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 [1–5]. 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.

Methods

Mosquito sample collection and identification

Specimens were collected resting inside houses from Karonga (10°19′S, 34°08′E) and Nkhota kota (12°55′S, 34°18′E) in Malawi, Chibuto (24°40′S, 33°33′E) in Mozambique and Nchelenge (9°21′S, 28°44′E) in Zambia. The samples were identified using the morphological keys of Gillies and Coetzee [2] and the molecular method of Koekemoer et al. [12]. All DNA samples were extracted from either single mosquitoes or available parts of mosquitoes using the Ballinger-Crabtree protocol [13]. The DNA templates were resuspended in TE buffer at volumes between 50 μL and 100 μL.

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 (MACROGEN) and two minor groove binding (MGB) probes (Applied Biosystems) were used. Primers Forward (5′- GCA GGA ACA GGA TGA ACA GT -3′) and Reverse (5′- GAA ATT CCT GCT AAA TGT AAT GAA A -3′) were used for binding the flanking region of both clade mutation sites in the COI. The probe clade I (5′- TCA GGA ATT GCT CAT GCT -3′) was labelled with 6-FAM for the detection of the clade I and the probe clade II (5′- TCA GGA ATT GCC CAT GCT -3′) were labelled with VIC for detection of the clade II. The 20 μL PCR reaction contained 1 μL of the genomic DNA of an individual mosquito, 10 μL of IQ™ Supermix (Bio-Rad), 0.8 μM of each primer and 0.4 μM of each probe. The PCR cycling conditions were as follows: an initial denaturation at 95°C for 10 minute, followed by 45 cycles of 95°C for 10 seconds and 63°C for 45 seconds. The increase in FAM and VIC fluorescence was monitored in real time by detecting fluorescence on the yellow channel (450–490 nm excitation and 515–530 nm detection) for clade I and the blue (515-535 nm excitation and 560-580 nm detection) for clade II. The Malawi and Mozambique results were confirmed with the sequence results to ensure that experimental error was limited.

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).

Figure 1
figure 1

Alignment from 3′ to 5′ end of the COI target region for probes. The alignment of An. funestus for the comparison were originated from GenBank (access No. AY423059). Mal and Moz represent Malawi and Mozambique respectively. Dots represent identity with respect to the An. funestus sequences. AG, CT and AT indicate polymorphic positions. The square indicates the sequences of the probes for clades I and II.

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Figure 2
figure 2

Hydrolysis probe analysis fluorescence results for clades I and II of An. funestus s.s. from Malawi and Mozambique. Scatter plot analysis (a) of A) clade I; B) clade II; C) negative control and quantitative peaks (b). Blue peaks are clade I and yellow peaks are clade II.

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Figure 3
figure 3

Hydrolysis probe analysis fluorescence results for clades I and II of An. funestus s.s. from Zambia. Scatter plot analysis (a) and quantitative peaks (b) of A) clade I; B) clade II; C) negative control. Blue peaks are clade I and yellow peaks are clade II. Positive controls (arrows) from the Figure 2 experiment are D) clade I and E) clade II.

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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 large-scale 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.