J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 Original Article Evaluation of the mtDNA-COII Region Based Species Specific Assay for Iden- tifying Members of the Anopheles culicifacies Species Complex *Arulsamy Mary Manonmani, Ashok Kumar Mathivanan, Candassamy Sadanandane, Purushothaman Jambulingam Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, India (Received 29 Feb 2012; accepted 15 May 2013) Abstract Background: Anopheles culicifacies, a major malarial vector has been recognized as a complex of five sibling spe- cies, A, B, C, D and E. These sibling species exhibit varied vectorial capacity, host specificity and susceptibility to malarial parasites/ insecticides. In this study, a PCR assay developed earlier for distinguishing the five individual species was validated on samples of An. culicifacies collected from various parts of India. Methods: The samples were initially screened using the rDNA-ITS2 region based primers which categorised the samples into either A/D group or B/C/E group. A proportion of samples belonging to each group were subjected to the mtDNA-COII PCR assay for identifying individual species. Results: Among the 615 samples analysed by rDNA-ITS2 PCR assay, 303 were found to belong to A/D group and 299 to B/C/E group while 13 turned negative. Among 163 samples belonging to A/D group, only one sample dis- played the profile characteristic of species A and among the 176 samples falling in the B/C/E group, 51 were identi- fied as species B, 14 as species C and 41 as species E respectively by the mtDNA-COII PCR assay. Samples exhib- iting products diagnostic of B/C/E, when subjected to PCR-RFLP assay identified 15 samples as species E. Conclusion: Validation of the mtDNA-COII PCR assay on large number of samples showed that this technique can- not be used universally to distinguish the 5 members of this species complex, as it has been designed based on mi- nor/single base differences observed in the COII region. Keywords: Anopheles culicifacies, COII PCR assay, rDNA-ITS2 PCR assay, Sibling species Introduction Anopheles culicifacies, an important mos- quito species occurring in the Indian sub- continent, is reported as a complex of five sibling species, A to E. All of them except species B are vectors of malaria (Sunil et al. 2004). Though morphologically similar, mem- bers of the complex show ecological and be- havioral differences which affect their vec- torial capacity (Subbarao 1998). Identifica- tion of different members is possible based on the variable banding pattern in the X chro- mosome and chromosome arm 2, and also structural differences in the male mitotic kar- yotypes. But these observations require speci- fically staged specimens and hence cannot be applied to the entire sample collection. Sev- eral PCR assays were developed based on the sequences of the D2, D3 and ITS2 region of the ribosomal DNA, which were able to categorize the 5 members into two groups, viz, A/D group and B/C/E group. Recently, a three step PCR assay was developed by Goswami et al. (2006), based on the differ- ences in the COII region of the mitochondrial DNA for identifying the individual members of this species complex. It was later reduced to a two step process by Manonmani et al. (2007), by replacing the ITS2 and RFLP steps by a single ITS2 group diagnostic assay. *Corresponding author: Dr Arulsamy Mary Manonmani, E-mail: ammanonmani@yahoo.com 154 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 This paper presents the findings of evalu- ation of this two step PCR assay using field specimens of An. culicifacies collected from various parts of India. Materials and Methods Mosquito collection Adult samples of An. culicifacies were col- lected from Malkangiri District, Orissa State, Alwar District, Rajasthan State, Tiruvanamalai and Ramanathapuram districts, Tamil Nadu state (Fig. 1) (Table 1). Daytime resting mos- quito collections were done between 6:00 to 8:00AM using aspirators, while all night col- lections were done between 6:00PM to 6:00AM by using light traps (Gunasekaran et al. 1994). The collections were done from human dwellings and cattle sheds. Samples of An. culicifacies from all the catches were identified