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.

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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 life cycle and the two categories (A and D and B, C and E) without can be identified only by the examination of species- the need of an intermediate RFLP assay, thus reducing specific fixed inversions in the polytene the number of steps involved in the available chromosomes. An. fluviatilis complex were identification assay and significantly cutting down the differentiated by an allele-specific polymerase chain time and cost. (Manonmani et al.,2007) reaction assay, which is based on differences in nucleotide sequences in D3 domain of 28S ribosomal Anopheles gambiae sensu lato (s.l) species complex DNA (Singh et. al., 2004 a). In addition, Singh et al. contains the most important mosquito vector of (2004b) developed an allele-specific polymerase chain malaria in Africa. Fast identification of the two main reaction to the D3 domain of 28S rDNA that species which vector malaria, Anopheles arabiensis discriminated Anopheles culicifacies species and An. gambiae sensu stricto, from the non-vector complex. The assay was further validated using species Anopheles quadriannulatus is frequently chromosomally identified specimens of Anopheles required as part of vector control programme. Taq- culicifacies from different geographical regions of Man assay was developed that distinguishes between India representing different sympatric associations. the main malarial vectors and non-vector. This method describes the rapid and sensitive assay that very Goswami et al. (2005) developed PCR-RFLP of effectively identifies the two main malaria vectors mitochondrial COII and ITS2 of rDNA markers for the from the non-vector sibling species (Chris Bass et al. identification of members of Anopheles culicifacies 2007). Moreover, Taq Man single nucleotide complex. Goswami et al. (2006) conducted a study to polymorphism genotyping proved for the identify all members of Anopheles culicifacies identification of An, gambiae s.l and An.arabiensis complex using allele specific polymerase chain with high success rate specific results (Edward D reaction assays. The PCR assays developed from the Walker et al, 2007) D3 and ITS2 regions of rDNA failed to identify all the members of the Anopheles culicifacies complex while Bass et al (2008) developed a new multiplex real time COII sequences showed single bp difference that have PCR assay for identifying members of An.gambiae been used to differentiate all the members of this complex. It uses three probes labelled with complex8 fluorophores with distinct emission and excitation spectra, allowing simultaneous detection of two main Anopheles culicifacies, a complex of five isomorphic malaria vectors from the non- vector sibling species. sibling species, is a major vector of malaria in India The TaqMan assay proved to be the most sensitive and and neighboring countries. The five species are specific for the identification of Anopheles parensis, provisionally designated as species A, B, C, D and E. Anopheles leesoni, Anopheles vaneedeni, Anopheles Polymerase chain reaction & polymerase chain

IRE 1701914 ICONIC RESEARCH AND ENGINEERING JOURNALS 48 © FEB 2020 | IRE Journals | Volume 3 Issue 8 | ISSN: 2456-8880 reaction-restriction fragment length polymorphism important vectors of malaria. It identified as species A, can differentiate the species A and D from species B, B, C, D by the AS – PCR method. C, and E. They reported allele-specific PCR assays is possible to identify individual specimens of any of the V. SPECIES- SPECIFIC –PCR species of this complex ( Geeta et al., 2006). Sibling species under A. gambiae complex were Distinction between members of An.nili group is characterized by polymerase chain reaction using difficult, mainly based on morphological characters. species specific single nucleotide polymorphism in the In order to achieve a quick and inexpensive tool for intergenic spacer region with primers specific for An. rapid identification of An.nili group is an allele gambiae s.s., An. arabiensis, An. melas, An. merus and specific PCR. That combine five primers were Anopheles quadriannulatus (Kabbale, 2016) developed. ITS2 region was used as template to design diagnostic PCR. Resulting PCR products differ from Six sibling species of the Anopheles maculipennis one another by at least 30 bp. So that they easily identified based on the second internal transcribed separated on agarose gel. The size of the diagnostic spacer of the ribosomal DNA. It was amplified and band is 188 bp for the typical An.nili, 357bp for sequenced for all six species. Based on differences in An.nili Oveng, 408 bp for An.carnevalei and 329 bp the nucleotide sequences, species specific primers for An.somalicus (Kengne et al, 2003). Moreover, were constructed for PCR amplification of mosquito Anopheles moucheti (Evans) s.l is a major malaria DNA that in combination with a universal primer vector in Africa. To differenciate the species, ITS2 of generate amplification products of different length, all An.moucheti s.l specimen was designed together each unique for one species (Proft et al., 1999). with three reverse primers AMou, Aber and ANig respectively, specific to An.m.moucheti, Cohuet et. al (2003) differentiated the five sibling An.m.bervoetsi and An.m.nigeriensis respectively. species of Anopheles funestus such as An. funestus, The size of the diagnostic band was 378 bp for An. leesoni, An. parensis, An. vaneedenip and An. An.m.bervoetsi, 312 bp for An.m.moucheti and 249 bp rivulorum. They used the species - specific PCR assay for An.m.nigeriensis based on this assay each which is supplemented by a primer specific to An. subspecies within An.moucheti were identified rivulorum and thus makes it possible to differentiate (Kengne et al., 2007). the five species of the An. funestus group.

