Rapid Discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) Analysis

Abstract

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.

INTRODUCTION

The mosquito An. gambiae Giles is the major vector of the malaria parasite Plasmodium falciparum in equatorial Africa, where its global impact is by far the most severe.^1,2 An. gambiae sensu lato (s.l.) is a complex that includes seven biological sibling species, which are genetically and behaviorally distinct and vary widely in their importance as malaria vectors. Particularly important are two members of the complex, An. gambiae sensu stricto (s.s.) and An. arabiensis Patton, which are morphologically indistinguishable as adults, are widely distributed, and have broadly overlapping geographical ranges.^2–5 Despite their sympatry, their roles in transmitting malaria often are different, partly reflecting differences in use of hosts, willingness to enter and rest in houses, and tolerance of dry climates. To complicate matters, the behavior of An. arabiensis is variable across its range,^2,6–8, and the distributions, abundances, and relative proportions of An. gambiae and An. arabiensis can shift seasonally⁹ or long-term, the latter perhaps a result of recent extensive use of insecticide-treated bed nets^10–13 or environmental change. To implement and evaluate vector-based malaria-control measures where these two species coexist, it is essential to have an identification method that is fast and simple.¹⁴

Identification of sibling species of An. gambiae s.l. was achieved originally by polytene chromosome analysis^3,4 and followed by other techniques, such as isoenzyme electrophoresis¹⁵ and HPLC of cuticular hydrocarbons.¹⁶ Current identification methods make use of PCR to detect specific DNA nucleotide differences in the intergenic spacer of the ribosomal DNA (rDNA).^17,18 In this paper, we have integrated the allele-specific (AS)-PCR and HRM analysis to discriminate An. gambiae s.s. from An. arabiensis rapidly and with high precision. HRM is not only rapid but a cost-effective technique compared with other genotyping methods, such as sequencing^17–19 and TaqMan single nucleotide polymorphism (SNP) typing.^20,21 Our method is simple, fast, and thus able to identify many samples to species level rapidly and accurately, allowing high-throughput processing of large field collections.

MATERIALS AND METHODS

Mosquito Collections

Specimens of An. gambiae s.l. were collected in the field by mouth aspirator and backpack aspirator during 2006–2008 from houses and resting pots at Mbita (00° 26–27′ S, 34° 12–13′ E) and Luanda (00° 28′ S, 34° 16–17′ E; Suba District, Nyanza, western Kenya), where An. gambiae s.s. and An. arabiensis have overlapping distributions and seasonally varying proportions. The specimens were held in a cold chest and carried to the laboratory within a few hours of collection and stored at −20°C in a freezer, except when it temporarily failed, possibly causing temporary thawing. They were later transferred to 95% ethanol until processed. Maintenance of mosquitoes has been approved under Institutional Review Board Permit 2004H0193 and Institutional Biosafety Committee Permit 2005R0020 from The Ohio State University (Columbus, OH, USA).

Control Samples

Specimens from previously identified colonies served as positive controls. The standards for An. gambiae s.s. originated from a local population in Mbita, Kenya. This Mbita strain was established in 2001 at the International Centre of Insect Physiology and Ecology (ICIPE; Kenya) and verified by molecular techniques.¹⁷ Specimens for analysis were taken from our Mbita-strain colony at The Ohio State University. Standard specimens of An. arabiensis (MRA-856 Dongola) were supplied by the Malaria Research and Reference Reagent Resource Center (MR4) from a colony maintained in Manassas, VA, USA.

DNA Purification and Quantification

Individual mosquito specimens from field and laboratory strains were prepared for identification by removing two legs from each specimen with sterile forceps and placing them into a sterile, 1.5-ml microtube. Each sample was ground and homogenized by a disposable pestle in a tube containing 180 μl PBS solution (pH 7.2, 50 mM potassium phosphate, 150 mM NaCl). DNA was purified with DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD, USA; Insect Protocol DY14, Aug. 2006). The concentration of DNA for a subset of samples was measured with a Qubit fluorometer (Invitrogen, Life Technologies, Carlsbad, CA, USA; according to the manufacturer's procedure for the high-sensitivity dsDNA kit; probes.invitrogen.com/qubit). The DNA yield from two legs of a mosquito was ca. 10 μg; however, the quality and quantity of DNA obtained from these specimens varied according to consistency of the storage conditions, with many below the detection limit of the Qubit fluorometer.

