Result
The only mosquito genera investigated in this research were Culexand Anopheles , although there is one species of mosquito that does not belong to any of the three discovered mosquito genera (Anopheles , Aedes , and Culex ); this newly morphologically recognized species is known as Dimelion . These different species of mosquitos were collected at different locations within Limpopo province (Figure 1). Among mosquito samples collected,Anopheles genera was more prevalent over the other genera,Culex . Only six mosquito samples were Culex of the 134 species collected, and they were all obtained from Makoxa. 128 Samples belonged to Anopheles genera and were collected from different regions in Limpopo (Table 2.). After Polymerase chain reaction amplification, DNA for different samples was run in a 1.5% agarose gel electrophoresis. To amplify samples Tube1- Tube42, which were labelled as T1, T2, etc, 18S r DNA was utilized. As seen in table 3, tubes (T1 -T42) contained a pool of mosquitos of the same species. The marker of interest previously mentioned (18S rDNA) produced varying amplicon sizes (900bp – 910bp) after running them on agarose gel electrophoresis as shown in gel images in figure 2. When repeated for the 3rd time 18S rDNA successfully amplified T26 which didn’t amplify at first place. 5ml of PCR mixture was added into each well for each sample and ran at 100 volts for 60 minutes and ChemiDoc imaging system was used to view DNA profiles. A 100bp ladder stained with purple dye was used as a reference to estimate sizes of PCR products. Prior to sequencing, amplicons were purified to eliminate any residues that may cause interference if not removed, such as non-specific bands.
Table 2. Various mosquito species, their numbers, and the area where they were collected in South Africa, Limpopo province.
Table 3. Pools of mosquito species per Tube ( T1-T42) and their corresponding geographic locations and coordinates.
Figure 2. T1-T42 mosquito samples’ 18S rDNA amplification profiles. Lane MW indicates a 100bp DNA ladder (New England Biolabs, China). It is also known as molecular weight marker (MWM). It was basically used to estimate PCR product sizes. Lane NC symbolizes the negative control. The obtained PCR products were then run at 100 volts on 1.5% agarose gel with 1X TAE running buffer. To obtain optimal separation of the DNA fragment on the agarose gel, the DNA agarose gel electrophoresis was allowed to run for 60 minutes. In certain mosquito species, the 18S rDNA amplicon is 900 bp in size, whereas in others, it is 910 bp. As a result, some tubes contained two bands (900 bp and 910 bp), showing that the pooled mosquitoes were not all from the same species.
The outcomes of multiple sequence alignment (MSA) in figures 3 do not show any discernible variation in the 18S rDNA region among the mosquito species under investigation. By comparing sequences found in this study with those on Genbank, this study was able to determine whether or not the Limpopo malaria institute’s morphological identification of mosquito species was accurate. Even though there are some gaps in the query sequences after alignment, they display little to no variation compared with the reference sequences. However multiple sequence alignment wouldn’t give reliable results alone, hence phytogenic diagram of 18S rDNA (Figure 4) was constructed to support MSA results by showing relationship that exist among the species of mosquitos under investigation. Multiple alignment sequences and phylogenetic analysis proved that the ”Dimelion ” species of mosquito, which was once believed to be an entirely novel species identified based only on its morphology, is in fact An.gambiae . An additional piece of evidence that Dimelion is not a new species of mosquito discovered in Limpopo, but rather An.gambiae , is that its sequence revealed a 99.65% identity with An.gambiae _OM350318.1 and no Dimelion species was observed in hits after blasting the query sequence. An.gambiae was not the only species that was mistakenly recognized morphologically; nonetheless, molecular identification disapproved other species’ names that were given to them by morphological procedures (Table 3). These mosquito species are believed to have been misclassified based on morphology because, when their 18S rDNA sequences were blasted, they displayed high percentage identities to other mosquito species, but the name assigned to them based on morphological features did not appear in possible hits. However, some sequences (T25_18S rDNA, T26_18S rDNA, and T28_18Sr_DNA) did not match any sequence when blasted on NCBI blast and were too divergent from other sequences to be included in the construction of a phylogenetic tree. As a result, they were not included in the MSA or phylogenetic construction. Five sequences were too short to be aligned with other sequences, leading in an error while attempting to match them with other sequences; hence, they were omitted from MSA. These sequences include T7_18S rDNA (An.gambiae ), T17_18S rDNA (An.gambiae ), T33_18S rDNA(An.listeri ), T36_18S rDNA (An.pretoriensis ), T42_18S rDNA (unknown).
Looking at the phylogenetic tree for 18S rDNA in figure 4, the existence of an elevated percentage of bootstrap support values, some of which are 100%, indicates that the data in table 3 is accurate. As a result, 18S rDNA phylogenetic analysis verified that the information acquired by comparing query sequences with reference sequences (Table 3) on Genbank through blasting is valid, implying that a large number of mosquitos were mistakenly classified based on their morphological traits. The 18S rDNA phylogenetic tree (figure 4) shows that tsetse flies which were used as outgroup are the most recent common ancestor and that two major clades diverged from the 95% node as a common ancestor. A high percentage of bootstrap support values guarantees that each of the involved species is closely linked. Regardless of what morphological traits  suggests, species within the same clade and closer to one another are thought to be genetically linked. Despite the six species reported by morphological identification, genetic identification confirmed only seven mosquito species and one non-mosquito species(Diaphorina ) among the samples collected. These mosquito species include An.gambiae , An.sundaicus , An.melas ,An.coluzzii , An.merus , An.maculipalpis , andAn.funestus . The phylogenetic tree (Figure 4) shows that mosquitos of the same species are closely related, suggesting that molecular identification was accurate. However, there are notable exceptions of species that are closely related to various other species due to some reasons that are highlighted in the discussion section.
Figure 3 : DNA bases of Multiple sequence alignment of 18S rDNA targeted in various species of mosquitoes collected from Limpopo. To generate alignment, the BioEdit program was utilized in conjunction with the Clustal W multiple alignment tool.
Table 4 : Molecular characterisation of different mosquito species through database searching and availability of 18S rDNA sequences.
Figure 4: Phylogenetic reconstruction of the various species of mosquitos using the 18S rDNA gene (900bp). Mega X software was used to generate phylogenetic tree, whereby the Maximum likelihood method inferred by kimura 2 parameter model after 100 replicates was utilized. Sequences from the present study start with letter “T”, T1-T41 . Bootstrap supports values are shown in percentage (%) , and the scale for the above phylogenetic tree is 0.2.