Detection of clade types (clades I and II) within Anopheles funestus sensu stricto by the hydrolysis probe analysis (Taqman assay)

Background Recent studies presented two clades (clades I and II) within the major malaria vector, Anopheles funestus s.s. on the mitochondrial DNA. We describe a hydrolysis probe analysis (Taqman assay) method for the rapid identification of these two clades. Findings A total of 53 An. funestus s.s. from Malawi and Mozambique were tested for detection of clade types using the hydrolysis probe analysis. Results were compared to DNA sequence analysis to verify the accuracy of the probes Taqman assay for this vector species. Analysis using the hydrolysis probe revealed that there were 21 individuals from Malawi and 13 individuals from Mozambique for clade I, and 19 individuals from Mozambique for clade II. The results were consistent with the results of DNA sequences. A field sample from northern Zambia revealed the presence of both clade types. Conclusion A diagnostic method using the hydrolysis probe analysis was developed to identify clade types within An. funestus s.s. This assay will be useful for screening clade types of field-collected An. funestus specimens accurately and efficiently in malaria vector research and control studies.

Keywords: Anopheles funestus, Clades, Malawi, Mozambique Findings Background One of the major African malaria vectors, Anopheles funestus Giles belongs to a group of at least eleven species, all of which are morphologically similar at the adult stage [1][2][3][4][5]. It is widespread in sub-Saharan Africa including Madagascar [1,6] and is the most anthropophilic and endophilic species of the group [1]. Michel et al. [7] reported that the population structure of An. funestus s.s. based on NADH Dehydrogenase subunit 5 (ND5) data revealed two cryptic subdivisions, clade I widespread throughout Africa and clade II known only from Mozambique and Madagascar. Choi et al. [8] also reported that An. funestus s.s. from Mozambique has two clades based on the DNA sequence analysis of ND5 and Cytochrome Oxidase I (COI).
The hydrolysis probe analysis combines PCR amplification reaction and visualisation into a single step. This method uses oligonucleotide probes dual-labelling with a fluorescent reporter dye and a quencher molecule. The probe-specific product is amplified and this leads to cleavage of the probe, generating an increase in reporter fluorescence. The reporter dye is released away from the quencher and cleavage of allele-specific probes can be observed in a single PCR by using different reporter dyes [9,10]. The hydrolysis probe analysis can be considered to be one of the best methods with regard to specificity and sensitivity for detecting one substitution such as the knockdown resistance (kdr) mutations [10,11].
In this article, we report on the use of a hydrolysis probe assay (Taqman assay) that is able to accurately and rapidly identify the two clade types of An. funestus s.s.

Hydrolysis probe analysis
PCR was performed using a CFX96™ Real-Time system (Bio-Rad). The primers and probes were designed from COI. The sequence information of COI was presented in Choi et al. [8]. Two standard oligonucleotides

Results and discussion
A total of 53 specimens were used for the assay, 21 from Malawi and 32 from Mozambique. The two specific probes for clades I and II were designed based on one substitution between clades in the partial region of COI (Figure 1). The first specific probe for the clade I allele was labelled with FAM and the second specific probe for the clade II allele was labelled with VIC. All 21 An. funestus individuals from Malawi were clade I type while 13 and 19 individuals from Mozambique were clades I and II respectively ( Figure 2). The results from the hydrolysis probe analysis in this study were 100% consistent with the results from the DNA sequencing. Subsequently, a field sample of 78 An. funestus individuals from Nchelenge in northern Zambia became available and were successfully analysed as clades I (n = 59) and II (n = 19), using the above sequenced specimens as positive controls (Figure 3). This assay will save time when routinely identifying collections of wild mosquitoes belonging to this important malaria vector species. Although this method is expensive compared to the PCR method, it is much more accurate than PCR, cheaper than DNA sequence analysis, and avoids the potential safety hazard presented by the use of ethidium bromide [10]. Similar results were reported by Bass et al. [10] who developed the hydrolysis probe analysis (Taqman assay) for detection of the kdr mutations in Anopheles gambiae based on one nucleotide substitution, concluding that it is the most specific and sensitive method compared with seven other diagnostic assays including the PCR method.
At present we do not know if the various molecular and chromosomal forms in An. funestus are associated with insecticide resistance or vector competence, nor whether "intergrades" can be found in different populations. It is also not clear whether the RFLP types [14], the clades [7] and the chromosomal forms [15] in An. funestus are themselves in any way associated. Since An. funestus is such an important malaria vector in Africa, it is vital that we understand the basic underlying genetics of the species as this will allow us to better comprehend its role in malaria transmission and the evolution and spread of insecticide resistance.

Conclusion
The application of the assay described here is expected to greatly improve the efficiency of screening largescale field-collected samples of An. funestus s.s. for clades I and II. Hence, this assay could help elucidate the role that each clade is playing in malaria transmission and whether there is any difference in insecticide resistance frequencies.