AS-PCR and HRM Analysis

Five microliters of unknown samples (∼5 μg DNA, based on 10% that had been randomly selected to quantify their DNA contents), along with the same volume of positive and negative controls, was placed in a 96-well PCR plate. The following Invitrogen primers were used in the amplification step, which generated a 165-bp-long amplicon: (1) universal forward 5′-GTGAAGCTTGGTGCGTGCT-3′ and (2) universal reverse 5′-GCACGCCGACAAGCTCA-3′.²⁰ They correspond, respectively, to the 623–641 and 772–788 positions of the 5′ end of the intergenic spacer region.²⁰ Each reaction consisted of 5 μl genomic DNA (gDNA) extract and 5 μl HRM master mix, which contained 1× of SsoFast EvaGreen supermix (Bio-Rad Laboratories, Hercules, CA, USA; P/N 172-5200) and 8 pmoles each of the primers listed above. All reactions were performed using the Bio-Rad CFX96 real-time PCR system with the following thermal cycle protocol: (1) 95°C for 180 s; (2) 92° for 15 s, 60° for 60 s, 40×; (3) 95° for 30 s; and (4) 65° for 10 s with a 0.2° increase for each repetition, 150×. Fluorescence data were collected during the 60° stage of Step #2 above and during all repetitions of Step #4 above. The resulting melting curves were analyzed with Precision Melt Analysis v1.0 (Bio-Rad Laboratories) with the following settings: 0.2 melt-curve shape sensitivity and 0.5 for melting temperature (T_m) difference threshold.

Sequencing of Amplicons and Alignment

All DNA sequencing reactions were performed with 1 μl of the resulting amplicon product from the AS-PCR/HRM procedure above, 4 pmoles primer, 2.0 μl 5× dilution buffer (Applied Biosystems, Life Technologies, Foster City, CA, USA), and 0.5 μl BigDye Terminator v3.1 master mix (Applied Biosystems, Life Technologies) in a 10.5-μl reaction with the following thermal cycling conditions: (1) 1 min at 95°C and (2) 95° for 10 s, 50° for 5 s, and 60° for 120 s (35×). Each amplicon was sequenced twice with each of the two primers described above, forward and reverse. The sequencing reactions were subsequently purified with Performa v3 dye terminator removal plates (Edge BioSystems, Gaithersburg, MD, USA), according to the manufacturer's protocol. The purified extension products were subjected to electrophoresis on the 3730 DNA Analyzer (Applied Biosystems, Life Technologies) and analyzed with Sequencing Analysis software v2.5 (Applied Biosystems, Life Technologies). The sequences were aligned with the program ClustalW2 and the slow default parameters (http://www.ebi.ac.uk/Tools/msa/clustalw2/). A cladogram was generated from the same software with default conditions.

RESULTS

Figure 1, shows the melt curves from the 17 samples listed in Table 1. Figure 1A is based on the raw fluorescence data for the controls (positive, negative, and no template) only, whereas Fig. 1B is the difference curve for normalized data of all samples. In Fig. 1A, the differences between the two species are obvious as a result of the shape of the curve and the difference in the T_m, i.e., 84°C for An. gambiae and 83°C for the An. arabiensis curves, although they have an identical number of bases. The samples included seven positive controls from maintained cultures and 10 randomly selected individuals (unknowns) obtained from the field (Table 1). Two negative control wells (Fig. 1A; triangles) were included that also had a dsDNA product, which is clearly not the same as the 165-bp product from the mosquitoes. The T_m for the negative control was 72°C as compared with 83°C for An. arabiensis and 84°C for An. gambiae and the melt-curve shape is indicative of two primers annealing, i.e., a primer dimer (data not shown). Primer dimers are not uncommon in the absence of template during PCR, and the melt curves did not indicate a secondary or primer-dimer peak in the presence of gDNA. Figure 1B includes the same samples as displayed in Fig. 1A, although shown after analysis for melt-curve differences. An. arabiensis traces are below −0.5 RFU, and the An. gambiae traces are at or above −0.05 RFU. An. arabiensis and An. gambiae clearly cluster together as indicated by the color of the lines—red and green, respectively. In addition, the Table 1 data, with their Percent confidence values, indicate how reliable the assignment is to a particular cluster, with all values >97%.