Sharpe et al. (1999) demonstrated that allele-specific Anopheles funestus is a major vector of malaria in amplification of the D3 variable region of the 28S Africa. It belongs to a group of sibling species that can rDNA gene distinguished An. minimus A rom C and be identified morphologically only at certain stages of single-strand conformation polymorphism (SSCP) of their development. A diagnostic polymerase chain the D3 amplified region discriminates four species, reaction based tool made it possible to differentiate An. varuna, An. aconitus, An. minimus A and C. five species of the group. This assay seems to be Unfortunately, this method is more time consuming relevant over all their distribution area for four of these and the results less easy to interpret than those from species: An. funestus, An. leesoni, An. parensis, and conventional polymerase chain reaction. An An. vaneedenip. The fifth species, An. rivulorum, is alternative approach was adopted by Van Bortel et al. the second most abundant species of the group. The (2000) who used PCR amplification of the rDNA species-specific PCR assay is supplemented by a internal transcriber spacer 2 (ITS2) region followed by primer specific to An. rivulorum-like and thus makes BsiZI restriction enzyme digestion to distinguish An. it possible to differentiate the five species of the An. aconitus, An. jeyporiensis, An. minimus A and C, An. funestus group and the newly defined taxon (Claire pampanai, An. varuna and subsequently An. Garros et al., 2004). culicifacies (Van Bortel et al., 2002). A polymerase chain reaction based diagnostic assay Walton et al (1999) developed AS-PCR to identify the was developed to differentiates the sibling species of members of Anopheles dirus complex which is most Anopheles claviger complex, An. claviger s.s. and An.

IRE 1701914 ICONIC RESEARCH AND ENGINEERING JOURNALS 49 © FEB 2020 | IRE Journals | Volume 3 Issue 8 | ISSN: 2456-8880 petragnani. The assay facilitates differences in the different molecular forms among the complex. One; internal transcribed spacer 2 ribosomal DNA species A, form comprise of morphologically different sequences to generate PCR products of specific length species A, B and C and the other; species B, consists for each of the two species. Based on the ITS2 of morphologically different species D. (Kothai, 2016) sequences, species-specific primers were selected and it produced DNA fragments of 269 base pairs for An. Morphological and molecular identification was found claviger s.s. and 367 base pairs for An. petragnani for species An. albimanus, An. aquasalis, An. darlingi when used in combination with the 5.8S primer. Helge and An. triannulatuss.l. However, disagreement was kampen et.al., (2003) designed specific primers for found for species An. nuneztovaris.l, An. two sibling species and also they tested PCR neomaculipalpus, An. apicimacula and An. conditions for yield of specific DNA fragments. punctimacula. The ITS2-PCR-RFLP assay proved valuable for discriminating species of northern and Manonmani et al. (2001) used species-specific western Colombia, especially those with overlapping differences in the nucleotide sequences of rDNA ITS2 morphology in the Oswaldoi Group (Cienfuegos et al., region to develop a diagnostic PCR assay for two 2011). sibling species of the Anopheles fluviatilis complex, members of which are major vectors of malaria in Anopheles longipalpis is morphologically similar to Central and Northern parts of India. the major African malaria vector Anopheles funestus at the adult stage although it is very different at the VI. RFLP – PCR ASSAY DIAGNOSTIC larval stage. Six species (An. funestus, An. funestus- like, An. parensis, Anopheles rivulorum, An. Diverse regions can be used to devise DNA vaneedeni and An. leesoni) in An. funestus group and hybridization or restriction fragment length An. longipalpis type C were subjected to RFLP assay. polymorphism (RFLP) assays (Collins et al, 1987, The enzyme, EcoRI digested only An. longipalpis type Beebe & Saul, 1995). In molecular biology, RFLP is C and An. funestus-like after species-specific An. a technique that exploits variations in homologous funestus group PCR assay. The An. longipalpis and DNA sequences. It refers to any variation in DNA An. funestus-like digestion profiles were characterized between individuals revealed by restriction enzymes by three fragments, 376 bp, 252 bp and 211 bp for An. of lengths in consequence of such variations. In RFLP longipalpis type C and two fragments, 375 bp and 15 analysis, DNA sample is digested into pieces bp for An. funestus-like. (Choi et al., 2010) by restriction enzymes and resulting restriction fragments are separated according to their lengths The Anopheles annular is a group of mosquitoes by gel electrophoresis. RFLP analysis is also an distributed in Southeast Asia. Four members of this important tool in genome mapping, localization of group are morphologically very similar and often genes for genetic disorders, determination of risk for difficult to distinguish. Comparison of the D3 disease and paternity testing sequences of the four species revealed two SmaI restriction sites in A. nivipes, but only one site in A. Sibling species of the Anopheles subpictus complex in philippinensis, A. annularis and A. pallidus. The ApaI Sri Lanka were identified by using a Restriction site was present in both A. philippinensis and A. Fragment Length Polymorphism – Polymerase Chain pallidus, while an NcoI site was present in A. pallidus Reaction assay based on the variations in the Internal only. Restriction digestion of PCR products of D3 Transcribed Spacer (ITS2) region. PCR assay was fragment individually with SmaI, ApaI and NcoI done with the available common forward primer ITS- produced a distinctive pattern for all four species. 2A (5' - TGT GAA CTG CAG GAC ACA T- 3') and They presented, a PCR-RFLP method to distinguish species-specific reverse primers ITS-2B (5'- TAT the four members of the A. annularis group of GCT TAA ATT CAG GGG GT - 3') in order to mosquitoes (Mohammad Tauqeer Alam et al., 2007a). amplify the ITS2 region. An analytical restriction digestion was performed by using NCo 1 restriction Anopheles minimus s.l., one of the most widespread digestion enzyme. The RFLP – PCR assay define two malaria vectors in South East Asia. It is a complex of