based on the taxonomic keys of Christophers (1933). DNA extraction and PCR assays DNA was extracted from the thoracic re- gion of each individual mosquito sample us- ing Genelute mammalian genomic DNA ex- traction kit (Sigma, St. Louis, MO, USA). The DNA pellet was dissolved in 50µ l of TE (Tris/EDTA) buffer and stored at -20 °C. Two PCR reactions were carried out on each DNA sample. Using the rDNA-ITS2-PCR assay described by Manonmani et al. (2007), samples were initially grouped either as A/D or B/C/E. These samples were then sub- jected to mtDNA-COII PCR assay reported by Goswami et al. (2006). Samples grouped as A/D were subjected to the AD-PCR assay while those grouped as B/C/E were sub- jected to the BCE-PCR assay. Details related to the primers used in these PCR assays and the expected products are given in Table 2. The amplified products were electrophoresed on an ethidium bromide stained 1.5% agarose gel, along with DNA molecular weight mark- er (Genei, Bangalore, India) and visualized under UV light. Amplification of the COII region The COII region of the mtDNA for 5 in- dividuals each of species B, C and E, identi- fied cytologically was amplified using a pair of primers, COIIF: 5’-AGAGCTTCTCCTTTAA TGGAACA-3’ and COIIR: 5’-CAATTGGTA TAAAACTATGATTTG-3’ respectively. The reaction mixture comprised 4.5 mM MgCl2, 200 µ M of each dNTP’s (GE healthcare UK limited, Buckinghamshire, UK), 10 µM of each primer (Metabion, Martinsried, Germany), 2.5 µ l of Taq buffer and 2 units of Taq polymer- ase (Finnzyme, Espoo, Finland). To this mix- ture was added 1/100th of the DNA from a whole mosquito and the reaction mixture was made up to 25 µ l volume using deionized water. These reactions were amplified in a Bio-Rad cycler PCR machine (Biorad, Cali- fornia, USA). PCR conditions were: denatura- tion at 94 °C for 4 min. followed by 40 cycles of denaturation at 95 °C for 40 sec, annealing at 50 °C for 1 min, extension at 68 °C for 1 min and a final extension step at 72 °C for 10 min. After checking a portion of each amplifi- cation product by gel electrophoresis, the re- mainder of the sample was purified using Qiaquick PCR purification kit (Qiagen, Hil- den, Germany). The concentration of DNA was quantified and subjected to sequencing in an ABI automatic sequencer (Applied Biosystems, California, USA), using the amplification primers to obtain sequence from both strands. PCR-RFLP Samples which exhibited products that are diagnostic of species B, C and E were subject- ed to COII PCR assay followed by DdeI di- gestion, which is reported to seperate species E from B and C (Goswami et al. 2005). Each 20µL digest consisted of 17.5µ L PCR product, 2µL 10x Buffer and 0.5 U of DdeI enzyme (Sigma, St. Louis, MO, USA). For each sam- ple, paired control reactions were set up that 155 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 contained only PCR product and 10x buffer. Reactions were incubated at 37 °C overnight, followed by heat inactivation at 65 °C for 10 minutes. Digested and undigested PCR prod- ucts were electrophoresed and observed as mentioned earlier. Results The results of the validation of the rDNA- ITS2 PCR assay and mtDNA-COII PCR as- say is given in Table 3. Collections from Tiruvanamalai District of Tamil Nadu repre- sented individuals belonging to BCE group in major proportion (86.8%) while those from Alwar District of Rajasthan represented indi- viduals belonging to AD group in major proportion (87.7%) (Table 3). All the An. culicifacies collections from Malkangiri dis- trict of Orissa and Ramanathapuram District of Tamil Nadu belonged to the BCE group. Thirteen (2.1%) of the samples did not be- long to either of the groups. About half the number of samples from each group, repre- senting different types of collections/ habi- tats/ villages were randomly selected and sub- jected to the COII-PCR assay developed by Goswami et al. (2006). Among the 163 sam- ples from Tiruvanamalai and Alwar districts, belonging to the AD group, one was found to be species A, 160 were species D and 2 be- longed to neither. The single sample belong- ing to species A originated from Tiruvanamalai District. The samples which belonged to the BCE group when subjected