MORPHOLOGY, SYSTEMATICS, EVOLUTION DNA Barcodes Can Distinguish Species of Indian Mosquitoes (Diptera: Culicidae) N. PRADEEP KUMAR,1 A. R. RAJAVEL, R. NATARAJAN, AND P. JAMBULINGAM Vector Control Research Centre (Indian Council of Medical Research), Medical Complex, Indira Nagar, Pondicherry, 605006 India J. Med. Entomol. 44(1): 1Ð7 (2007) KEY WORDS DNA barcodes, Culicidae, taxonomy, India Description and study of biodiversity is an important aspect to understand the intricate evolutionary trends of life. Linnaean classiÞcation of animals and plants remains as an important step made toward this goal, and with that commenced the systematics based on morphological characteristics, which is being followed for more than two centuries. However, only ⬇10% of extant species on earth (10 Ð15 million) are known to science so far (Besansky et al. 2003, Pennisi 2003). Recent studies on DNA-based approaches show a promising trend in the rapid description of biodiversity (Hebert et al. 2003a; Hebert and Gregory 2005). HebertÕs approach to DNA barcode the entire animal kingdom based on single gene could be advantageous, being economic and practicable, provided the analysis of the DNA sequences of this gene may yield a useful tool for species identiÞcation in different species of the Animal Kingdom. The mitochondrial genome was selected for this approach, owing to its advantages such as maternal lineage, lack of recombination, lack of “indels,” and higher mutation rates (Saccone et al. 1999). Among the mitochondrial genes, cytochrome c oxidase subunit 1 (COI) is reported to be the most conserved gene in the amino acid sequences and hence has distinct advantage for taxonomic studies (Knowlton and Weigt 1998). However, differences of opinion exist in adopting this technology toward spe1 Corresponding author, e-mail: kumarnp@yahoo.com. cies identiÞcation, and the ongoing studies on the construction of barcodes of life (Ballard and Whitlock 2004, CBOL 2005). So far, this methodology had been used to testify the identiÞcation of widely varied animals as Lepidoptera (phylum Invertebrata, class Insecta) (Hebert et al. 2003a,b, 2004a; Hajibabaei et al. 2006), birds (phylum Vertebrata, class Aves) (Hebert et al. 2004b), and tachinid parasitoids successfully. Also, this methodology is being currently followed for barcoding animals of different groups such as Þshes and primates (CBOL 2005, Lorenz et al. 2005). However, no studies exist on the utility of DNA barcodes for identiÞcation of mosquitoes, which comprise ⬇3,500 species globally. The morphological identiÞcation keys used currently for identiÞcation of mosquitoes are mainly speciÞc to few developmental stages (imaginal and fourth instars) only, which makes it difÞcult to identify other stages of development collected in the Þeld, without rearing them in the laboratory. Also, many adult specimens collected in routine disease surveillance programs get damaged (they loose important identiÞcation characteristics such as bristles and scales) and hence are impossible to identify. In addition, existence of sibling species has further complicated the species identiÞcation of mosquitoes. These sibling species are morphologically indistinguishable and could be identiÞed only by cytotaxonomically using polytene chromosomes, speciÞc to certain tissues in particular developmental stages. 