Acta Tropica 114 (2010) 49–54
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Spatial distribution and r-DNA second internal transcribed spacer characterization of Anopheles dirus (Diptera: Culicidae) complex species in north-east India Anil Prakash, D.K. Sarma, D.R. Bhattacharyya, P.K. Mohapatra, K. Bhattacharjee, K. Das, J. Mahanta ∗ Regional Medical Research Centre, NE (Indian Council of Medical Research), Post Box No. 105, Dibrugarh 786001, Assam, India
a r t i c l e
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Article history: Received 1 December 2009 Received in revised form 29 December 2009 Accepted 5 January 2010 Available online 13 January 2010 Keywords: An. baimaii ASPCR Dirus complex r-DNA ITS2 Malaria vector Sibling species Species X of China
a b s t r a c t The identity and distribution of the prevalent member species of the Anopheles dirus complex mosquitoes in the north-eastern region of India was investigated in a cross-sectional study. We altogether collected 267 individuals of An. dirus s.l. from 27 forested/forest fringed locations spread across the seven northeastern states, identified the species using a ribosomal DNA (r-DNA) second internal transcribed spacer (ITS2) based allele specific polymerase chain reaction (ASPCR) method and sequenced the ITS2 locus in a sub set of mosquitoes. An. baimaii was identified as the main, almost exclusive (266/267), species of the Dirus complex throughout the north-east India with no intraspecific variation in the 479 base pair long ITS2 sequences in 59 of the 60 individuals sequenced. Ribosomal DNA of one individual from Assam state did not amplify in the ASPCR, possessed 786 base pair long ITS2 sequence and showed 99.7% similarity with the sequence of An. dirus species D (An. baimaii) from Yunnan province of China, later referred to as species X of the Dirus complex. These observations suggest the presence of another, possibly the new, species of the Dirus complex, sympatric with An. baimaii, in Assam warranting investigations on its distribution, biology and role in human malaria transmission in north-east India. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Malaria with average annual morbidity of 0.2–0.3 million reported cases and 100–150 deaths is a major public health concern in the north-eastern region of India (Prakash et al., 2003). All the four species of human malaria parasites, with the predominance of Plasmodium falciparum, are prevalent in this part of India (Mohapatra et al., 2008). Malaria, mostly concentrated in the forested hilly and foot hill regions, is mainly vectored by members of Anopheles dirus, An. fluviatilis and An. minimus complex mosquitoes in north-east India (Prakash et al., 2006). An. dirus complex includes several efficient human malaria vectors associated with the sylvatic environment of the South-east Asian region along with India (Obsomer et al., 2007). In India, though the distribution of the Dirus complex mosquitoes encompasses tropical rain forest areas of north-east India, Andaman-Nicobar islands and Western Ghats in south-western peninsular India, these are implicated in transmitting malaria only in the north-east India (Bhat, 1998). Taxonomically, the Dirus complex belongs to the Leucosphyrus subgroup under Anopheles (Cellia) leucosphyrus group and includes
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at least seven formally named isomorphic species: An. dirus s.s. (=dirus species A), An. cracens (=dirus species B), An. scanloni (=dirus species C), An. baimaii (=dirus species D), An. elegans (=dirus species E), An. nemophilus (=dirus species F) and An. takasagoensis (Sallum et al., 2005). Of these, four member species viz. An. dirus, An. cracens, An. scanloni and An. baimaii are the proven vectors of human malaria in different South-east Asian countries while the remaining three species perhaps only transmit simian malaria (Obsomer et al., 2007). The fact that like any species complex, members of the Dirus complex too have different ecologies, biological attributes, vectorial capacities and responses to control measures (Manguin et al., 2002), makes the correct species identification necessary for a better understanding of their potential roles in malaria transmission in areas of their occurrence. Moreover, different species of a complex may exhibit seasonal spatial variations in their abundance, thus, making the prospects of vector control a difficult preposition (Baimai et al., 1988). It further emphasizes the need to correctly identify and determine spatial distribution of member species of the Dirus complex to achieve greater understanding of its bionomics and effective control. Although the presence of An. baimaii of the Dirus complex in north-east India has been documented (Prakash et al., 2006), no systematic effort was ever made to determine the species composition of the Dirus complex at a larger spatial scale in this region. Here we report the spatial distribution of the An. dirus complex species in the seven north-east Indian states,
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Fig. 1. Map showing Dirus complex mosquito collection sites in north-east India (site numbers are shown in black and white colours for contrast purpose).