to the species di- agnostic mt-DNA PCR assays showed that all the 3 sibling species were present in the 4 dis- tricts, though species C was seen in lesser num- bers in Tiruvanamalai, Alwar and Malkangiri districts respectively. However, samples ex- hibiting products diagnostic of all the 3 spe- cies were found in sizeable numbers (36.4%) (Fig. 2). Sequencing of the mtDNA-COII region showed that polymorphism was noticed in the nucleotide positions which were diagnos- tic. With primer CR designed to amplify spe- cies C, it was noticed that the base ’A’ at po- sition 171 which is the only diagnostic point differentiating species C from B was found to be ’G’ in all the species C samples se- quenced by us (Fig. 3). With primer ER, the diagnostic point ‘T’ and ‘C’ in species B and E was found to be ‘T’ in one of our samples belonging to species E (Fig. 4). With the pri- mer BCR, the only diagnostic point ’G’ at position 334 differentiating species B and C from E was found to exhibit polymorphism by being either ’G’ or ’A’ (Fig. 5). Samples which exhibited products diag- nostic of species B, C and E were subjected to the PCR-RFLP assay. Amplification of the mtDNA-COII region gave a band of size 573bp. This product, when subjected to di- gestion with the restriction enzyme, DdeI produced 3 fragments of sizes 300, 156 and 117bp respectively for species E and 2 frag- ments of sizes 456 and 117bp respectively for species B and C. These fragments were as per the predicted restricted sites of this en- zyme. However, only 15 samples out of 64 yielded fragments corresponding to E, 15 pro- duced fragments corresponding to species B and C and the products of 34 samples re- mained uncut (Table 3). 156 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 Fig. 1. Map showing the collection sites of Anopheles culicifacies s.l. 157 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 Fig. 2. COII species diagnostic PCR assay for Anopheles culicifacies. Lane 1–3: sibling D (359bp+166bp), Lane 4: sibling A (359bp), Lane 5: 100bp ladder, Lane 6, 11: B/C/E (248bp+178bp+95bp), Lane 8: sibling C (248bp+95bp), Lane 10: sibling B (248bp), Lane 7, 9, 12–15: sibling E (248bp+178bp). Fig. 4. ER primer site of the mtDNA-COII sequences for species B, C and E of the Anopheles culicifacies complex. Accession numbers AJ518810, AJ519493 and AJ534646 (Goswami et al. 2006). Accession numbers COXIIAcSL-B1 and COXIIAcSL-E1 (Su- rendran et al. 2006). Accesssion numbers HQ377221- HQ377228: mtDNA COII sequences of species B, C and E generated in the current study. Fig. 3. CR primer site of the mtDNA-COII sequences of species B and C of the Anopheles culicifacies complex. Accession numbers AJ518810 and AJ519493 Goswami et al. (2006). Accession numbers HQ377221-HQ377225: mtDNA-COII sequences of B and C generated in the current study. Fig. 5. BCR primer site of the mtDNA-COII se- quences for species B, C and E of the Anopheles culicifacies complex. Accession numbers AJ518810, AJ519493andAJ534646 (Goswami et al. 2006). Ac- cession numbers COXIIAcSL-B1 and COXIIAcSL- E1 (Surendran et al. 2006). Accession numbers HQ377221-HQ377228: mtDNA COII sequences of species B, C and E generated in the current study. Fig. 6. ITS2 region based phylogenetic tree (Neighbor joining method) for Anopheles culicifacies showing two dis- tinct clades of A/D and B/C/E with an out group Anopheles aconitus 158 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 Table 1. Details of collected Anopheles culicifacies samples District and State Geographical co-ordinates Village Habitat Type of collection No. Tiruvanamalai Tamilnadu Lat: 11°55'–13°15'N Long: 78°20'–79°50'E Kolamanjanur Cattle shed Light trap 3 Kolamanjanur Cattle shed Hand catch 141 Kolamanjanur Human dwelling Hand catch 34 Labour colony Cattle shed Hand catch 22 Labour colony Human dwelling Hand catch 5 Ramanathapuram Tamilnadu Lat: 9°05'–9°50'N Long: 78°10'–79°27'E Karaiyur Human dwelling Hand catch 13 Rajakoil Human dwelling Hand catch 11 Tharavaithoppu Human dwelling Hand catch 36 Alwar Rajasthan Lat: 27°03'–28°14'N Long: 76°07'–77°13'E Balana Cattle shed Hand catch 20 Balana Human dwelling Hand catch 27 Lalpura Cattle shed Hand catch 109 Lalpura Human