0022-2585/07/0001Ð0007$04.00/0 䉷 2007 Entomological Society of America Downloaded from http://jme.oxfordjournals.org/ by guest on January 17, 2017 ABSTRACT Species identiÞcation of mosquitoes (Diptera: Culicidae) based on morphological characteristics remains often difÞcult in Þeld-collected mosquito specimens in vector-borne disease surveillance programs. The use of DNA barcodes has been proposed recently as a tool for identiÞcation of the species in many diverse groups of animals. However, the efÞcacy of this tool for mosquitoes remains unexplored. Hence, a study was undertaken to construct DNA barcodes for several species of mosquitoes prevalent in India, which included major vector species. In total, 111 specimens of mosquitoes belonging to 15 genera, morphologically identiÞed to be 63 species, were used. This number also included multiple specimens for 22 species. DNA barcode approach based on DNA sequences of mitochondrial cytochrome oxidase gene sequences could identify 62 species among these, in conÞrmation with the conventional taxonomy. However, two closely related species, Ochlerotatus portonovoensis (Tiwari & Hiriyan) and Ochlerotatus wardi (Reinert) could not be identiÞed as separate species based on DNA barcode approach, their lineages indicating negligible genetic divergence (Kimura two-parameter genetic distance ⫽ 0.0043). 2 JOURNAL OF MEDICAL ENTOMOLOGY Hence, standardization of tools that could identify mosquito species even from a small piece of tissue from any developmental stage would be of importance for the taxonomy of mosquitoes. Construction of DNA barcodes for each species of mosquito would provide an important tool for identiÞcation of mosquito species and may enable description of the species biodiversity of this important group of insects. Here, we present the Þrst report on construction of DNA barcodes for 63 species of mosquito species prevalent in India. Multiple specimens, collected from different regions of the country also were included in the study. Together, data on 111 DNA sequences of mosquito specimens collected from different regions of India was used in the study. Materials and Methods Þnal extension at 72⬚C for 10 min. The 50-l reaction included 1.5 U of Thermus aquaticus polymerase, 5 l of 10⫻ PCR buffer, 2.5 mM magnesium chloride, 2.5 l of Q solution (QIAGEN GmbH, Hilden, Germany), and 0.5 l of 10 pmol each of forward and reverse primers, along with the DNA of the mosquito species. A Blast search with the conserved DNA primers proposed by Hebert et al. (2003a) did not yield desirable similarity for different sequences of COI region of mosquitoes available with the GenBank. Hence, initially the primer set C1J-1718 (forward) and C1N-2191 (reverse) described by Simon et al. 1994 was used in the study. However, this DNA primer set could amplify only ⬇500 bp of COI DNA, and there was difÞculty in amplifying COI region of different genera used in the study. Hence, as proposed in the DNA Primer Design section of Laboratory Protocol for COI AmpliÞcation (CBOL 2005), a consensus DNA primer which could amplify ⬇700 bp of the COI was designed using Primer3 software (Whitehead/MIT Center for Genome Research, Cambridge, MA). Because the ampliÞed product of these DNA primers spans the region ampliÞed by the earlier primers (Simon et al. 1994) and that of HebertÕs 648-bp region, this DNA primer set was used subsequently in the study. Details of both the primer sets used in the study are as follows: 1) DNA primers (Simon et al. 1994): forward primer, 5⬘-GGAGGATTTGGAAATTGATTAGTT-3⬘ and reverse primer, 5⬘-CCCGGTAAAATTAAAATATAAACTTC-3⬘; and 2) DNA primers designed in the current study: forward primer, 5⬘-GGATTTGGAAATTGATTAGTTCCTT-3⬘ and reverse primer, 5⬘ AAAAATTTTAATTCCAGTTGGAACAGC 3⬘. These DNA primers were custom synthesized by Metabion (Martinsried, Germany) and were high-performance liquid chromatography puriÞed. DNA Sequencing and Analysis. The ampliÞed fragments were run