identified using a ribosomal DNA ITS2 based molecular method to enrich the existing knowledge on the species distribution of this complex as an aid to the National Vector Borne Diseases Control Programme in its quest for malaria control. Another aim of this study was to characterise and sequence the ITS2 region of the Dirus complex species present in north-east India to elaborate intra and interspecies differences, if any. 2. Material and methods 2.1. Mosquito collections A cross-sectional study was carried out during 2006–2009 covering all seven states of the north-east India (22◦ 04 and 29◦ 31 N and 89◦ 48 and 97◦ 25 E) viz. Assam, Arunachal Pradesh, Manipur, Meghalaya, Mizoram, Nagaland and Tripura. Mosquito collections were done mainly during the malaria transmission season i.e., April–October, on average from 6 to 7 places in each state (Fig. 1) selected on the basis of technical and operational considerations. Each collection spot was georeferenced using hand held GPS instrument. Host seeking adult females were captured from dusk-to-dawn (18:00–6:00 h) in human dwellings using Centres for Disease Control (CDC) miniature light traps. Trapped anophelines were identified morphologically next morning using the keys of Das et al. (1990). Members of the Dirus complex were identified sensu lato, kept individually in capped beam capsules/plastic vials along with silica gel and stored at 4 ◦ C till further processing. In addition, mosquito immatures were collected, wherever feasible, from the potential breeding habitats of the Dirus complex mosquitoes and reared in the laboratory till emergence, identification and processing.
(Sambrook and Russel, 2001). The ASPCR method of Walton et al. (1999) was employed with slight modification for identifying species of the Dirus complex mosquitoes. This method, developed on mosquitoes of Thailand and later successfully applied in north-east India (Prakash et al., 2006), is capable of identifying unambiguously five species of the Dirus complex viz. An. dirus, An. cracens, An. scanloni, An. baimaii and An. nemophilous. Final optimized ASPCR reaction conditions used were: D–U primer at 0.5 M, D–D primer at 0.8 M, D–AC, D–B and D–F primers at 0.28 M each, 2 mM MgCl2 , 160 mM dNTPs, 10% DMSO, 20 mM (NH4 )2 SO4 , 75 mM Tris–HCl (pH 9.0), 0.01% (w/v) Tween, 0.25 U of Taq DNA polymerase and 0.5 l of mosquito template DNA in a reaction volume of 25 l. After initial denaturation of 5 min at 94 ◦ C, and 35 amplification cycles of 94 ◦ C for 30 s, 58 ◦ C for 30 s and 72 ◦ C for 1 min, final extension at 72 ◦ C for 10 min was used in a thermal cycler (Bio Rad MJ Mini-model PTC 1148). 2.3. Amplification of r-DNA ITS2 Since ITS2 sequences are likely to be fixed within the species and vary between closely related species due to the high intraspecific homogeneity and interspecific variability (Walton et al., 1999), we amplified the ITS2 region of the ribosomal DNA of all An. dirus s.l. mosquitoes to verify the ASPCR identification results. The primers of Beebe and Saul (1995) and the reaction conditions similar to Walton et al. (1999) with minor modifications were used. The reaction was performed in a 50 l volume containing 1 l of genomic DNA, 1 M of each primer, 200 m dNTPs, 4 mM MgCl2 , 20 mM (NH4 )2 SO4 , 75 mM Tris–HCl, 0.01% (v/v) ‘Tween’, 1.25 units of Taq DNA polymerase and 10% DMSO. DNA sample was initially denatured by heating at 94 ◦ C for 5 min prior to 39 cycles of amplification at 94 ◦ C for 1 min, 51 ◦ C for 1 min and 72 ◦ C for 2 min followed by a final extension step of 10 min at 72 ◦ C.
2.2. DNA extraction and ASPCR assays for species identification 2.4. Sequencing of r-DNA ITS2 Genomic DNA of An. dirus s.l. mosquitoes was extracted individually, in most instances from the 2 legs and in few cases using the whole body, by the standard phenol–chloroform method
ITS2 PCR amplicons of a few randomly chosen, ASPCR identified, specimens of the Dirus Complex from all the collection sites,
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Table 1 Collection sites and molecular identification of the Dirus complex mosquitoes in north-east India. State
District
Area
Site no.
Coordinates (N/E)
No. of An dirus s.l. collected An.