dwelling Hand catch 101 Indok Cattle shed Hand catch 2 Sawar Cattle shed Hand catch 40 Sawar Human dwelling Hand catch 25 Malkangiri Orissa Lat: 17°45'–18°40'N Long: 81°10'–82°00'E Teakguda Cattle shed Hand catch 26 Total samples col- lected 615 Lat: Latitude, Long: Longitude Table 2. Details of primers used in PCR assays S. No Primer Name/ Sequence (5’-3’) Tm value GC Content PCR product size (bp) 1 5.8S - ATCACTCGGCTCATGGATCG 60 55 AD group: 409 BCE group: 253 2 Sp AD - CAGTGCTGCAAACCACCACTTAT 64 45.8 3 Sp BCE - TGTTAGTAGGCTGCCGGGGTTC 66 59.1 4 ADF - CTAATCGATATTTATTACAC 48 25 Species A: 359 Species D: 359+166 5 ADR - TTACTCCTAAAGAAGGC 48 41.2 6 DF - TTAGAGTTTGATTCTTAC 45 27.8 7 BCEF - AAATTATTTGAACAGTATTG 46 20 Species B: 248 Species C: 248+95 Species E: 248+1788 BCR - TTATTTATTGGTAAAACAAC 46 20 9 CR - AAGGAGTATTAATTTCGTCT 49 31.6 10 ER - GTAAGAATCAAATTCTAAG 47 26.3 1–3: Primers for rDNA-ITS2 group diagnostic PCR assays (Manonmani et al. 2007) 4–10: Primers for mtDNA-COII species diagnostic PCR assays (Goswami et al. 2006) 159 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 Table 3. Evaluation of PCR assays for identification of individual members of Anopheles culicifacies Area rDNA-ITS2 PCR assay mtDNA-COII PCR assay PCR-RFLP A/D B/C/E T ot al c ol le ct ed A /D gr ou p B /C /E gr ou p -v e N u m b er s p ro ce ss ed A D -v e N u m b er s p ro ce ss ed B C E B /C /E -v e B /C /E E B /C Tiruvanamalai 205 19 178 8 19 1 17 1 81 25 3 17 32 4 32 7 7 Ramanathapur am 60 0 58 2 0 0 0 0 43 15 9 7 11 1 11 3 1 Alwar 324 284 38 2 144 0 14 3 1 27 4 1 10 12 0 12 4 1 Malkangiri 26 0 25 1 0 0 0 0 25 7 1 7 9 1 9 1 6 Total 615 303 299 13 163 1 16 0 2 176 51 1 4 41 64 6 64 1 5 15 -ve = negative Fig. 7. D3 region based phylogenetic tree (Neighbor joining method) for Anopheles culicifacies showing two dis- tinct clades of A/D and B/C/E with an out group Anopheles aconitus Discussion Accurate identification and knowledge about the distribution of vector and non-vector members of species complexes is required for planning effective control measures. Hence, development of molecular diagnostics for the individual species of the An. culicifacies com- plex was felt necessary as 4 of these species have been incriminated as vectors of malaria in India. Goswami et al. (2006) have devel- oped a three step PCR assay for the identify cation of 5 individual members of this species complex, which involved (1) amplification of the rDNA-ITS2 region, (2) restriction diges- tion of the ITS2 product and (3) PCR assay based on the COII region of the mtDNA. This three step PCR assay was later reduced by developing a PCR assay based on the rDNA- ITS2 region (Manonmani et al. 2007) which grouped the 5 species into the same 2 cate- gories as obtained after the first two steps of Goswami et al. (2006). In the present study this two step PCR assay was evaluated on 160 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 samples of An. culicifacies collected from dif- ferent regions of India. Of the 615 samples evaluated, 49.3% were categorized as A/D while 48.6% were B/C/E. Selected samples (163) from the A/D group when subjected to mtDNA-COII PCR assay showed that 0.6% were species A and 98.2% were species D. Likewise, when 176 samples belonging to the B/C/E category were analyzed, 29%, 8% and 23.3% exhibited products diagnostic for spe- cies B, C and E respectively, while 28% ex- hibited products diagnostic of all the 3 species. The specificity of the COII-PCR assay in identifying the individual member within the 2 groups was low. This can be attributed to single base differences in the COII region which has been used for the design of spe- cies diagnostic assay. Similar problem was encountered when single base differences of the rDNA-ITS2 region was used for design- ing PCR assay for species A and C of the An. minimus complex (Garros et al. 2004). The major role played by members of the An. culicifacies complex in malaria trans- mission in several regions of India has com- pelled the authors to use the minor differ- ences in designing these primers. Hence, the basic requirements such as 18–24 nucleotide length, Tm value between 56–62 and GC content between 45–60% could not be