IRE 1701914 ICONIC RESEARCH AND ENGINEERING JOURNALS 50 © FEB 2020 | IRE Journals | Volume 3 Issue 8 | ISSN: 2456-8880 at least two isomorphic species and based on that could differentiate all five sibling species of An. morphology, members of the complex are difficult to culicifacies in India. A number of RAPD primers were distinguish from closely related species. An screened and genetic fingerprints for all 5 sibling identification method was developed for An. minimus species of An. culicifacies were developed using a species A and C and four related species, An. aconitus, selected primer. A total of 34 loci representing these An. pampanai, An. varuna and An. jeyporiensis. The sibling species using selected primer were entire ITS2 rDNA fragments, including partial polymorphic and able to differentiate the sibling sequence of 5.8S and 28S of six identified species species and showed 100% polymorphism (Varun et were sequenced. The fragments exhibited distinct al., 2015) differences between species and based on these sequences the restriction endonuclease BsiZI was used Wilkerson et al. (1993) differentiated the two and it distinguishes the all six species. However, it did morphologically indistinguishable taxa within not give proper results for An. pampanai. Moreover, Anopheles gambiae complex, An.gambiae Giles and MspI also used for identification of An. aconitus, An. An.arabiensis Patton. Formerly, these two taxa have varuna and An. Jeyporiensis. But it falis to been distinguished by polythene chromosome discriminate the members of the An. minimus species banding. But RAPD analysis proved as more efficient complex (Van Bortel et.al., 2000). tool in discrimination of two species of An.gambiae and An.arabiensis. PCR-RFLP-ITS2 assay performed with AluI, FspI or DraIII is useful for identification of species Kengne et al. (2001) described use of random belonging to the Oswaldoi Group of Anopheles amplified polymorphic DNA markers for the subgenus. As such, amplified ITS2 products were identification of species in the An. minimus group. selected for the enzyme restriction analysis and RAPD fragments specific for species A and C were digested. In addition to single restriction digests, cloned and sequenced. From these sequences, specific double restriction digests with AluI/FspI were used to primer pairs were designed to carry out multiplex PCR discriminate An. nuneztovari s.l. from An. able to identify five species, An.minimus A and C, An. benarrochi s.l. (Cienfuegos et al., 2011, Matson et al., aconitus, An. pampanai and An. varuna. In this assay, 2008). PCR products from hybrids of An. minimus A and C were different from both parents. VII. RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) VIII. DNA – BARCODING

