Material and Methods
Collection and processing of samples.
A total of one hundred and thirty-four (134) mosquito samples were utilized in this investigation. These specimens were obtained from different regions in Limpopo province including Masisi, Mutale, Tshivaloni, Xikukwani, Makoxa, Thomo, Seloane, Domboni and Musunda as shown in figure 1 and these regions have a unique geographic coordinate (Table 1). Samples were obtained using two different techniques: pits or larvae collecting methods. Stored samples acquired by the Limpopo Malaria Institute were transferred safely and securely from Limpopo to the University of KwaZulu-Natal and stored in the freezer at Genetic Laboratory 3, Dr Moses’s laboratory before DNA extraction. In order to prevent DNA mixing in case that mosquitoes were able to feed prior to collection, the abdomen was removed from all samples. Only the legs, head, and thorax were utilized for DNA extraction. The abdomen was removed while the specimen was viewed under the microscope. PCR was used to amplify regions of interest where the successful amplification was confirmed by presence of either one or more bands per tube. The majority of tubes contained a pool of three mosquitoes, however some tubes contained fewer or more than three mosquitoes. The main purpose of this was to simply determine whether the quantity of mosquitoes had any impact on the concentration of DNA after extraction. In this investigation, a total of 42 tubes containing pooled mosquitoes were employed. According to the absorbance at 260/280, which was at least 1.6 for all tubes, the DNA produced was of excellent quality.
Figure 1: South Africa Map showing different geographic areas where mosquito samples were collected.
Table 1 : Different regions of collection and their corresponding geographic coordinate
DNA extraction
Some of the samples that were gathered were undocumented, and those samples were utilized to evaluate the best DNA extraction tool. The first kit used to extract DNA from a pool of mosquitoes was the Promega Wizard® Genomic DNA Purification Kit, but this kit produced very little DNA yield as it employs a micro pestle, which doesn’t crush mosquitoes very effectively. However, the beads utilized by the Zymo-Research Quick-DNA Tissue/Insect Miniprep Kit for crushing mosquitos properly crushed the mosquitos, resulting in the best DNA yield suitable for PCR. As a result, the Zymo-Research Quick-DNA Tissue/Insect Miniprep Kit was employed for DNA extraction in this investigation. As previously stated, DNA was obtained from the legs, thorax, and head of mosquitos, with the abdomen left out to avoid DNA mixing if mosquitos were allowed to feed before collection. In this study, 134 mosquito samples were examined; however, mosquitos were pooled, with the majority of tubes holding a pool of three mosquitos, resulting in 42 tubes containing diverse DNA samples. DNA was successfully extracted according to manufacturer’s instruction; no modifications were made. The quantity and purity of the extracted DNA were evaluated using a NanoDrop 2000 UV Visible Spectrophotometer, and the DNA was then kept at -20 C until it was utilized for genetic analysis by polymerase chain reaction (PCR).
Amplification of 18S rDNA gene using PCR .
The polymerase chain reaction was employed to amplify the 18S rDNA region in DNA obtained from pooled mosquitos. Primers that were utilized to amplify 18S rDNA were; Forward primer 5’ GAG GGA GCC TGA GAA ACG GCT AC 3’ and Reverse primer 5’ CCT TCC GTC AAT TCC TTT AAG TTT C 3’ (Beebe et al. 1996). The Bio-Rad T100 Thermal Cycler was employed for PCR amplification. A 25µL PCR reaction was set up as follows: 12.5µL of Dream Taq Master Mix, 1µL of each primer (1.9 µM concentration), 1 µL of genomic DNA, and 9.5µL of nuclease-free water. Since the conditions often employed by prior research didn’t work well for the primer, it needed to be optimized. As a result, 62.2°C annealing temperature yielded the best results, thus the following PCR conditions were used to amplify 18S rDNA: Denaturation: 94°C for 4 minutes, 1 cycle; annealing: 95°C for 1 minute, 62.2°C for 1 minute, and 72°C for 1 minute, 35 cycles; final extension: 72 C for 7 minutes, 1 cycle; hold: 4 C.
Agarose gel electrophoresis and viewing of amplified PCR products of 18S rDNA.
Availability of target DNA region was checked using agarose gel electrophoresis. A 1.5% agarose gel stained with EtBr was used to run 5 µL of each sample. This type of agarose gel was made by just mixing 100 mL of 1X TAE buffer (Bio Concept, Switzerland) with 1.5g of agarose powder (Cleaver scientific, United Kingdom) in a 1000ml beaker. The powder was dissolved in a buffer by heating the mixture in the microwave for approximately three minutes. The mixture was then allowed to cool before being poured into a gel casting tray with a comb in  to produce wells. Then after, the mixture was allowed solidified to form a gel that was used to run 5µL of PCR product in electrophoresis. A hundred bp (100bp) molecular weight marker (New England Biolabs, China) was employed as a reference for comparing DNA fragment sizes of PCR products. One µL of the molecular weight marker was combined with two µL of 6X purple dye. This dye aided in the visualization of a molecular weight marker. Electrophoresis was performed at 100 volts for 60 minutes utilizing Enduro gel XL electrophoresis system (Labnet, United States of America). The ChemiDoc MP imaging machine (Bio-Rad, United States of America) was used to visualize the DNA bands in the gel.
Phylogenetic Analysis.
The 18S ribosomal DNA mosquito samples were sent to Stellenbosch for sequencing. The base calling and trimming were done utilizing FinchTV. The sequences were aligned using the Clustal W multiple alignment function of the BioEdit program, version 7.2.5. MEGA version 6 (Tamura et al., 2013) was used to create phylogenetic trees using the maximum likelihood (ML) technique.