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.