Identified as
Arunachal Pradesh
Changlang
Miao Jairampu r Pukke TR Deomali Kameng Bari
1 2 3 4 5
N-27◦ 29 , E-96◦ 10 N-27◦ 20 , E-96◦ 00 N-27◦ 03 , E-92◦ 36 N-27◦ 09 , E-95◦ 28 N-27◦ 04 , E-92◦ 25
2 7 3 31 2
2 7 3 31 2
Nil Nil Nil Nil Nil
Sonitpur
Soraipung Bokakhat Kaziranga NP Titabor Jatinga Umrangsu Nameri NP
6 7 8 9 10 11 12
N-27◦ 23 , E-95◦ 34 N-26◦ 34 , E-93◦ 14 N-26◦ 36 , E-93◦ 28 N-26◦ 36 ,E-94◦ 17 N-25◦ 11 , E-93◦ 02 N-25◦ 31 , E-92◦ 47 N-26◦ 90 , E-92◦ 95
29 4 16 9 14 1 3
29 4 16 9 13 1 3
Nil Nil Nil Nil 1 (Sp. X) Nil Nil
Tamenglong
Ziribam
13
N-24◦ 47 , E-93◦ 12
baimaii
East Kameng Tirap West Kameng Assam
Dibrugarh Golaghat Jorhat N.C.Hills
Manipur
◦
◦
Others
3
3
Nil
Meghalaya
E. Garo Hills W. Garo Hills W. Khasi Hills
W. Nagar Tura Aradonga
14 15 16
N-25 32 , E-90 37 N-25◦ 51 , E-90◦ 22 N-25◦ 55 , E-90◦ 56
4 1 3
4 1 3
Nil Nil Nil
Mizoram
Kolasib Lunglei Mamit Serchip W. Phailang
Rengtelui Tlabung Lengpui Thenzawl Dumpa TR
17 18 19 20 21
N-24◦ 14 , E-92◦ 42 N-22◦ 53 E-92◦ 45 N-22◦ 83 , E-92◦ 63 N-23◦ 19 , E-92◦ 45 N-23◦ 42 , E-92◦ 29
4 18 7 1 28
4 18 7 1 28
Nil Nil Nil Nil Nil
Nagaland
Dimapur Wokha
Aiyomkum Bhandari
22 23
N-25◦ 54 , E-93◦ 44 N-26◦ 17 , E-94◦ 10
5 55
5 55
Nil Nil
Tripura
Dhalai N. Tripura S. Tripura W. Tripura
Ambasa Kailasahar Sabroom Kathaliya
24 25 26 27
N-23◦ 55 , E-91◦ 51 N-24◦ 20 , E-92◦ 01 N-23◦ 0 , E-91◦ 43 N-23◦ 19 , E-91◦ 19
2 6 8 1 267
2 6 8 1 266
Nil Nil Nil Nil 1
Total
were cleaned using High Pure PCR Product Purification kits (RocheDiagnostics GmbH, Germany) and sequenced in both directions using Big Dye Terminator V3.1 kit on a 16 capillary automated DNA sequencer (Applied Biosystems, USA 3130 XL Genetic Analyzer) as per the manufacturer’s recommendations. 2.5. Handling of r-DNA sequences The forward and reverse sequences of each individual were checked and edited manually using BioEdit v7.0.9 software (Hall, 1999). All edited sequences were aligned using default parameters in clustal W2 (Larkin et al., 2007) and examined for any sequence variation. Neighbour-Joining phylogeny based on ITS2 sequences was constructed (Tamura et al., 2004) using Kimura 2 parameters distance matrix with 1000 bootstrap replicates to infer the relationship among the member species of the Dirus complex in MEGA version 4.1 (Tamura et al., 2007).
10 (Jatinga area, N.C. Hills district, Assam) did not amplify in ASPCR, suggestive of a species other than An. dirus, An. cracens, An. scanloni, An. baimaii and An. nemophilus. ITS2 of the 266 ASPCR identified individuals of An. baimaii amplified to a band of 540 base pair size, characteristic for this species (Walton et al., 1999), thus, corroborating the ASPCR identification results. However, in case of DA-70, ITS2 band of about 850 base pair was amplified (Fig. 2) confirming it a different species than An. baimaii. ITS2 region of the 60 randomly chosen individuals (59 ASPCR confirmed An. baimaii and DA-70), representing all the seven north-east Indian states, were sequenced. After manual editing and
3. Results Collections attempted at 52 locations in the seven north-east Indian states yielded 267 adults of An. dirus s.l. from 27 sites (Table 1). Of these, 255 individuals were captured in light traps and 12 emerged from larvae collected from 3 sites in Assam (site nos. 8 and 10) and Arunachal Pradesh (site no. 4). Altogether, 20 anopheline species were captured in these collections including An. dirus s.l. and An. minimus s.l., the two well known malaria vectors in the north-east India. The Dirus complex mosquitoes were found in all the seven states, though relatively less frequently in Manipur, Meghalaya and Nagaland. In ASPCR, 266 of the 267 An. dirus individuals produced a 306 base pair band hence were identified as An. baimaii. One specimen (code no. DA-70), emerged from the immatures collected at site no.
Fig. 2. r-DNA ITS2 PCR amplification of ASPCR confirmed An. baimaii in north-east India (M, 100 base pair ladder; B, blank; lane 1, dirus A positive control; lane 2, dirus B positive control; lanes 3–16, representative field collected samples from different NE states (3 and 4 – Arunachal Pradesh, 5 and 6 – Assam, 7 and 8 – Manipur, 9 and 10 – Meghalaya, 11 and 12 – Mizoram, 13 and 14 – Nagaland, 15 and 16 – Tripura); lane 17, DA-70 from Jatinga, Assam).