FIGURE 1.

HRM analysis of Anopheles sp. based on X-chromosome-based rDNA amplicons. The data from the same samples are displayed in A and B. (A) Raw melt-curve data. Green lines with circles are from An. gambiae, red lines with diamonds are from An. arabiensis, and black lines with triangles are from water (negative control). (B) The difference-melt curves normalized to the cluster An. gambiae. The negative control is not present in B as a result of the absolute signal being too low for comparison. RFU, Relative fluorescence units.

TABLE 1.

Results of High-Resolution Melt Analysis

Sample	Content	Cluster	Color	Percent confidence
1992	Unkn	Arabiensis #2	Red	98.1
2119	Unkn	Arabiensis #2	Red	98.7
2142	Unkn	Arabiensis #2	Red	98.5
2159	Unkn	Arabiensis #2	Red	99.1
Arabiensis #5	Pos Ctrl	Arabiensis #2	Red	98.7
4184	Unkn	Arabiensis #2	Red	99.6
2271	Unkn	Arabiensis #2	Red	99.7
5194	Unkn	Arabiensis #2	Red	98.9
Arabiensis #1	Pos Ctrl	Arabiensis #2	Red	99.6
Arabiensis #2	Pos Ctrl	Arabiensis #2	Red	99.1
Arabiensis #8	Pos Ctrl	Arabiensis #2	Red	98.8
2226	Unkn	Arabiensis #2	Red	97.5
5711	Unkn	Arabiensis #2	Red	97.8
2063	Unkn	Arabiensis #2	Red	98.8
Gambiae #2	Pos Ctrl	Gambiae #2	Green	98.4
Gambiae #1	Pos Ctrl	Gambiae #2	Green	97.9
Gambiae #3	Pos Ctrl	Gambiae #2	Green	97.8

Results of 10 unknown (Unkn) Anopheles individuals collected from the field, as well as six individuals (Pos Ctrl) from laboratory-maintained colonies. The Percent confidence is reported by the Precision Melt Analysis software as to how similar/dissimilar the cluster is to adjacent clusters.

To confirm that the clustering pattern presented in the difference-melt curves represented two different species, both strands of each amplicon were sequenced, with the resulting consensus sequences listed in Fig. 2. Two control sequences were included from the GenBank database, which had been submitted in conjunction with additional published research when the sequences were aligned.²² An. arabiensis is represented by Submission Number EU091306.1 and An. gambiae by AF470116.1. The sequences from the positive control and unknown samples clearly align with GenBank controls as well as each other, confirming that the HRM analysis worked correctly for all of the samples. The relationship between the individuals is demonstrated in the cladogram (Fig. 3). The An. gambiae individuals clearly group together with AF470116.1, and the An. arabiensis samples, as well as the 10 unknowns, clearly group together with EU091306.1. The clustering of sequences also was confirmed with SeqScape software (Applied Biosystems, Life Technologies), which uses a different algorithm from ClustalW2 (data not shown). In total, there are 13 SNPs between An. gambiae and An. arabiensis whereas there is one SNP, A/T, at Position 92 within the An. arabiensis group, and some individuals are heterozygous with a W (an A or T).

FIGURE 2.

Alignment of X-chromosome-based rDNA sequences. Each individual can be placed in one of four groups listed above with the corresponding sequence. Among the An. arabiensis groups, only one base differs, at position 89, whereas the An. gambiae group has 13 differences from the An. arabiensis groups, as indicated by the letters in row G. Reference sequences from GenBank submissions are AF470116.1 for An. gambiae and EU091306.1 for An. arabiensis. Name–number combinations are specimens from identified colonies. Un-named numbers are record codes of field-collected samples previously unidentified.

FIGURE 3.

Cladogram of X-chromosome-based rDNA sequences. The same relationship is confirmed with the HRM analysis and sequence alignment. Control sequences from GenBank submissions are AF470116.1 for An. gambiae and EU091306.1 for An. arabiensis. Name–number combinations are specimens from identified colonies. Un-named numbers are record numbers of field-collected unknowns.

