Journal of Mosquito Research 2024, Vol.14, No.2, 87-99 http://emtoscipublisher.com/index.php/jmr 88 2 Methods of Mosquito Species Identification 2.1 Morphological identification techniques Morphological identification of mosquito species has traditionally been the cornerstone of entomological studies and vector control programs. This method relies on the examination of physical characteristics such as wing patterns, leg markings, and body size. However, it is often challenged by the need for expert knowledge and the potential for misidentification, especially when dealing with morphologically similar species or damaged specimens. For instance, the identification of mosquitoes from Mexico State using morphology was complemented by DNA barcoding to overcome these limitations, highlighting the necessity of integrating molecular techniques for accurate species identification (Adeniran et al., 2020). 2.2 Molecular identification methods DNA barcoding, particularly using the mitochondrial cytochrome c oxidase subunit I (COI) gene, has become a widely adopted method for mosquito species identification. This technique involves sequencing a short, standardized region of the genome, which provides a unique genetic fingerprint for each species. Studies have demonstrated the efficacy of COI barcoding in identifying mosquito species across various regions, including Mexico (Adeniran et al., 2020), the UK (Hernández-Triana et al., 2019), and Thailand (Chaiphongpachara et al., 2022). The method has proven effective in distinguishing species with high accuracy, even in cases where morphological identification is challenging due to damaged specimens or cryptic species complexes. Polymerase Chain Reaction (PCR) and Single Nucleotide Polymorphism (SNP) genotyping are other molecular techniques used for mosquito identification. PCR-based methods can amplify specific DNA regions to detect and identify mosquito species and their associated pathogens. For example, a targeted amplicon sequencing panel was developed to simultaneously identify mosquito species and Plasmodium presence across the Anopheles genus, demonstrating the utility of multiplex PCR in large-scale genetic surveillance (Makunin et al., 2021). SNP genotyping, on the other hand, focuses on identifying genetic variations at single nucleotide positions, which can be used to generate species-specific barcodes. A decision tree-based barcoding algorithm utilizing SNPs has been proposed for the rapid and reliable identification of Anopheles species (Swain et al., 2019). 2.3 Advancements in high-throughput sequencing technologies High-throughput sequencing technologies have revolutionized mosquito species identification and phylogenetic studies. These technologies allow for the simultaneous sequencing of multiple genetic loci or entire genomes, providing comprehensive insights into genetic diversity and evolutionary relationships. For instance, a new method of DNA metabarcoding was developed to detect microsporidian infections and identify host mosquito species using next-generation sequencing, showcasing the potential of high-throughput approaches in uncovering hidden biodiversity (Trzebny et al., 2020). Additionally, the use of multilocus amplicon sequencing has enabled the identification of mosquito species and the detection of malaria parasites in a single sequencing run, highlighting the efficiency and scalability of these methods for vector control programs (Makunin et al., 2021). High-throughput genomic sequencing has also facilitated detailed phylogenetic analyses, as demonstrated in studies on the Culex pipiens complex and the Neotropical mosquito fauna. These studies have provided robust support for the monophyly of various mosquito subfamilies and tribes, offering valuable insights into the evolutionary history and diversification of mosquitoes (Adeniran et al., 2020; Lorenz et al., 2021). The integration of high-throughput sequencing with advanced analytical tools continues to enhance our understanding of mosquito phylogenetics and species identification, paving the way for more effective vector control strategies. 3 Phylogenetic Analysis in Mosquitoes 3.1 Importance of phylogenetics in mosquito research Phylogenetic analysis is crucial in mosquito research as it helps elucidate the evolutionary relationships and divergence times among mosquito species, which are essential for understanding their role in disease transmission and developing control strategies. For instance, the study of the mitochondrial genomes of 102 mosquito species revealed significant insights into the evolutionary history of the Culicidae family, including the divergence of the
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