on a 1% agarose gel to check the integrity of the fragments and the PCR product was puriÞed by QIAGEN GmbH PCR puriÞcation kit. The puriÞed products were eluted to 20 l of deionized water, and a portion of it was lyophilized in a Speed Vac concentrator (Thermo Electron Corporation, Waltham, MA) and was shipped to MWG (Edersberg, Germany/Microsynth, Balgach, Switzerland) for custom sequencing. Both reads (from forward primer as well as reverse primer) were done, and the sequences were analyzed as follows. The DNA sequences were subjected to alignment using ClustalW. Sequence divergences among individuals were quantiÞed by using the Kimura two-parameter distance model (Kimura 1980). A neighborhood joining (NJ) tree of K2P distances was created to provide a graphic representation of the clustering pattern among different species (Saitou and Nei 1987, Hajibabaei et al. 2006). These analyses of the sequences were conducted using MEGA version 3.1 software (Kumar et al. 2004). Results and Discussion All DNA extractions were done using adults, of which 70 were female and 41 were male. Of these Downloaded from http://jme.oxfordjournals.org/ by guest on January 17, 2017 Mosquito Specimens. Mosquito specimens used for constructing DNA barcodes were from collections made from different states for mosquito biodiversity study in India. Larval and adult collections of mosquitoes were done in the Þeld. When larvae were collected, they were reared individually to adults, and associated larval and pupal skins were mounted. The emerged adults were identiÞed morphologically and assigned a museum identiÞcation number and were used for DNA extraction with the corresponding larval and pupal skins deposited as voucher specimens in the Vector Control Research Centre Mosquito Museum (Rajavel et al. 2005). When collected as adults, females were isolated for oviposition, and the F1 generation adults were used for DNA extraction, with the corresponding larval and pupal skins deposited in the museum. In a few adults, DNA was extracted using legs of one side, with the adult retained in the museum as voucher specimen. When male specimens were used for DNA extraction, the genitalia was mounted and deposited in the museum. Species identiÞcation was done based on taxonomic keys (Christophers 1933, Barraud 1934, Huang 1972, Sirivanakarn 1976, Harrison 1980, Reuben et al. 1994). In sibling species complexes, specimens used were sensu lato, except for Anopheles subpictus complex, which included both sensu lato and morphologically identiÞed specimens (Suguna et al. 1994). DNA Extraction and Polymerase Chain Reaction (PCR). Total DNA from individual mosquitoes was extracted following a modiÞed method proposed by Collins et al. (1987). The DNA were pelleted, dissolved in water, and subjected to a phenol chloroform isoamyl extraction followed by chloroform. DNA were precipitated using NaAc/ethanol and dissolved in 30 l of deionized water (Sigma-Aldrich, St. Louis, MO). PCR was performed to amplify the 5⬘ COI region of mitochondrial DNA by using the following cycle in a Bio-Rad iCycler. PCR conditions were as follows: an initial denaturation of 5 min (95⬚C) was followed by Þve cycles of 94⬚C for 40 s (denaturation), 45⬚C for 1 min (annealing), and 72⬚C for 1 min (extension) and 35 cycles of 94⬚C for 40 s (denaturation), 51⬚C for 1 min (annealing), 72⬚C for 1 min (extension), and a Vol. 44, no. 1 Species Aedeomyia (Aedeomyia) catasticta Aedes (Aedimorphus) vexans Aedes (Diceromyia) iyengari Aedes (Fredwardsius) vittatus Aedes (Lorrainea) fumidus Aedes (Stegomyia) aegypti Aedes (Stegomyia) aegypti Aedes (Stegomyia) albopictus Aedes (Stegomyia) albopictus