To further test the usefulness of this technique, a larger set of individuals, previously collected from the field, was subjected to HRM analysis. In total, 88 individuals (Fig. 4), were analyzed, and two of them produced an insufficient fluorescence signal to be assigned by the Precision Melt software, although from the amplification curves from the PCR step, we know that some DNA amplification did occur (data not shown). The same occurred for the negative control reactions, i.e., water instead of gDNA, and in this case, the products were again primer dimers. The six positive-control reactions (three for each species) performed correctly and as expected. Of the 86 mosquitoes that generated measurable data, four were found to be An. gambiae, 81 were An. arabiensis, and one was a putative hybrid, as the curve lies midway between the An. gambiae and An. arabiensis clusters. Based on a score of 85 successful, confirmed identifications out of 86 samples, HRM analysis had an accuracy of ∼99%.

FIGURE 4.

Difference curves for multiple individuals from the field, including positive control samples for An. arabiensis and An. gambiae. Of the 86 unknowns that were tested, four are An. gambiae, i.e., at or above 0.0; 81 are An. arabiensis, i.e., below 0.0; and one is a putative hybrid (black line), in between the two clusters. Each color, six in total, represents a different cluster, with each member having a confidence value of 0.95 or higher on a scale of 0.0 to 1.0. Temperature is Celsius.

DISCUSSION

HRM analysis is an established technique for determining genotypic variation, as indicated by a recent search of the National Center for Biotechnology Information database that yielded over 2000 published research articles. For example, HRM can readily distinguish as subtle a genetic difference as one SNP.^23,24 This technique was used to determine whether it would be effective in distinguishing field-captured An. gambiae from An. arabiensis, as these species are morphologically inseparable and overlap geographically but have distinctive behaviors. To monitor the melting process in real-time, we used a fluorescent dye, EvaGreen, which binds specifically to dsDNA and has several advantages over the older and commonly used dye, SYBR Green.^25,26 For example, EvaGreen intercalates into the DNA molecule and therefore, provides greater resolution during melting, as compared with SYBR Green, which binds in the minor groove of the helix in a less-consistent manner.

The An gambiae genome has 278 million base pairs.²⁷ So far, among its many regions used in studies of An. gambiae s.l. population genetics, only the X-linked rDNA portion contains molecular variations that reliably differentiate among chromosomal forms.²⁸ This region has been used by many researchers, starting in 1993,¹⁷ through the use of AS-PCR. The primers reported in that article were considered to be the “gold standard” for many years and were tried initially in our work but unfortunately, with unsatisfactory success, as the fluorescently labeled fragment pattern following electrophoresis from many individuals often indicated it was both species simultaneously (data not shown). Yet, cytogenetic studies and more recent molecular studies indicate that hybrids are rare.^29–31

After further review of the literature, we used the primers and PCR conditions from the Walker laboratory,²⁰ with some modifications, to take advantage of local instrumentation and with the goal of lowering costs. Previously, the detection method relied on a TaqMan assay³² to interrogate the SNPs in this 165-bp region. So, the dual-labeled oligonucleotide (TaqMan) probe was replaced with EvaGreen as the detection method. Therefore, increased fluorescence, caused by the release of a quenched dye by exonuclease activity, was replaced with the measurement of the melting characteristics by a decrease in fluorescence as the dye is released by the separating DNA strands. The two techniques have similar accuracy (∼98%) and use the same instruments and bench techniques, but HRM has a significant advantage over TaqMan: a lower cost by ∼25%/reaction. HRM does not require the use of a relatively expensive, dual-labeled, sequence-specific probe but instead, uses a saturating fluorescent dye that costs less.

Although a real-time PCR system is expensive, the cost and portability of these instruments are decreasing and increasing, respectively, each year, as the technology improves with the use of light-emitting diode technology, single-dye platforms, and more robust cooling/heating systems. With the placement of instrumentation in local laboratories in areas indigenous to An. gambiae s.s., identification can be achieved for hundreds of individuals in 1 day by one technician at a cost of approximately $0.50/sample for reagents, i.e., plates, films, EvaGreen kit, primers, and others. This cost includes neither the technician's labor nor the DNA extraction. For example, with sonication (which adds only the cost of labor), it takes minutes to extract DNA from each set of samples and ∼90 min to analyze 96 samples in the real-time PCR instrument. It is possible to process samples stored at −20°C or in ethanol, but fresh samples are better by having a higher yield of gDNA (unpublished data).

In conclusion, HRM analysis, in combination with AS-PCR, is an effective and efficient method to distinguish between An. gambiae and An. arabiensis.