The randomly amplified polymorphic DNA- PCR DNA barcoding is a molecular method that is (RAPD-PCR) which was developed by Williams et al. increasingly become a popular for the identification of in 1990 belongs to the first category. Here, arbitrary animal species based on the partial mitochondrial regions of the genome are amplified using a single DNA sequences (Hebert et al., 2003). decider primer. The assay based on species specific differences in hyper variable regions of the genome in Weeraratne et al. (2017) described the identification of all species. Anopheleline species mosquitoes based on the DNA barcoding technique which are major mosquitoes Favia et al (1994) examined RAPD which is rapid and group of interest in Sri Lanka. 15 species of reliable tool for genome analysis in the malaria vector mosquitoes species were collected such as Anopheles of Anopheles gambiae. This technique identified the aconitus, Anopheles annularis, Anopheles different mosquito strains of An. gambiae s.s. and An. barbirostris, Anopheles culicifacies, Anopheles arabiensis. jamesii, Anopheles karwari, Anopheles maculatus, Anopheles nigerrimus, Anopheles pallidus, Anopheles Anopheles culicifacies is a complex of 5 isomorphic peditaeniatus, Anopheles pseudojamesi, Anopheles types A, B, C, D and E in India with varying biological subpictus, Anopheles tessellatus, Anopheles vagus, characteristics. Varun et al developed RAPD markers and Anopheles varuna. This study reflects the

IRE 1701914 ICONIC RESEARCH AND ENGINEERING JOURNALS 51 © FEB 2020 | IRE Journals | Volume 3 Issue 8 | ISSN: 2456-8880 importance and feasibility of COI and ITS2 genetic negligible genetic divergence. Alam et al. (2007a) markers in identifying anophelines and their sibling reported the sequence analysis and a method for their species. molecular identification of the rDNA ITS2 and D3 regions of the four members of the Anopheles Rubio et al. (2016) distinguished the An.neivai from annularis group- Anopheles nivipes, Anopheles sympatric species and indicates genetic variability philippinensis, Anopheles annularis and Anopheles within the species through DNA barcoding pallidus from Assam and Andaman and Nicobar techniques. Islands. Alam et al. (2007b) analyzed the distribution of Anopheles annularis complex collected from In Abigal chan et al (2014) they explored the Sonapur (Assam), Jabalpur (Madhya Pradesh), Ranchi applicability of mitochondrial cytochrome C oxidase (Jharkhand), and Ghaziabad (Uttar Pradesh) and subunit I (COI) gene based DNA barcoding as an developed a molecular method using the rDNA ITS2 alternative tool to identify the mosquito species. COI and Domain 3 for identification purposes. based DNA barcoding achieved a 100% of success rate in identifying the mosquito species of Aedes Alam et al. (2008) described the sequencing of rDNA aegypti, Anopheles sinensis, Culex vishnui and Culex ITS2 and D3 loci of Anopheles stephensi stephensi mimulus. COI based DNA barcoding is a useful tool (type form) and Anopheles stephensi mysorensis. for identification of mosquito species. They also revealed that these populations showed identical sequences at both rDNA loci. Prakash et al. (2006) identified members of the Anopheles minimus and Anopheles dirus complexes CONCLUSION from north eastern region of India using sequences for the ITS2 of rDNA. They differentiated Anopheles The study of Anopheles taxonomy is greatly benefits minimus (species A) of the Anopheles minimus from the powerful information provided by DNA complex and Anopheles baimaii (species D) of the sequences. The identification and detection of Anopheles dirus complex from Arunachal Pradesh, Anopheles species specially sibling species are readily Assam, Meghalaya and Nagaland. Alam et al. (2006) discriminated by use of molecular identification reported sequence analysis of rDNA ITS2 and D3 assays. All species of Anopheles mosquitoes have regions of the four members of Anopheles annularis examined having species-specific sequences that can group- Anopheles nivipes, Anopheles philippinensis, form the basis of the diagnostic assays. Anopheles annularis and Anopheles pallidus for their molecular identification from Andaman and Nicobar The present review reveals that morphologically Islands. They found that there was no intraspecific identified Anopheles complex could be distinguished sequence variation among the specimens and easily by different molecular techniques such as interspecific sequence differences were greater for random amplified polymorphic DNA (RAPD), ITS2 than the D3 regions restriction fragment length polymorphisms (RFLP). DNA barcoding, Polymerase chain reaction, Allele- Singh et al. (2006) examined the conspecificity of Specific PCR and DNA – Barcoding method. And also Anopheles fluviatilis S and Anopheles minimus C by it minimizes the time and money in the laboratory and analyzing the DNA sequences of nuclear ribosomal also ensure the data reliability. In addition, it ITS2 and D2-D3 domain of 28S rDNA. minimizes time and ensuring more reliability in molecular characterization. Kumar et al. (2007) studied DNA barcodes for several species of mosquitoes belonging to 15 genera, REFERENCES prevalent in India which included major vector species. However, two closely related species, [1] Abigail Chan, Lee-Pei Chiang, Hapuarachchige C Ochlerotatus portonovoensis and Ochlerotatus wardi Hapuarachchi, Cheong-Huat Tan, Sook-Cheng could not be identified as separate species based on Pang, Ruth Lee, Kim-Sung Lee, Lee-Ching Ng and DNA barcode approach as their lineages indicated Sai-Gek Lam-Phua, DNA barcoding:

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complementing morphological identification of funestus group an Anopheles longipal type C by mosquito species in Singapore, Parasites & par-RFLP, Malaria Journal, 9 (2010) 316. Vectors, 7 (2014) 569. [10] Cienfuegos AV, Gomez GF, Cordoba LA, [2] Alam MT, Bora H, Das MK, Sharma YD. The Luckhart S, Conn JE, Correa MM. Diseno y type and mysorensis forms of the Anopheles evaluacion de metodologias basadas en PCR- stephensi (Diptera: Culicidae) in India exhibit RFLP de ITS2 para la identification molecular de identical ribosomal DNA ITS2 and domain-3 mosquitos Anopheles spp. (Diptera: Culicidae) de sequences, Parasitology Research; 103 (2008) la Costa Pacifica de Colombia. Rev Biomed. 19:35 943-948. (2008) 44.

[3] Alam MT, Das MK, Dev V, Ansari MA, Sharma [11] Cienfuegos A.V., Rosero D.A., Naranjo N., YD. (2007a) PCR-RFLP method for the Luckhard S., Conn J.E. and Correa M.M., identification of four members of the Anopheles Evaluation of a PCR RFLP ITS2 assay for annularis group of mosquitoes (Diptera: discrimination od Anopheles species in northern Culicidae), Transactions of the Royal Society of and western Colombia, Trans R Soc Trop Med Tropical Medicine and Hygiene, 10 (2007a) 239- Hyg.118(2) (2011)128-135. 244. [12] 12. Claire Garros, Lizette L. Koekemoer, [4] Alam MT, Das MK, Dev V, Ansari MA and Maureen Coetzee, Marc Coosemans, And Sylvie Sharma YD. Identification of two cryptic species Manguin., A single multiplex assay to identify in the Anopheles (Cellia) annularis complex using major malaria vectors within the African ribosomal DNA PCR RFLP, Parasitology Anopheles funestus and the oriental An. minimus Research, 100 (2007b) 943-948. groups., Am. J. Trop. Med. Hyg., 70(6) (2004) 583–590 [5] Alam MT, Das MK, Dev V, Ansari MA, Sharma YD. PCR-RFLP method for the identification of [13] Cohuet A., Simard F., Toto J.C., Kengne P., four members of the Anopheles annularis group of Coetzee M. and Fontenille D.,Species mosquitoes (Diptera: Culicidae), Transactions of identification within the Anopheles funetud group the Royal Society of Tropical Medicine and of malaria vectors in Cameroon and evidence for Hygiene, 101 (2006) 239-244. new species, Am.J.Trop.Med. Hyg, 69(2) (2003) 200-205. [6] Besansky, N. J., Finnerty V. and Collins F. Molecular perspectives on the genetics of [14] Corey L.Brelsfoard, Gary N. fritz and Roberto mosquitoes. Advances in Genetics 30 (1992) 123- Rodriguez, DNA based metods for identifying 184. malaria mosquitoes, Eastern Illinois University, Biological science Research poster (2005) [7] Bass C.,Williamson M. S., Wilding C.S., Donnelly M.J. and Field L.M., Identification of the main [15] Dusfour I., Blondeau J., Harbach R.E., malaria vectors in the Anopheles gambiae species Vythilingham I., baimai V., Trung H.D., Sochanta complex using a TaqMan real- time PCR assay, T., Bangs M.J. and manguin S., Polymerase Chain Malaria journal, 6 ( 2007) 155. Reaction Identification of Three Members of the Anopheles sundaicus (Diptera: Culicidae) [8] Bass C., Williamson M. and Field L.M., Complex, Malaria Vectors in Southeast Asia, 44(5) Development of multiplex real- time PCR assay (2007) 723-731. for identification of members of the Anopheles gambiae species complex, Acta Tropica, 107(1) [16] Edward D Walker, Alisha R Thibault, Annettee P (2008) 50-53. Thelen, Blair A Bullard, Juan huang, Maurice R Odiere, Nabie m bayoh, Elizabeth E Wilkins and [9] Choi K.S., Coetzee M., Koekemoer L.L., john M Vulule. Identification of field caught Simultaneous identification of the Anopheles Anopheles gambiae s.s and Anopheles arabiensis