met while designing the 7 species diagnostic pri- mers (Table 3). GC content, melting and an- nealing temperatures being dependent on one another play a crucial role in the efficiency and sensitivity of PCR assays (Rychlik et al. 1990, Dieffenbach et al. 1993, He et al. 1994). In the case of AD-PCR assay, species A should exhibit a single product of size 359bp while species D should produce 2 products of size 359 and 166 bp respectively. But in this study, all samples produced products characteristic of species D. Goswami et al. (2006), while evaluating this assay found that the AD-PCR results did not agree with the earlier reports on the distribution of these members in Kheda and Sonepat, India. They attributed this to the polymorphic nature of the i1 inversion in species A in these areas and hence samples collected from these re- gions, whether homozygous or heterozygous for this inversion, have been identified as species D. This might be the reason for all samples being identified as species D in our study also. The i1 homozygotes could be spe- cies D or polymorphic forms of species A. Hence, the AD-PCR assay will not be useful in areas where the i1 inversion is polymor- phic. Vasantha et al. (1991) using i1 inversion for detecting species D found that in certain areas, presence of species D becomes evident only due to the deficiency of heterozygotes. Hence, it appears that cytotaxonomy also, can- not be used universally in the identification of species A and D of this species complex. In our study, sequencing of the mtDNA- COII region for species B, C and E showed polymorphism in the nucleotide positions used for designing of diagnostic primers. Similar observations were made by Surendran et al. (2006) while characterizing species B and E of this species complex from Srilanka. Though they observed acrocentric and submetacentric Y chromosomes in species B and E as re- ported by Kar et al. (1999), they were not able to correlate these results with those ob- tained by the PCR-RFLP assay (Goswami et al. 2005). This was because several bases of the COII region were found to exhibit poly- morphism. In particular, the base ‘C’ at posi- tion 255 in the COII gene which is the only diagnostic point differentiating species E from B as well as serving as the restriction site for the enzyme, Dde I, itself exhibited polymor- phism. This base which was ’T’ and ’C’ in spe- cies B and E respectively, in the samples stud- ied by Goswami et al. (2006) was found to be `C’ in both the species from Srilanka. Hence, the species diagnostic PCR assay and PCR- RFLP cannot be used with 100 % accuracy for distinguishing species B and E. While using cytotaxonomical methods for identifying species B and C of the An. 161 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 culicifacies complex, B/C heterozygotes were seen in forested and deforested villages of Northern Orissa, which represented inter- specific hybrids obtained due to breakdown of premating barriers between species B and C (Nanda et al. 2000). Phylogenetic analysis of the ITS2 and D3 region sequences (Figs. 6, 7) showed that the 5 members fell into 2 clades, one represent- ing species B, C and E and the other repre- senting species A and D. Similar categoriza- tion has been made by Dassanayake et al. (2008) and Raghavendra et al. (2009) based on the sequences of the ITS2 and D2 regions of the rDNA respectively. Hence, species A and B of An. culicifacies reported by Green and Miles (1980) appears to be the two dis- tinct sibling species of this malaria vector while the others namely species D and species C and E seems to have diverged very recently from species A and species B respectively. Further, the fact that the members within the 2 groups have not yet developed post mating barriers (Raghavendra et al. 2009) shows that the speciation process among the members of the 2 groups is not yet complete. The present study has shown that the mtDNA-COII PCR assays cannot be used universally to distinguish the members of An. culicifacies complex, as has been observed by Surendran et al. (2006) in Srilanka as well. Acknowledgements The authors are grateful to Dr SL Hoti, Sci- entist `F’ and Dr AR Rajavel, Scientist ’D’, Vector Control Research Centre, Puducherry for critically reviewing the manuscript. The technical assistance rendered by Mr S Murugaraj and staff of the Vector Biology and Control section is gratefully acknowl- edged. This study was supported by a re- search grant from the Department of Science and Technology (DST), Govornment of In- dia. The authors declare that there is no con- flict of interests. References Christophers SR (1933) The fauna of British In- dia. Diptera, Vol. IV. Family-Culicidae. Tribe Anophelini. Today and Tomor- row’s Printers and Publishers, New Delhi. Dassanayake RS, Gunawardene YI, Silva BD (2008) ITS-2 secondary structures and phylogeny of Anopheles culicifacies spe- cies. Bioinformation. 2: 456–460. Dieffenbach CW, Lowe TMJ, Dveksler DS (1993) General concepts for PCR pri- mer design. Genome Res. 3: S30–S37. Garros C, Koekemoer LL, Coetzee M, Coosemans M, Manguin S (2004) A Single multiplex assay to identify ma- jor malaria vectors within the African Anopheles funestus and the oriental Anopheles minimus groups. Am J Trop Med Hyg. 70: 583–590. Goswami G, Raghavendra K, Nanda N, Gakhar SK, Subbarao SK (2005) PCR–RFLP of mitochondrial cytochrome oxidase subu- nit II and ITS2 of ribosomal DNA: mark- ers for the identification of members of Anopheles culicifacies complex (Diptera: Culicidae). Acta Trop. 95: 92–99. Goswami G, Singh OP, Nanda N, Raghavendra K, Gakhar SK, Subbarao SK (2006) Identification of all members of the Anopheles culicifacies complex using allele-specific polymerase chain reac- tion assays. Am J Trop Med Hyg. 75: 454–460. Green CA, Miles (1980) Chromosomal evi- dence for sibling species of the malaria vector Anopheles (Cellia) culicifacies Giles. J Trop Med Hyg. 83: 75–78. Gunasekaran K, Jambulingam P, Sadanandane C, Sahu SS, Das PK (1994) reliability of light trap sampling for Anopheles fluviatilis, a vector of malaria. Acta Trop. 58: 1–11. He Q, Marjamaki M, Soini H, Mertsola J, Viljanen MK (1994) Primers are decisive 162 J Arthropod-Borne Dis, December 2013, 7(2): 154–163 AM Manonmani et al.: Evaluation of the … http://jad.tums.ac.ir Published Online: August 31, 2013 for sensitivity of PCR. Biotechniques. 17: 82–87. Kar I, Subbarao SK, Eapen A, Ravindran, J, Satyanarayana TS, Raghavendra K, Nanda N, Sharma VP (1999) Evidence for a new malaria vector species, spe- cies E within the Anopheles culicifacies complex (Diptera: Culicidae). J Med Entomol. 36: 595–600. Manonmani AM, Sadanandane C, Sahu SS, Mathivanan A, Jambulingam P (2007) rDNA-ITS2-PCR assay for grouping the cryptic species of Anopheles culicifacies complex. (Diptera: Culicidae). Acta Trop. 104: 72–77. Nanda N, Yadav RS, Subbarao SK, Joshi H, Sharma VP (2000) Studies on Anopheles fluviatilis and Anopheles culicifacies sib- ling species in relation to malaria in forested hilly and deforested riverine ecosystems in northern Orissa, India. J Am Mosq Control Assoc. 16: 199–205. Raghavendra K, Cornel AJ, Reddy BP, Collins FH, Nanda N, Chandra D, Verma V, Dash AP, Subbarao SK (2009) Multiplex PCR assay and phylogenetic analysis of sequences derived from D2 domain of 28S rDNA distinguished members of the Anopheles culicifacies complex into two groups, A/D and B/C/E. Infect Genet Evol. 9: 271–277. Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temper- ature for DNA amplification in vitro. Nucleic Acids Res. 18: 6409–6412. Subbarao SK (1998) Anopheline species com- plexes in South East Asia. Technical Publication, Serial Number 18, World Health Organization, Regional Office for South East Asia, New Delhi. Sunil S, Raghavendra K, Singh OP, Malhotra P, Huang Y, Zheng L, Subbarao SK (2004) Isolation and characterization of microsatellites markers from malaria vec- tor, Anopheles culicifacies. Mol Ecol Notes. 4: 440–442. Surendran SN, Hawkes NJ, Steven A, Hem- ingway J, Ramasamy R (2006) Molec- ular studies of Anopheles culicifacies (Diptera: Culicidae) in Sri Lanka: sibling species B and E show sequence iden- tity at multiple loci. Euro J Entomol. 103: 233–237. Vasantha K, Subbarao SK, Adak T, Sharma VP (1991) Anopheles culicifacies complex: population cytogenetic evidence for spe- cies D (Diptera: Culicidae). Ann Entomol Soc Am. 84: 531–536. 163