Aedes (Stegomyia) albopictus Aedes (Stegomyia) albopictus Anopheles (Anopheles) barbirostris Anopheles (Anopheles) peditaeniatus Anopheles (Cellia) aitkeni Anopheles (Cellia) annularis Anopheles (Cellia) culicifacies s.l. Anopheles (Cellia) culicifacies s.l. Anopheles (Cellia) fluviatilis s.l. Anopheles (Cellia) fluviatilis s.l. Anopheles (Cellia) fluviatilis s.l. Anopheles (Cellia) fluviatilis s.l. Anopheles (Cellia) fluviatilis s.l. Anopheles (Cellia) fluviatilis s.l. Anopheles (Cellia) fluviatilis s.l. Anopheles (Cellia) jamesi Anopheles (Cellia) jeyporiensis Anopheles (Cellia) jeyporiensis Anopheles (Cellia) jeyporiensis Anopheles (Cellia) jeyporiensis Anopheles (Cellia) jeyporiensis Anopheles (Cellia) maculatus Anopheles (Cellia) minimus s.l. Anopheles (Cellia) pallidus Anopheles (Cellia) pallidus Anopheles (Cellia) splendidus Anopheles (Cellia) stephensi Anopheles (Cellia) stephensi Anopheles (Cellia) stephensi Anopheles (Cellia) stephensi Anopheles (Cellia) stephensi Anopheles (Cellia) subpictus s.l. Anopheles (Cellia) subpictus s.l. Anopheles (Cellia) subpictus s.l. Anopheles (Cellia) subpictus s.l. Anopheles (Cellia) subpictus A Anopheles (Cellia) subpictus B Anopheles (Cellia) subpictus B Serial no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Pathirapuliyur Periakattupalayam Thannerbagi Moratandi Coringa Paari nagar Ennorekuppam Gorimedu Uppandara Saharpur Vikhroli Kizhoor Kedar road Saharpur Purthidigia Purthidigia Rameswaram Mamalapusi Attavalasa Govindapally Attavalasa Attavalasa Attavalasa Attavalasa Mailam Attavalasa Attavalasa Govindapally Attavalasa Attavalasa Mamalapusi Mamalapusi Gopalankadai Purthidigia Badakamurda Bommiapalayam Nagapattinam Pattukottai Bommiapalayam Pattukottai Mailam Mamalapusi Konthamur Moorthikuppam Moorthikuppam Bahoor Bahoor Locality State 12⬚ 07.197⬘ 79⬚ 36.282⬘ 11⬚ 50.927⬘ 79⬚ 48.295⬘ 12⬚ 53.580⬘ 74⬚ 48.920⬘ 11⬚ 58.502⬘ 79⬚ 47.460⬘ 16⬚ 52.972⬘ 82⬚ 15.070⬘ 11⬚ 57.056⬘ 79⬚ 48.632⬘ 13⬚ 13.784⬘ 80⬚ 19.578⬘ 11⬚ 57.096⬘ 79⬚ 47.613⬘ 12⬚ 52.189⬘ 74⬚ 52.103⬘ 21⬚ 35.695⬘ 85⬚ 24.936⬘ 19⬚ 05.774⬘ 72⬚ 56.430⬘ 11⬚ 53.344⬘ 79⬚ 41.010⬘ 11⬚ 59.413⬘ 79⬚ 28.283⬘ 21⬚ 35.877⬘ 85⬚ 24.959⬘ 22⬚ 09.337⬘ 85⬚ 33.759⬘ 22⬚ 09.337⬘ 85⬚ 33.759⬘ 09⬚ 15.24⬘ 79⬚ 16.44⬘ 21⬚ 33.358⬘ 85⬚ 28.697⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 18⬚ 30.363⬘ 82⬚ 15.397⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 12⬚ 07.555⬘ 79⬚ 36.694⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 18⬚ 30.363⬘ 82⬚ 15.397⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 18⬚ 30.326⬘ 82⬚ 01.179⬘ 21⬚ 33.463⬘ 85⬚ 28.650⬘ 21⬚ 33.358⬘ 85⬚ 28.697⬘ 11⬚ 55.704⬘ 79⬚ 45.767⬘ 22⬚ 09.337⬘ 85⬚ 33.759⬘ 22⬚ 11.626⬘ 85⬚ 31.510⬘ 11⬚ 59.638⬘ 79⬚ 51.055⬘ 10⬚ 52.467⬘ 79⬚ 50.119⬘ 11⬚ 14.46⬘ 79⬚ 43.36⬘ 11⬚ 59.638⬘ 79⬚ 51.055⬘ 11⬚ 14.46⬘ 79⬚ 43.36⬘ 12⬚ 07.555⬘ 79⬚ 36.694⬘ 21⬚ 33.370⬘ 85⬚ 28.670⬘ 12⬚ 08.068⬘ 79⬚ 43.507⬘ 11⬚ 47.364⬘ 79⬚ 47.690⬘ 11⬚ 47.364⬘ 79⬚ 47.690⬘ 11⬚ 47.667⬘ 79⬚ 45.687⬘ 11⬚ 47.667⬘ 79⬚ 45.687⬘ GPS coordinates Collection details of specimens Pondicherry Pondicherry Karnataka Pondicherry Andhra Pradesh Pondicherry Tamil Nadu Pondicherry Karnataka Orissa Maharashtra Pondicherry Pondicherry Orissa Orissa Orissa Tamil Nadu Orissa Orissa Orissa Orissa Orissa Orissa Orissa Pondicherry Orissa Orissa Orissa Orissa Orissa Orissa Orissa Pondicherry Orissa Orissa Pondicherry Tamil Nadu Tamil Nadu Pondicherry Tamil Nadu Pondicherry Orissa Pondicherry Pondicherry Pondicherry Pondicherry Pondicherry Details of mosquito specimens used for DNA barcoding and analysis VCRC Museum no. A11437 A11529 A10198 A11499 A11451 A11491 A13663 A11475 A10211 A10456 A11176 A11462 A11517 A10486 A11511 A11512 DB173 A11505 