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by TaqMan single nucleotide polymorphism Anopheles funestus and An. rivulorum, and genotyping, Malaria Journal, 6 (2007) 23. uncover a cryptic taxon, Insect Mol Biol 9 (2000) 369–374. [17] Erlank E., Koekemoer L.l. and Coetzee M., The importance of morphological identification of [25] Hebert P.D, Cywinska A, Ball SL, De Waard JR., African Anopheline mosquitoes (Diptera: (2003) Biological identifications through DNA Culicidae) for malaria control programmes, barcodes, Proc Biol Sci, 270 (2003) 313 321. Malaria Journal, 17 (2018) 43. [26] Helge Kampen, Anja Sternberg, Jana Proft, Sandra [18] Favia G., DimopouiosG. and Louis C., Analysis of Bastian, Francis Schaffner, Walter A Maier and the Anopheles gambiae genome using RAPD Hanns M. Seitz., Polymerase chain reaction- based markers, Insect Molecular Biology, 3(3) (1994) differentiation of the mosquito sibling species 149-157. Anopheles claviger s.s and Anopheles petragnani, The American society of Tropical Medicine and [19] Fettene.M., Koekemoer L.L., Hunt R.H., and Hygiene, 69(2) (2003) 195-199. Coetzee M., (2002) PAR assay for identification of Anopheles quadriannulatus species B from [27] Hill, S. M. and Crampton, J. M., DNA-based Ethiopia and other sibling species of the Anopheles methods for the identification of insect vector, gambiae complex, Medical and Veterinary Annals of Tropical Medicine Parasitological 88 Entomology, 16, (2002) 214-217. (1994) 227-250.

[20] Geeta Goswami, O. P. Singh, Nutan Nanda, K. [28] https://www.who.int/news-room/fact- Raghavendra, S. K. Gakhar, And Sarala K. sheets/detail/vector-borne-diseases Subbarao., Identification of all members of the [29] https://www.cdc.gov/malaria/malaria_worldwide/ anopheles culicifacies complex using allele- impact.html specific polymerase chain reaction assays., Am. J. Trop. Med. Hyg., 75(3) (2006) 454–460. [30] http://www.searo.who.int/srilanka/areas/malaria/ malaria-free-sri-lanka.pdf [21] Gillies M.T, Coetzee M, A supplement to the Anophelinae of Africa South of the Sahara [31] Kabbale F.G., Akol A.M., Kaddu J.B., Matovu E., (Afrotropical Region) (publication no. 55). Kazibwe A., Yadouleton A. and Onapa W., (2016) Johannesburg, South African Institute of Medical Molecular identification of Anopheles gambiae Research. 55 (1987) sensu stricto Giles (formerly Anopheles gambiae Savannah Form) in Kamuli District, Uganda, [22] Goswami G, Raghavendra K, Nanda N, Gakhar African Jpurnal of Biotecnology, 15(39), (2016) SK, Subbarao SK. PCR-RFLP of mitochondrial 2124-2131. cytochrome oxidase subunit II and ITS2 of ribosomal DNA: Markers for the identification of [32] Kengne P., Awono-Ambene,P., Antonio- members of the Anopheles culicifacies complex Nkondjio, C., Simard, F., & Fontenille, D., (Diptera: Culicidae), Acta Tropical, 95 (2005) 92- Molecular identification of the Anopheles nili 99. group of African malaria vectors, Medical and Veterinary Entomology, 17 (2003) 67-74. [23] Gunathilaka N. and Karunaraj P., (2015) Identification of sibling species status of [33] Kengne P., Antonio-Nkondjio, C., Awono- Anopheles culicifacies breeding in polluted water Ambene, H.P., Simard, F., awolola, T.S. & bodies in Trincomalee district of Sri Lanka, Fontenille, D. (2007), Molecular identification of Malaria Journal, 14 (2015) 215. three closely related members of the mosquito species complex, Anopheles moucheti, by [24] Hackett B.J, Gimnig J, Guelbeogo W, Costantini micochondrial and ribosomal DNA C, Koekemoer LL, Coetzee M, Collins FH, polymorphism, Medical and Veterinary Besansky NJ, (2000) Ribosomal DNA internal Entomology, 21, 177-182. transcribed spacer (ITS2) sequences differentiate

