Discussion
The present investigation portrays genetic characterisation of several
mosquito species that have been morphologically defined by the Limpopo
Malaria Institute. According to morphological characterization, the
species under examination are from the Anopheles and Culexsubfamilies, and there has never been a study like this one in Limpopo.
The main aim of this investigation was just basically to confirm or
disprove Limpopo Malaria Institute’s morphological categorization of
mosquito species, as well as to investigate the accuracy of genetic
markers in identifying closely related species. The Limpopo Malaria
Institute employed the two techniques of collecting mosquitos indicated
in 3.2.1 (pits or larvae), and after collection, they utilized standard
morphology-based taxonomy approach to identify mosquitos
morphologically, and two genera (Anopheles and Culex ) were
detected. As previously demonstrated (Table 2), the majority of species
identified by morphological features belonged to Anopheles (102
species), only 6 were classified as Culex, and 26 were thought to be new
species (Dimelion ) that had not been classified previously,
however molecular characterization disproved that.
Gel images show positive results for all samples when the 18S rDNA
region were amplified; however, some samples displayed multiple bands
per sample, which was thought to be caused by the presence of various
species in the pool of mosquitos. However, sequencing only produced one
sequence per sample, i.e., one sequence per sample for 18S rDNA marker,
with no varying sequences per sample as an indication of various species
present in a pool of mosquitoes. Therefore, gel pictures that displayed
more than one DNA band per well may be an indication that non-specific
bands have been amplified (Bovo et al., 1999). DNA barcoding using 18S
rDNA was able to identify species that were mistakenly classified
morphologically. The occurrence of high percentage identity (81%-100%)
between query and reference sequences on GenBank is evidence of
successful molecular characterisation and validation of morphological
identification of distinct mosquito species (Tables 3.3). Some sequences
of samples that were morphologically described
as An.rufipes (T25, T26, and T28) matched no sequence on GenBank,
suggesting that these are new haplotype of An.rufipes found by
the current study using 18S rDNA.
The results reported in this study (Table 3) demonstrates that many
samples were incorrectly categorized morphologically. This was proven by
molecular analysis, which makes use of highly specific molecular
techniques (Wilson et al., 2000). Damage to essential distinguishing
features, human error, the occurrence of novel or cryptic species, the
presence of species showing overlapping or unreported traits, and
intraspecific morphological changes are all potential causes of
misclassification of mosquito species (Zhang et al., 2022). Despite the
fact that the majority of mosquito species were incorrectly classified
morphologically, Molecular characterisation using 18S rDNA revealed that
certain species were accurately identified. The identification of
samples T5, T8, T9, T18, T19, T20, and T21 as An.gambiae by 18S
rDNA and morphological characterization indicates that the morphological
characterization of these species was valid. Individual pools of
mosquito species were represented by highly supported clades, with high
percentages of bootstrap values up to 100%, validating the
morphological identification of diverse mosquito species investigated.
According to morphological characterization of mosquito species, six
different species of mosquitos were obtained; however, molecular
characterization by 18S rDNA demonstrated that there were actually seven
different mosquitos studied plus one non-mosquito species, and among
these species of mosquitos, some had been overlooked by morphological
characterization and others were mischaracterized (Table 3). These eight
species found by molecular characterisation comprise species that were
mistakenly categorized by morphology as well as species that were
verified to be appropriately classified morphologically by molecular
analysis. Seven species of mosquitos found by molecular characterization
using 18S rDNA includes An.gambiae , An.sundaicus ,An.melas , An.culuzzi , An.merus ,An.maculipalpis , and An.funestus . In addition to these
species, 18S rDNA also discovered a non-mosquito species ,Diaphorina , which was thought to be Culex species, and
this serves as a proof that morphological identification alone cannot be
trusted when it comes to characterization of various species of
mosquitos. Unknown species (T42) were recognized by 18S rDNA asAn. gambiae . The 18S rDNA based molecular characterization of
samples that were not documented , T42(Unknown), discovered that the
pool of mosquitos in this sample belong to An.gambiae and this
was supported by a very high percentage similarity of 18S rDNA query
sequence to MG753768.1 (An.gambiae ) reference (81.32%) and a
being relatively small e value (3e-95). The accuracy of 18S rDNA
characterization results (Table 3) was supported by a close relationship
that exist between mosquitos of the same species and their reference
sequences in the phylogenetic construction (Figure 4)
In a phylogenetic tree, species in the same clade and near to one
another are genetically connected, i.e., the closer the species are to
one another, the closer their genetic relationship is (HAMZAOLU et al.,
2017). Other studies made an effort to modify the aforementioned claim
in order to conform to the world of science; they claimed that two
species are more connected if they share a most recent common ancestor
and less related if they share a less recent common ancestor (Lo et al.,
2003; Gregory,2008). Furthermore, the presence of a node with a high
bootstrap value close to 100% suggests that the species that diverged
from that node, also known as a common ancestor, are closely related to
one another and that their genetic makeup is identical or contains very
little variation. Since previous studies have shown that a very high
bootstrap value up to 100% gives researchers confidence in concluding
that particular species are siblings. Hence, high bootstraps support
values in the 18S rDNA phylogenetic construction and relatively small
scale (0.2) imply a close relationship between the species being
studied. Phytogenic trees (Figures 4) investigated variation well
because it shows that mosquitos of the same species are more closely
related, sharing clades and most recent common ancestors.
