not fully interchangeable as usually assumed, and highlight limits of the actual definition of the two molecular forms, which might not fully correspond to the two A. gambiae incipient species in their entire geographical range. These limits are discussed and operational suggestions on the choice of the most convenient method for large-scale M- and S-form identification are provided, also taking into consideration technical aspects related to the epidemiological characteristics of different study areas.

Figure 1

Sequence and alignment of M and S Anopheles gambiae molecular form specific diagnostic primers. a) Primer sequences, restriction enzymes and M and S Anopheles gambiae molecular form specific products from PCR-RFLP⁵⁸¹[16] and PCR-RFLP⁶⁹⁰ [16,17]; b) primer sequences and molecular form-specific products as in AS-PCR [13] and IMP-PCR [14]; c) location of primer pairs and restriction sites utilized in AS-PCR and PCR-RFLPs are reported on the 28S (from 41 to 400) IGS sequence (from 401 to 1321) (AF470093-AF470116; [29]).

Figure 2

Location of collection sites. Black and white pies indicate the exclusive presence of either M or S Anopheles gambiae molecular forms, respectively. Black/white pies indicate sites where both molecular forms were sampled.

Figure 3

Box-plots of CNP scores of IGS⁵⁸¹ SNP (a) and IGS⁶⁹⁰ SNP (b) in Anopheles gambiae specimens. Specimens are classified based on results from IGS⁵⁸¹/IGS⁶⁹⁰ PCR-RFLPs. The underlined SS/SS (N = 9) and MM/MM (N = 15) groups correspond to S-form and M-form specimens from Burkina Faso and Angola, while the not-underlined groups correspond to specimens from The Gambia and Guinea Bissau (SS-SS: N = 15; MS-SS: N = 14; MS-MS: N = 36; MM-MS: N = 11; MM-MM: N = 15).