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.