[34] Kengne, P., Trung, H.D., Baimai, V., Coosemans, Multiplex PCR assay for malaria vector Anopheles M. & Manguin, S. A multiplex PCR-based method minimus and four related species in the Myzomyia derived from random amplified polymorphic DNA series from Southeast Asia. Medical and (RAPD) markers for the identification of species of Veterinary Entomology, 17 (2003) 423-428. the Anopheles minimus group in Southeast Asia. [43] Prakash A, Walton C, Bhattacharyya DR, Insect Molecular Biology, 10 (2001) 427–435. Loughlin SO, Mohapatra PK, Mahanta J., [35] Koekemoer, L.L., Kamau, L., hunt, R.H., and Molecular characterization and species Coetzee, M. A cocktail polymerase chain reaction identification of the Anopheles dirus and assay to identify members of the Anopheles Anopheles minimus complexes in north-east India funestus group, The American Society of Tropical using rDNA ITS-2. Acta Tropica, 100 (2006)156- Medicine and Hygiene, 6(6), (2001) 804-811. 161

[36] Kothai Kaveevendan, Development of Cleaved [44] Proft J., Walter A. and Kampen H., Identification Amplified Polymorphic DNA (CAP) assay to of six sibling species of the Anopheles distinguish the subpictus of Anopheles subpictus in maculipennis complex (Diptera: Culicidae) by a Sri Lanka, M.Sc. thesis, Post Graduate Institute of polymerase chain reaction assay, Parasitol Res, 85 Science, University of Peradeniya, Sri Lanka, (1999) 837-843. 2016. [45] Rubio A.L., Vasco J.s., Marcet P.L., Molina N. R., [37] Kumar N. P, Rajavel AR, Natarajan R, Caceres L., Porter C. and Uribe s., (2016) Use of Jambulingam P. DNA Barcodes Can Distinguish DNA barcoding to distinguish the malaria vector Species of Indian Mosquitoes (Diptera: Culicidae). Anopheles neivai in Colombia, Zoo taxa, 4175(4) Journal of Medical Entomology, 44(1) (2007) 1-7. (2016) 377-389.

[38] Matson R, Tong Rios C, Banda Chavez C, Gilman [46] Rueanghiran C, Apiwathnasorn C, Sangthong P, R, Florin D, Lopez Sifuentes V, Cardenas Greffa Samung Y, Ruangsittichai J., Utility of a set of R, Fernandez R, Yori P, Velasquez Portocarrero D, conserved mitochondrial cytochrome oxidase Vinetz J, Kosek M. Improved molecular technique subunit I gene primers for Mansonia annulata for the differentiation of neotropical Anopheline identification. Southeast Asian J Trop Med Public species. Am J Trop Med Hyg., 78 (2008) 492–498. Health, 42 (2011) 1381 1387

[39] Manonmani AM, Sadanandane C, Sahu SS, [47] Saiki, R.K., Scharf, S.J., Faloona, F., Mullis, K.B., Mathivanan A, Jambulingam P. rDNA-ITS2-PCR Horn, G.T., Erlich, H.A. and Arnheim, N., assay for grouping the cryptic species of Enzymatic amplification of ß-globin genomic Anopheles culicifacies complex (Diptera: sequences and restriction site analysis for Culicidae). Acta Tropica, 104 (2007) 72-77 diagnosis of sickle cell anemia. Science 230 (1985)1350. [40] Manonmani A, Townson H, Adeniran T, Jambulingam P, Sahu S, Vijayakumar T. (2001) [48] Scott J.A., Broodon W.B. and Collins R.H., rDNA-ITS2 polymerase chain reaction assay for Identification of single specimens of the the sibling species of Anopheles fluviatilis. Acta Anopheles gambiae complex by the Polymerase Tropica, 78 (2001) 3-9. Chain Reaction, Am.J.Trop. Med. Hyg, 49(4) (1993) 520- 529. [41] Paskewitz, S.M. & Collins, F.H., Use of the polymerase chain reaction to identify mosquito [49] Sharpe, R.G., Hims, M.M., Harbach, R.E. & species of the Anopheles gambiae complex. Butlin, R.K. (1999) PCR-based methods for Medical and Veterinary Entomology, 4 (1990) identification of species of the Anopheles minimus 367–373. group: allele-specific amplification and single- strand conformation polymorphism. Medical and [42] Phuc,H.K., Ball, A.J., Son, L., Hanh, N.V., Veterinary Entomology, 13(3) (1999) 265–273. Tu,N.D., Lien, N.G., Verardi,A., & Townson, H.,