A12467 A12478 A12482 A12471 A12452 A12501 A11440 A12479 A12500 A12502 A12506 A12507 A10474 A11510 A11442 A11509 A11508 A11459 A13574 A13667 A11467 A13668 A11438 A11506 A11457 A11538 A11537 A11536 A11535 Habit/habitat Quarry pit Casuarina pit Tyre Cement tank Tree hole Pot Well Tar tin Tree hole Bamboo Resting on root Canal Road side pool Stream Resting in cattle shed Resting in cattle shed Resting in human dwelling Resting in human dwelling Resting in cattle shed Resting in human dwelling Resting in cattle shed Resting in cattle shed Resting in cattle shed Resting in cattle shed Pond Resting in cattle shed Resting in cattle shed Resting in cattle shed Resting in cattle shed Resting in cattle shed Paddy Þeld Resting in human dwelling Paddy Þeld Resting in cattle shed Light trap Well Well Well Well Well Pond Light trap Cement tank Resting in cattle shed Resting in human dwelling Resting in cattle shed Resting in cattle shed AY729969 AY917213 DQ431717 AY834246 AY729978 AY729987 DQ424949 AY729984 AY834241 DQ310142 DQ424959 AY729982 DQ149237 AY917209 AY917197 AY917198 DQ424962 AY917202 DQ154155 DQ154156 DQ154158 DQ310150 DQ317595 DQ317596 AY729972 DQ154157 DQ154159 DQ317591 DQ317592 DQ317593 DQ267690 AY917196 AY729974 AY917212 AY917207 AY729980 DQ154166 DQ310143 DQ310148 DQ317594 AY729970 AY917203 DQ267688 DQ310145 DQ310146 DQ310147 DQ310149 GenBank accession no. KUMAR ET AL.: DNA BARCODES OF INDIAN CULICIDAE Downloaded from http://jme.oxfordjournals.org/ by guest on January 17, 2017 Table 1. January 2007 3 Species Anopheles (Cellia) vagus Anopheles (Cellia) varuna Armigeres (Armigeres) subalbatus Armigeres (Armigeres) subalbatus Armigeres (Armigeres) subalbatus Culex (Culex) bitaeniorhynchus Culex (Culex) bitaeniorhynchus Culex (Culex) fuscocephala Culex (Culex) gelidus Culex (Culex) hutchinsoni Culex (Culex) pseudovishnui Culex (Culex) pseudovishnui Culex (Culex) quinquefasciatus Culex (Culex) quinquefasciatus Culex (Culex) sitiens Culex (Culex) sitiens Culex (Culex) sitiens Culex (Culex) sitiens Culex (Culex) sitiens Culex (Culex) tritaeniorhynchus Culex (Culex) tritaeniorhynchus Culex (Culex) tritaeniorhynchus Culex (Culex) tritaeniorhynchus Culex (Culex) vishnui Culex (Culex) vishnui Culex (Culex) whitmorei Culex (Culiciomyia) nigropunctatus Culex (Culiciomyia) pallidothorax Culex (Eumelanomyia) brevipalpis Culex (Eumelanomyia) brevipalpis Culex (Eumelanomyia) brevipalpis Culex (Eumelanomyia) malayi Culex (Eumelanomyia) pluvialis Culex (Lophoceraomyia) infantulus Culex (Lophoceraomyia) infantulus Culex (Lophoceraomyia) minor Culex (Lophoceraomyia) minutissimus Culex (Lophoceraomyia) rubithoracis Culex (Lutzia) fuscanus Ficalbia minima Heizmannia (Heizmannia) chandi Heizmannia (Mattinglyia) discrepans Malaya genurostris Mansonia (Mansonioides) annulifera Mansonia (Mansonioides) uniformis Mimomyia (Mimomyia) chamberlaini Ochlerotatus (Finlaya) cogilli Serial no. 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 Continued Aranganur Kurumpuram Auroville Thallapadi Chorao Nanjappa nagar Mettupalayam Edainchavadi Mettupalayam Kedar road Kizhoor Pallinelianur Kattukuppam Panchavadi Koraikuppam Srinivasapuram Wandoor Kanyapuram Moorthikuppam Pinnachikuppam Kizhoor Moorthikuppam Maravakadu Gopalankadai Katterikuppam Baluguda Mettupalayam Arakku Gorimedu Mamalapusi Londamundasi Alagramam Navohar Mettupalayam Purthidigia Badagosda Kurumpuram Kizhoor Gorimedu Bahoor Saharpur Dharmasthala Badagosda Pinnachikuppam Veerampattinam Manghakuppam Uppandara Locality 11⬚ 49.982⬘ 79⬚ 44.958⬘ 12⬚ 13.253⬘ 79⬚ 53.447⬘ 11⬚ 59.119⬘ 79⬚ 47.489⬘ 12⬚ 45.695⬘ 74⬚ 51.857⬘ 15⬚ 30.741⬘ 73⬚ 52.098⬘ 10⬚ 36.46⬘ 92⬚ 36.30⬘ 11⬚ 56.696⬘ 