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Discrimination of the Anopheles Sp. Using Different Molecular Techniques © FEB 2020 | IRE Journals | Volume 3 Issue 8 | ISSN: 2456-8880 Discrimination of the Anopheles Sp. Using Different Molecular Techniques KOTHAI KAVEEVENDAN Eastern University, Sri Lanka Abstract- Malaria is one of the most malaria free Sri Lanka in 2016. (World Health common infectious diseases and public health Organization, 2016). problems around the globe. Malaria parasite is transmitted by Anopheles mosquitoes classified Human malaria is exclusively transmitted by under Family Culicidae of Order Diptera and. The Anopheles spp. mosquitoes in Sri Lanka and other part Anopheles complex has been particularly of the island. Mostly all key malaria mosquito vectors incriminated as one of the important malaria vectors are members of complexes or groups comprising in Sri Lanka and other part of the island. The present morphologically indistinguishable sibling species. review provides basic aspects to differentiate Mostly identification based on the morphological morphologically identified sibling species of the criteria fails when sibling species and species with Anopheles complex by different DNA based overlapping morphological characters are involved. molecular techniques such as random amplified polymorphic DNA, restriction fragment length The correct identification of disease vectors is the first polymorphisms. DNA barcoding and Polymerase step towards implementing an effective control chain reaction. Moreover, it is particularly useful programme. Traditionally, malaria control was based when using small amount of starting materials from on the morphological differences observed in adults particular specimen or from immature stages. and larvae between different mosquito species. However, the discovery of species complexes meant Molecular technique provides a powerful tool for that genetic tools were needed to separate the sibling effective examination on the population dynamics of species and now a day there are standard molecular mosquitoes. It enables more detailed understanding techniques that are used to identify the two major on the relationships between the vectorial capacity, malaria vector groups of mosquitoes. genetic makeup and geographical origin for a particular species of Anopheles, more detailed and Advanced DNA based molecular technology have precise taxonomy, as well as evolutionary studies. facilitated the development of simple and rapid molecular tools for the identification of sibling Indexed Terms- Anopheles, Malaria, Mosquitoes, species. Various genetic techniques are available for Molecular techniques, DNA identification of isomorphic species (Besansk et al.,1992, Hill &Crampton 1994) like random I. INTRODUCTION amplified polymorphic DNA (RAPD), restriction fragment length polymorphisms (RFLP). DNA In worldwide, around a billion people become infected barcoding, Polymerase chain reaction or sequencing with vector-borne diseases such as malaria, dengue method have revolutionized population genetics and fever, yellow fever and West Nile virus in every year. insect systematics. (WHO, 2017). Malaria is one of the most common infectious diseases and a great public health II. JUSTIFICATION problem around the world, of which more than 91 countries were declared as an epidemic regions (CDC, Anopheles species has been particularly incriminated 2017). Malaria threat has been reduced significantly as one of the malaria vectors in island wide. All the during the last few years in Sri Lanka and declared as members of the complex should be studied in detail to IRE 1701914 ICONIC RESEARCH AND ENGINEERING JOURNALS 46 © FEB 2020 | IRE Journals | Volume 3 Issue 8 | ISSN: 2456-8880 formulate effective vector control strategies. Scott et al (1993) identified the An. gambiae, An. Morphological properties are widely used to identify Arabiensis, An. quadriannnulatus and either An.melas and classify mosquito species without the need for in western Africa or An. melas in eastern and southern specialized and expensive equipment in field studies. Africa by the ribosomal DNA polymerase chain Since reported morphological characteristics are not reaction. Dusfour et al. (2007) developed a multiplex useful, a simple DNA based technique is more suitable polymerase chain reaction assay based on the two to identify the members of Anopheles complex. In this mitochondrial DNA markers to clearly identify the background this study suggests that various molecular members of An. sundaicus s.s, An. epiroticus and An. techniques for identification of Anopheles sp sundaicus. III. POLYMERASE CHAIN REACTION (PCR) H.K.Phuch et al. (2003) described the development of single multiplex PCR for identifying An.minimus Polymerase chain reaction (PCR) based assay which species A and C and distinguishing the