Introduction
Anopheles mosquitoes are essential vectors in the distribution of
the malarial parasite, Plasmodium , hence monitoring mosquito
populations is a crucial factor in predicting malaria risk in a
geographic region of interest. The number of mosquito species is
estimated to be 3500, and they are divided into 41 genera (Zheng, 2020).
Anopheles, Aedes, and Culex are three of these 41 genera that are
thought to be crucial in the spread of human illness caused by
mosquitoes (Adugna et al., 2021). Of the 530 species ofAnopheles , only 30 to 40 naturally spread malaria (Nicoletti et
al., 2020). Various Anopheles mosquito species have been found to prefer
distinct habitats; thus, the plasmodium parasite is spread by
different Anopheles species depending on geographic locations (Nicoletti
et al., 2020).
According to Adugna et al., (2020), identification of Anophelesspecies is crucial since each species has a different treatment or
preventative strategy. However, identification of Anophelesmosquitoes in Limpopo has mainly concentrated on morphological
characterization, which is likely to misidentify Anophelesspecies due to high similarity of phenotypic features (Jones et al.,
2021), leading to assignment of certain insecticides or treatment to
incorrect Anopheles species which may be the reason why some
insecticides do not kill some Anopheles mosquitoes present in the
geographic region of interest. As a results, previous studies found it
more convenient to confirm morphological characterisation with molecular
characterization through studying genetic diversity of Anophelesmosquito species present in the area of interest. Geneticists
predominantly believe in molecular characterization since it employs
extremely precise molecular procedures such as PCR, Agarose gel
electrophoresis, DNA sequencing, and so on, which employ very sensitive
components such as genetic markers. Genetic markers are DNA sequences
with known positions on chromosomes. They play crucial role in the study
of populations, cell identification, as well as species classification
(Safdar, 2011).
One of the genetic markers of interest employed in this study’s
molecular analysis of the Anopheles mosquito species is 18S
ribosomal DNA (18S rDNA). According to Carranza et al., (1996), all
prokaryotic and eukaryotic cells use rDNA as a critical component of
their protein production machinery. Due to its high conservation among
species and the presence of variable regions, 18S rDNA has been
frequently employed for identifying and assessing the genetic diversity
of eukaryotes (Kounosu et al., 2019). This sequence conservation is
thought to represent functional limitations on the molecules necessary
for best translational effectiveness. According to Rackevei et al.,
(2022), V1 to V9 variable regions of 18S rDNA are commonly used in
biodiversity studies. Despite being a member of a multigene family, the
18S rDNA has undergone concerted development to keep all of its copies
homogenous(Carranza et al., 1996). This marker is also being used to
determine phylogenetic connections between living organisms (Carranza et
al., 1996). There are lot of reason for this maker to be used for
studying phylogenetic analysis and those reason are detailed in a review
by Woese (1987); Sogin (1991); and Adoutte et al., (1993).
PCR, Agarose gel electrophoresis, Multiple sequence alignment (MSA), and
phylogenetic analysis were used to examine the genetic variance of
distinct mosquito species. Genetic variation is simply the variance in
genomic DNA sequence between individuals within a community/same species
(Al-Koofee et al., 2020). PCR was mainly utilized to amplify the areas
of interest, and the existence of the target region was determined by
agarose gel electrophoresis. Following Agarose gel electrophoresis,
samples must be further analyzed with MSA. This method is used to
determine the evolutionary links and common patterns among target genes
(Sofi et al., 2022). According to a genetic concept, MSA is the
alignment of at least three genomic DNA sequences of same length.
Computational methods are used to produce and analyze these alignments
(Sofi et al., 2022).Identification of a novel protein members after
comparing them to related sequences is an additional role played by MSA
(Shukla et al., 2022). Since further analysis of research is dependent
on the outcome of the MSA, the accuracy of the MSA plays a crucial role.
Consequently, creating trustworthy and accurate MSA tools will always be
a worthwhile endeavor(Shukla et al., 2022). These techniques, along with
targeted genetic markers, are considered to be exceedingly sensitive and
very specific, which is why they were chosen to measure mosquito
diversity and give trustworthy findings.