79⬚ 45.412⬘ 11⬚ 59.844⬘ 79⬚ 50.260⬘ 11⬚ 56.696⬘ 79⬚ 45.412⬘ 11⬚ 59.413⬘ 79⬚ 28.283⬘ 11⬚ 53.280⬘ 79⬚ 40.920⬘ 11⬚ 54.464⬘ 79⬚ 39.087⬘ 11⬚ 47.532⬘ 79⬚ 46.432⬘ 12⬚ 00.578⬘ 79⬚ 46.322⬘ 13⬚ 22.881⬘ 80⬚ 19.934⬘ 13⬚ 00.993⬘ 80⬚ 16.623⬘ 11⬚ 35.407⬘ 92⬚ 37.100⬘ 11⬚ 41.34⬘ 92⬚ 42.47⬘ 11⬚ 47.364⬘ 79⬚ 47.690⬘ 11⬚ 48.753⬘ 79⬚ 45.763⬘ 11⬚ 53.280⬘ 79⬚ 40.920⬘ 11⬚ 47.364⬘ 79⬚ 47.690⬘ 10⬚ 18.844⬘ 79⬚ 27.055⬘ 11⬚ 55.704⬘ 79⬚ 45.767⬘ 12⬚ 00.364⬘ 79⬚ 47.690⬘ 18⬚ 17.033⬘ 82⬚ 56.323⬘ 11⬚ 56.696⬘ 79⬚ 45.412⬘ 18⬚ 15.388⬘ 83⬚ 01.870⬘ 11⬚ 57.096⬘ 79⬚ 47.613⬘ 21⬚ 33.496⬘ 85⬚ 28.740⬘ 22⬚ 06.397⬘ 85⬚ 28.598⬘ 12⬚ 09.780⬘ 79⬚ 35.701⬘ 12⬚ 54.453⬘ 75⬚ 04.464⬘ 11⬚ 56.696⬘ 79⬚ 45.412⬘ 22⬚ 09.512⬘ 85⬚ 33.835⬘ 21⬚ 35.180⬘ 85⬚ 25.261⬘ 12⬚ 13.253⬘ 79⬚ 53.447⬘ 11⬚ 53.344⬘ 79⬚ 41.010⬘ 11⬚ 57.096⬘ 79⬚ 47.613⬘ 11⬚ 47.667⬘ 79⬚ 45.687⬘ 21⬚ 35.695⬘ 85⬚ 24.936⬘ 12⬚ 56.253⬘ 75⬚ 08.774⬘ 21⬚ 35.087⬘ 85⬚ 25.327⬘ 11⬚ 48.753⬘ 79⬚ 45.763⬘ 11⬚ 53.079⬘ 79⬚ 49.049⬘ 12⬚ 04.415⬘ 79⬚ 52.896⬘ 12⬚ 52.189⬘ 74⬚ 52.103⬘ GPS coordinates Collection details of specimens Pondicherry Tamil Nadu Pondicherry Karnataka Goa Andamans Pondicherry Pondicherry Pondicherry Pondicherry Pondicherry Tamil Nadu Pondicherry Pondicherry Tamil Nadu Tamil Nadu Andamans Andamans Pondicherry Pondicherry Pondicherry Pondicherry Tamil Nadu Pondicherry Pondicherry Andhra Pradesh Pondicherry Andhra Pradesh Pondicherry Orissa Orissa Pondicherry Karnataka Pondicherry Orissa Orissa Tamil Nadu Pondicherry Pondicherry Pondicherry Orissa Karnataka Orissa Pondicherry Pondicherry Pondicherry Karnataka State VCRC Museum no. A11500 A11527 A11490 A9802 A10597 A12558 A11427 A11515 A11428 A11522 A11501 A11532 A11448 A11460 A13575 A13577 A12560 A12561 A11528 A11443 A11502 A11444 A8364 A11441 A11530 A12348 A11445 A12334 A11481 A10397 A5601 A11519 A9239 A11430 A11507 A10490 A11523 A11461 A11478 A11434 A10454 A10197 A10450 A11439 A11494 A11458 A10120 Habit/habitat Paddy Þeld Stream pool Cement tank Coconut shell Biting outdoor Ground pool Resting in well Ground pool Resting in well Ground pool Paddy Þeld Canal Cement tank Cess pit Backwater pool Well Backwater pool Paddy Þeld Resting in cattle shed Canal Paddy Þeld Pond Resting in crab hole Paddy Þeld Canal Light trap Unused well Paddy Þeld Cement tank Tree hole Outdoor resting Ground pool Resting in cement tank Resting in well Outdoor resting Stream pool Pit shelter Canal Cement tank Resting in Pistia plant Bamboo Bamboo Leaf axil Canal Pond Ground pool Tree hole AY834247 DQ149241 AY729986 AY834244 DQ424958 DQ154162 DQ267687 DQ149236 AY729965 DQ149239 AY834248 AY917215 AY729977 DQ267689 DQ154160 DQ154161 DQ154163 DQ310144 DQ317598 AY729975 AY834249 AY917206 DQ424952 AY729973 AY917214 DQ154167 AY729976 DQ154154 AY834238 DQ424960 DQ424961 DQ149238 DQ317597 AY729966 DQ267691 AY917211 DQ149240 AY729981 AY729985 AY729967 AY917208 AY834242 AY917204 AY729971 AY729988 AY729979 AY834240 GenBank accession no. JOURNAL OF MEDICAL ENTOMOLOGY Downloaded from http://jme.oxfordjournals.org/ by guest on January 17, 2017 Table 1. 4 Vol. 44, no. 1 DQ154153 DQ424953 DQ424957 DQ154164 DQ154165 DQ424948 DQ424951 AY917200 AY917210 AY917201 DQ424954 AY917205 AY917199 AY834245 DQ424950 DQ424955 DQ424956 A12344 A8089 A8356 A12563 A12564 A13607 A8551 A10579 A10487 A11421 A8321 A10451 A10551 A11497 A8541 A4606 A8173 Rock pool Resting in crab hole Resting in crab hole Paddy Þeld Swamp Swamp Resting in crab hole Bamboo Bamboo Tree hole Resting in crab hole Tree hole Crab hole Ground pool Biting outdoor Biting outdoor Biting outdoor GPS, global positioning system; VCRC, Vector Control Research Centre. Ochlerotatus (Finlaya) pseudotaeniatus Ochlerotatus (Rhinoskusea) portonovoensis Ochlerotatus (Rhinoskusea) portonovoensis Ochlerotatus (Rhinoskusea) wardi Ochlerotatus (Rhinoskusea) wardi Ochlerotatus (Rhinoskusea) wardi Ochlerotatus (Rhinoskusea) wardi Orthopodomyia anopheloides Tripteroides (Rachionotomyia) aranoides Uranotaenia (Pseudoficalbia) atra Uranotaenia (Pseudoficalbia) atra Uranotaenia (Pseudoficalbia) bicolor Uranotaenia (Pesudoficalbia) recondita Verrallina (Neomacleaya) indica Verrallina (Verrallina) lugubris Verrallina (Verrallina) lugubris Verrallina (Verrallina) lugubris 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 Muliaguda bridge Muthupet Maravakadu Wandoor Santhipur Rameswaram Coringa Badagosda Saharpur Zuari Maravakadu Mamalapusi Saharpur Moratandi Coringa Chorao Muthupet Andhra Pradesh Tamil Nadu Tamil Nadu Andamans Andamans Tamil Nadu Andhra Pradesh Orissa Orissa Goa Tamil Nadu Orissa Orissa Pondicherry Andhra Pradesh Goa Tamil Nadu 18⬚ 14.316⬘ 83⬚ 01.768⬘ 10⬚ 20.314⬘ 79⬚ 32.313⬘ 10⬚ 18.844⬘ 79⬚ 27.055⬘ 11⬚ 35.407⬘ 92⬚ 37.100⬘ 12⬚ 20.005⬘ 92⬚ 46.256⬘ 09⬚ 15.24⬘ 79⬚ 16.44⬘ 16⬚ 49.287⬘ 82⬚ 17.989⬘ 21⬚ 35.581⬘ 85⬚ 25.169⬘ 21⬚ 35.695⬘ 85⬚ 24.936⬘ 15⬚ 24.870⬘ 73⬚ 54.628⬘ 10⬚ 18.844⬘ 79⬚ 27.055⬘ 21⬚ 33.496⬘ 85⬚ 28.740⬘ 21⬚ 35.877⬘ 85⬚ 24.959⬘ 11⬚ 59.402⬘ 79⬚ 49.051⬘ 16⬚ 49.287⬘ 82⬚ 17.989⬘ 15⬚ 31.668⬘ 73⬚ 51.886⬘ 10⬚ 20.314⬘ 79⬚ 32.313⬘ VCRC Museum no. GPS coordinates Collection details of specimens State Locality Species Serial no. Continued 5 adults, 65 were reared to adults from larval and three from pupal collections, 25 were F1 adults obtained from females collected in the Þeld, and 18 were adults from the Þeld and from which only the legs were used for DNA extraction. Larval habitats from which specimens were obtained ranged from groundwater habitats to both natural and artiÞcial container habitats, whereas adults were collected resting in various sites, those landing on humans to bite, and those caught in light traps. The specimens used in this study represent distribution in eight states and union territories of India. In total, 111 DNA sequences were deposited with the GenBank. Collection details including habitat, geocoordinates of localities, voucher specimens, and GenBank accession numbers of specimens are given in Table 1. Gene Sequences, Nucleotide and Amino Acid Diversity, and Genetic Distances. The DNA sequences ampliÞed using the primers C1J-1718 and C1N-2191 were ⬇500 bp, whereas the sequence with the primer MTFN and MTRN designed by us was ⬇700 bp. The latter sequence included the former sequences and hence 500 bp was taken for the analysis. This region corresponded to the 5⬘ region of COI gene. The sequences were AT rich for the mitochondrial genome, the G ⫹ C content being 0.316. The mean genetic distance (K2P) computed for the different species of Culicidae belonging to 15 genera studied was found to be 0.1469. The NJ tree showed 62 species clusters clearly among the sequences studied, thus identifying 62 species among the specimens analyzed (Fig. 1). The maximum intra-speciÞc K2P values recorded among the species clusters was for Anopheles pallidus Theobald (0.0184). The interspeciÞc K2P values ranged from 0.0587 (Anopheles fluviatilis James s.l. and Anopheles minimus Theobald) to 0.2565 [Verrallina indica (Theobald) and Anopheles jamesi Theobald] (Table 2, supplemental data available online). Hence, this study denoted that the K2P genetic distances were ⬎0.02 between different species studied for Culicidae, as cited elsewhere for other group animals (Hebert et al. 2003a,b). However, the K2P value between two very closely related species, Ochlerotatus wardi (Reinert) and Ochlerotatus portonovoensis (Tewari & Hiriyan), was only 0.0043; thus, they were