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.