[50] Singh O.P, Dinesh Chandra, Nutan Nanda, K. [57] Walton C., Handley J.M., Kuvangadilok C., Raghavendra, Sujatha Sunil, S. K. Sharma, V. K. Collins F.H., Harbach R., Baimai V. and Butlin Dua, and Sarala K. Subbarao., Differentiation of R.K., Identification of five species of the members of the anopheles fluviatilis species Anopheles dirus complex from Thailand, using complex by an allele-specific polymerase chain allele-specific polymerase chain reaction, Medical reaction based on 28s ribosomal DNA sequences, Veterinary Entomology, 13 (1999) 24-32. Am. J. Trop. Med. Hyg., 70(1) (2004a) 27–32 [58] 58. Weeraratne. T.C, Surendran. S.N, Lisa J. R, [51] Singh O.P, Chandra D, Nanda N, Sharma SK, Htun Charles S. W, Devika B.M.P, Catherine W. and PT, Adak T., On the conspecificity of Anopheles Karunaratne.S.H.P., (2017), Molecular fluviatilis species S with Anopheles minimus characterization of Anopheline (Diptera: species C. Journal of Biosciences, 31(5) (2006) Culicidae) mosquitoes from eight geographical 671 -677. locations of Sri Lanka, Malaria Journal,16 (2017) 234. [52] Singh O.P, Goswami G, Nanda N, Raghavendra K, Chandra D, Subbarao SK., An allele -specific [59] 59. Welsh, J. and McClelland, M. (1990) polymerase chain reaction assay for the Fingerprinting genomes using PCR with arbitrary differentiation of members of the Anopheles primers. Nucl Acids Res 18: 7213- 721 8. culicifacies complex. Journal of Biosciences, 29 [60] 60. Wesson, D.M., Porter, C.H. and Collins, (2004b) 275 -280. F.H., Sequence and secondary structure [53] Van Bortel, W., Trung,H. D., Roelants, P., comparisons of ITS rDNA in mosquitoes (Diptera: Harbach, R. E., Backelijau, T., & Coosemans, M., Culicidae). Mol Phyl Evoll: 253 (1992) 269. Molecular identification of Anopheles minimus s.l [61] 61. Wilkerson, R.C., Parsons, T.J., Albright, beyond distinguishing the members of the species D.G., Klein, T.A., & Braun, M.J., Random complex. Insect Molecular Biology, 9 (3) (2000) amplified polymorphic DNA (RAPD) markers 335-340. readily distinguish cryptic mosquito species [54] Van Bortel, W., Sochanta, T., Harbach, R.E., (Diptera: Culicidae: Anopheles), Insect Molecular Socheat, D., Roelants, P., Backeljau, T. & Biology, 1 (4) (1993) 205-211. Coosemans, M., Presence of Anopheles [62] 62. Williams, J.G.K., Kubelik, A.R., Livak, K.J., culicifacies B in Cambodia established by the PCR Rafalski, J.A. and Tingey, S.V., DNA RFLP assay developed for the identification of polymorphisms amplified by arbitrary primers are Anopheles minimus species A and C and four useful as genetic markers. Nucl Acids Res 18 related species. Medical and Veterinary (1990) 6531-6535. Entomology, 16 (2002) 329–334.

[55] Varun Tyagi, Ajay K. Sharma, Ruchi Yadav, T. Adak, D. Sukumaran, O. P. Agrawal, Vijay Veer., Implication of Random Amplified Polymorphic DNA method for differentiating Anopheles culicifacies sibling species, European Journal of Biotechnology and Bioscience, 8 (2015) 47-54.

[56] Vezenegho S.B., Bass C., Puinean m., Williamson M.S., Field L.M., Coetzee M. and Koekemoer L.L., Development of multiplex real – time PCR assays for identification of members of the Anopleles funestus species group, Malaria Journal, 8 (2009) 282.

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