not identiÞed as separate species. More samples of these species may have to be analyzed before arriving at a conclusion regarding their species status. The average K2P value of 0.1469 exhibited by Culicidae is similar to that recorded for Lepidoptera (Hajibabaei et al. 2006). Also, the COI gene was found to be very conserved, as described previously (Knowlton and Weigt 1998), the deduced amino acid variability being only 0.0329. This value indicated the utility of this gene for taxonomy of Culicidae, because it provides ample nucleotide variability toward species identiÞcation and its conserved nature for higher orders of taxonomic strata (Hebert et al. 2003a). The seven specimens of Anopheles subpictus Grassi used in the study included four sensu lato specimens, two specimens identiÞed as species B and one specimen identiÞed as species A based on egg ridge num- Downloaded from http://jme.oxfordjournals.org/ by guest on January 17, 2017 Table 1. KUMAR ET AL.: DNA BARCODES OF INDIAN CULICIDAE Habit/habitat GenBank accession no. January 2007 6 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 1 Downloaded from http://jme.oxfordjournals.org/ by guest on January 17, 2017 Fig. 1. NJ phylogenetic tree based on Kimura two-parameter genetic distances of the COI gene sequences of mosquitoes prevalent in India. bers. The nucleotide diversity between the specimens identiÞed as species A and species B was 11.3%, indicating them to be very distinct from each other. The sensu lato specimens matched with the clade of species A and hence could be the same species. Thus, this study evinced that the DNA barcode approach could distinguish members of sibling species complexes in insects as reported elsewhere (Hebert et al. 2004a). Unfortunately, the issue of sibling species complexes could not be addressed further in this study, due to January 2007 KUMAR ET AL.: DNA BARCODES OF INDIAN CULICIDAE lack of identiÞed specimens for not only other members of An. subpictus complex but also for sibling species complexes such as An. culicifacies, An. fluviatilis, and An. minimus. We propose to deal with this aspect in the future. The current study indicates the utility of using single-gene sequences (5⬘ region of mitochondrial cytochrome oxidase subunit one gene), toward identiÞcation of the mosquito species. The NJ tree computed was in general agreement with the taxonomy based on morphology as reported previously (Hebert et al. 2003a,b. 2004a; Hajibabaei et al. 2006). Acknowledgments References Cited Ballard, J. W., and M. C. Whitlock. 2004. The incomplete natural history of mitochondria. Mol. Ecol. 13: 729Ð744. Barraud, P. J. 1934. The fauna of British India including Ceylon and Burma. Diptera vol. V. Family Culicidae Tribes Megarhinini and Culicini. Taylor & Francis, London, United Kingdom. Besansky, N. J., D. W. Severson, and M. T. Ferdig. 2003. DNA barcoding of parasites and invertebrate disease vectors: what you donÕt know can hurt you. Trends Parasitol. 19: 545Ð546. CBOL [Consortium for the Barcode of Life]. 2005. A global standard for identifying biological specimens. 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Rajavel, and V. Dhanda. 1994. Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex. Med. Vet. Entomol. 8: 88 Ð94. Received 24 March 2006; accepted 7 September 2006. Downloaded from http://jme.oxfordjournals.org/ by guest on January 17, 2017 We are indebted to P. K. Das and K. Balaraman, Vector Control Research Centre, Pondicherry, India, for encouragement in conducting the study. The technical assistance provided by Regna Kumari Packirisamy and N. Krishnamoorthy Laboratory technicians is acknowledged. 7