Date Published: April 19, 2019
Publisher: Public Library of Science
Author(s): Joseph Chabi, Arjen Van’t Hof, Louis K. N’dri, Alex Datsomor, Dora Okyere, Harun Njoroge, Dimitra Pipini, Melinda P. Hadi, Dziedzom K. de Souza, Takashi Suzuki, Samuel K. Dadzie, Helen P. Jamet, Ruslan Kalendar.
The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72°C for An. arabiensis, 75°C for An. gambiae and 86°C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.
The Anopheles gambiae sensu lato (An. gambiae s.l.) complex comprises at least seven mosquito species originally defined by polytene chromosome analysis [1, 2]. All the An. gambiae s.l. species are currently identified by PCR-diagnostic assays based on specific DNA nucleotide differences in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) [3–6]. Detailed analyses of the IGS region of rDNA further revealed nucleotide substitutions that differentiated between the two forms within the Anopheles gambiae s.s previously designated as S and M molecular forms , and were recently named An. gambiae and An. coluzzii . These two species can be identified by PCR and gel electrophoresis showing the presence or absence of a diagnostic Short Interspersed Nuclear Element (SINE) on the X-chromosome. .
Rapid and reliable identification of species and sub-species of malaria vector populations is an important part of malaria vector control programmes. PCR-RFLP and SINE PCR methods have been developed for this purpose and have both shown to successfully identify An. arabiensis, An. gambiae and An. coluzzii out of the complex of eight species [4, 10, 11]. However, both methods involve at least two PCR steps with gel staining that require precision and time to identify the species. In addition, inconsistent identification of An. gambiae, An. coluzzii and their hybrids has been reported by either PCR using form-specific primers or PCR-RFLP genotyping carried out in different laboratories . Mis-identification of An. gambiae vs An. arabiensis based on gel-bands is even more likely since both the SINE200 and the 28S IGS techniques produce similar-sized amplicon sizes (223 vs 249 bp and 315 bp vs 390 bp respectively) which only separate when gels are run sufficiently long (the An. gambiae /An. coluzzii distinctive HhaI digest  is usually not performed in Eastern Africa). The 36% error rate in western Kenyan samples clearly demonstrates the risk of mis-interpreting bands on gels, and consequently the value of the melt-curve approach described here. From Scott et al. 1993 to date, SINE PCR has shown greater reliability among all the protocols allowing the differentiation of An. gambiae and An. coluzzii. Moreover, each species identification protocol allows partial identification of the whole An. gambiae complex (Table 2). In addition to the benefits outweighed given the fastest and more reliable results, the current SYBR green melt-curve technique allows the full identification of the three main malaria vectors, An. gambiae, An. coluzzii and An. arabiensis species simultaneously.
The SYBR green real time PCR techniques showed an additional option for the characterization of both sibling species and An. arabiensis while all the other real time PCR protocol of species differentiation could not allow the characterization of the sub-species such as An. gambiae and An. coluzzii. The assay designed in this study is a new tool to help researchers and particularly malaria vector control entities to identify clearly the subspecies within the An. gambiae s.l. complex in the context where each of the species has unique behaviour and impact on malaria.