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.