It is evident from a detailed examination of the 18S rDNA phylogenetic
tree that mosquitoes belonging to the same species are genetically
related to one another. This tree diagram demonstrates two significant
branches that diverged from the 95% node that was determined to
represent the most recent common ancestor of all the species analyzed in
the current study, and that divergence resulted in the formation of two
major clades. An. gambiae species are found to be closely linked
to one another in the top clade, and their tight relationship is
supported by the occurrence of a high bootstrap value of 99% where all
of these species diverged from. Regardless of An.maculipalpis being
closely related to each other , but their divergence from a common
ancestor with An.gambiae , supported by 85% bootstrap value,
provides evidence that An.maculipalpis and An.gambiae are
genetically linked. Additionally, this tree diagram was able to show
that An. sundaicus are related to each other, An. melas as
a reference species, and An.maculipalpis , however they are indeed
distantly related to An.gambiae . Diaphorina , a
non-mosquito species, was demonstrated to be genetically linked toAn.gambiae , An.maculipalpis , An. melas , andAn.sundaicus by sharing a clade and diverging from a common
ancestor. This relationship is supported by a 100% bootstrap support
value. Looking at the bottom branch, there are An. gambiae andAn.maculipalpis species that are closer to one another and
their reference sequences, providing proof that these species are in
fact An.gambiae and An.maculipalpis . These species,
however, are distantly related to the same species in the upper clade,
and the occurrence of these species in various geographical locations
may be the cause of this variability (Marcus et al., 2017). There is
also the presence of An.funestus reference species in this clade,
which is distantly linked to T22 An.funestus from this study, and
this variation might be attributable to a variety of factors, including
the one previously stated, mutation, geneflow, or sexual reproduction
(Barton, 2010 ). This reasoning also applies to other species that are
distantly linked to their phylogenetic references, such asDiaphorina , An.gambiae , and others. This information
combined offers a proof that 18S rDNA molecular identification was more
accurate than morphological identification, as it can seen that the
species stated to be linked by molecular characterisation appear to be
connected in the phylogenetic tree too (Figure 4).
It is possible that there is still a significant danger of malaria
transmission in Limpopo given the presence of so many distinctAnopheles species in this province. According to 18S rDNA, this
study has discovered a possibly novel haplotype of the Anophelesspecies (T25, T26, and T28 and T32). The main reason behind this fact is
that the sequences for these sample show no match when compared to
sequences on Genbank. Anopheles species are constantly being
discovered via the use of molecular methods; for example, new species ofAnopheles nuneztovari have been discovered in Brazil (Scarpassa
et al., 2016). Based on the results, it can be inferred that there is a
significant and widespread group of mosquitoes in all of the Limpopo
regions that are close to the coast and along the border (Table 1). This
is in line with earlier research which showed that the majority of
Anopheline mosquito species are found in temperate and tropical climates
(Schäfer et al., 2001). All this together causes a rapid spread of
malaria in Limpopo and contributes to a rise in malaria cases and
malaria-related deaths. According to earlier research, An.
funestus , An. arabiensis , An.pretoriensis ,An.quadriannulatus , and An.gambiae are the only species of
Anopheles present and in charge of transmitting malaria in South Africa,
specifically in Limpopo (Burke et al., 2019; Dahan-Moss et al., 2020;
Braack et al., 2020; Dahan et al., 2020). However, molecular
characterisation carried out in the present study discovered numerous
more Anopheles species that were assumed to be missing in South
Africa, including as An.sundaicus , An.melas ,An.coluzzii , An.merus , An.maculipalpis ,An.triannulatus , and An.darlingi . These Anopheline
mosquito species may have been imported to South Africa by immigrants
via luggage or flights from various regions of the world. The rising
incidence of arbovirus (malaria) epidemics in Limpopo, and the rapid
propagation of such diseases, as well as high volume of the public
health consequences (Charrel et al., 2007; Enserink, 2007; Semenza et
al., 2014), have prompted multiple calls for South Africa to practice
greater vigilance regarding arboviruses, as well as an associated need
to comprehend the population status of existing or potential vector
mosquitoes (Cornel et al., 2018). Hence prior research investigated the
diversity of mosquito species since some scientists observed that
mosquitos are treated based on the type or kind of species found in a
specific location. Presence of these various species as vectors of
malaria which were not known to be present in Limpopo really contributed
to increasing cases of malaria in Limpopo due to the fact that they are
given wrong treatment. The necessity of understanding the genetic makeup
of different mosquito species is demonstrated by the information
presented here, and it also made it clear that morphological
characterisation of mosquito species alone does not provide trustworthy
findings due to its numerous restrictions and lack of specificity.