four related was developed in 1985 by Saiki et al., (1985) become species, An.aconitus, An.jeyporiensis, An.varuna and popular for species identification as the technology is An.pampanai. The rDNA ITS2 region was amplified vastly improved and reagent cost is also reduced. The using conserved 5.8s and 28s primers modified from PCR based assay essentially requires variation in those used by Paskewitz &Collins (1990). Based on nucleotide sequence across species. Information the species-specific differences, six primers were gathered has revealed that two features of the ITS2 designed as reverse primers. These reverse primers region of Anopheles species that are diverse region for were mixed with 5.8S forward primer to amplify the developing PCR- based species diagnostic assays. whole ITS2 region (Manonmani et al., 2001). This First, these regions are relatively short (generally less diagnostic produced a 184 bp PCR product with than 1 kb), making PCR amplification of the template DNA from An. minimus A, a 252 bp intervening ITS2 from flanking conserved primers band for An. varuna, a 306 bp band for An. aconitus, relatively simple. Second, intra species variation in a 346 bp band for An. jeyporiensis, a 452 bp band for ITS2 sequences is lower than interspecies variation, An. pampanai and a 509 bp band for An. minimus C. thus permitting the development of PCR diagnostic This species- specific diagnostic assay produced assays (Wesson et al, 1992). The use of diagnostic significantly different size of products. length for the identification of diagnostic sequences for cryptic Anopheles species complexes is a valuable Two multiplex PCR was developed to identify the two technique. sets of morphologically similar species of malaria mosquitoes incorporating species- specific primers Erlank et al (2018) described the diagnostic of three derived from direct sequencing of the ITS2 region. species (Anopheles pretoriensis, A rufipes and A One PCR reaction identified Anopheles rhodesiensis) amplified members of the An. funestus oswaldoi and an undescribed species (species C) and group and four species (An. pretoriensis, An. rufipes, second PCR reaction identified mosquitoes as Anopheles listeri and Anopheles squamosus) belonging to a complex of four species, which includes amplified members of the An. gambiae complex by An.deanorum, An.albitarsis A, An.albitarsis B and Standard PCR assay. An.marajoara. (Corey and Brelsfoard et al., 2005) Gunathilaka et al (2015) performed the PCR assay to The PCR-SSCP assay diffrentiate A. rivulorum, A. distinguish the members of Anopheles culicifacies funestus, A. vaneedeni, and A. leesoni from each sibling species A, B, C, D and E and following band others (Hackett et al., 2000). However, this method sizes were obtained 359 bp, 248 bp, 95 + 248 bp, 166 fails to distinguish between A. vaneedeni and A. + 359 bp and 178 + 248 bp to compare the sibling parensis. These two species are morphologically species of A, B, C, D and E respectively. Fettene et al similar to A. funestus (Gillies et al., 1987). Hackett (2002) distinguished the sibling species A and B of and others used the ITS2 PCR to distinguish A. An.quadriannulatus s.l as well as other members of funestus and A. rivulorum only. An.gambiae complex by modified PCR assay. IRE 1701914 ICONIC RESEARCH AND ENGINEERING JOURNALS 47 © FEB 2020 | IRE Journals | Volume 3 Issue 8 | ISSN: 2456-8880 Koekemoer et al (2002) identified five of most rivulorum and Anopheles funestus s.s. This assay very commonly found members of An.funestus group. such effectively identified all five members of the as A. funestus, A. vaneedeni, A.parensis, A.leesoni An.funestus group (Vezenegho et al, 2009). and A.rivulorum. Primers were designed using sequence of ITS2 fragments. After PCR production IV. ALLELE- SPECIFIC PCR ASSAY (AS-PCR) lengths of amplified species – specific products were verified such as A. leesoni ≈146 bp, A. funestus ≈505 The generalization of partial or complete sequencing bp, A. vaneedeni ≈587 bp, A. parensis ≈252 bp, and of many genomes allowed the development of A. rivulorum ≈411 bp. Here, all six primers can be identification assays based on a single step easier to included in a cocktail PCR reaction mix implement. These assay named as allele- specific (AS- simultaneously without any effect on amplification. PCR) or PCR amplification of specific alleles (PASA) Each unknown specimen can be identified without Anopheles fluviatilis, one of the major vectors of performing five separate PCR reactions. malaria in India, is a complex of at least three cryptic species provisionally designated as species S, T, and rDNA-ITS2 PCR assay was developed to group the U. Species S, T, and U are morphologically five species of Anopheles culicifacies complex into indistinguishable at any stage of their
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