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Fig. 3. Pairwise alignment of ITS2 sequences of species D/X of China (GenBank accession no. U60411) and DA-70 from Jatinga, Assam (each dot indicates identity with the first sequence).
Fig. 4. Unrooted Neighbour-Joining phylogenetic tree based on ITS2 sequences of the member species of An. dirus complex. Only Boot strap values ≥50% are shown.
defining the 5.8S and 28S borders, sequences of all the 59 confirmed An. baimaii were found 479 base pair long with no intraspecific variation and 100% similarity with ITS2 sequences of An. baimaii from Thailand (Walton et al., 1999). However, the edited ITS2 sequence of DA-70 was found 786 base pair long. BLAST search (www.ncbi.nlm.nih.gov) of this sequence revealed 99% identity with the ITS2 sequence of species D of An. dirus complex (=An. baimaii) reported from the Yunnan province of China (GenBank accession no. U60411), subsequently referred to as species X by Walton et al. (1999), with 2 differences (i) substitution of G (DA-70) at position 705 in place of A (species X) and (ii) deletion at position 730 in DA-70 against the insertion of C in species X (Fig. 3). The Neighbour-Joining phylogenetic tree, constructed using the representative ITS2 sequences of An. baimaii (=Dirus D) generated in this study from all the seven north-east Indian states, DA-70, species X of China (GenBank accession no. U60411) and five member species of the Dirus complex from Thailand (drawn from Walton et al., 1999), revealed complete similarity of An. baimaii of northeast India with that of Thailand. Species X was found relatively closer to species A (=An. dirus s.s.) and formed a sister clade with
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DA-70 indicating their close relatedness (Fig. 4). Except Dirus D (=An. baimaii), all other species of the complex, including DA-70, formed a common phyletic group suggesting that An. baimaii is a distantly related member species in the Dirus Complex.
4. Discussion Successful control of vector mosquitoes is dependant on the precise information on the biological attributes of the involved species which in turn is dependant on the accurate identification and information on the distribution of the specific vectors (Trung et al., 2005). Proper identification together with information on geospatial distribution of malaria vectors and their sibling members is important to malaria control programmes for targeted intervention and proper utilization of economic resources (Marrelli et al., 2006). Knowledge on the distribution of the member species of a complex is all the more important since different species may exhibit differences in ecology, vectorial capacity and response to control measures (White, 1972). Ecology of the Dirus complex members, comprising of at least seven species varying from highly competent vectors to non-vectors of human malaria parasites, is associated with the forests (forested hills, foot hills, deep forests, forest fringes) through out its range of distribution in South-east Asian countries and north-east India (Manguin et al., 2002). We demonstrated that An. baimaii is the most prevalent species of the Dirus complex in the north-eastern region of India. Though the presence of An. baimaii in north-east India has been reported earlier (Baimai, 1989; Prakash et al., 2006) this is, by far, the most comprehensive investigation covering 27 populations across the seven north-eastern states. Though our results show near exclusiveness of An. baimaii in the north-eastern region of India yet to conclude the absence of other member(s) of the Dirus complex would be erroneous. Present data indicate that at least two sympatric species of the Dirus complex are present in the state of Assam. In the Jatinga area of N.C. Hills district, Assam, in addition to An. baimaii, one substantially different specimen (DA-70) than An. baimaii at molecular level was found. This individual did not amplify in the Dirus complex specific ASPCR, had much longer ITS2 (786 base pairs) compared to An. baimaii (479 base pairs) from Thailand and India (Walton et al., 1999; Prakash et al., 2006) and recorded 99% ITS2 sequence similarity and 0.00 value of the pairwise distance with An. dirus species D (=An. baimaii) from China (Xu and Qu, 1997). The pairwise distances between ITS2 sequence of this individual and An. dirus, An. cracens, An. scanloni, An. baimaii (of Thailand and India) and An. nemophilus were 0.0129, 0.0476, 0.0151, 0.115 and 0.033 respectively (data not shown) suggesting distant genetic relationship of DA-70 with these species compared to An. baimaii of China. Presence of An. dirus s.s. in Hainan province and An. baimaii in Yunnan province of China has been reported (Xu and Qu, 1997; Xu et al., 1998). However, ITS2 sequences generated from the specimens of the Dirus complex (presumably An. baimaii) collected in Yunnan province (Xu and Qu, 1997) were found substantially different than that of An. baimaii from Thailand, thus, possibly representing a different species in the complex, which was later termed as the ‘species X’ by Walton et al. (1999). In view of very little sequence variation with species X and considerable divergence from the other species of the Dirus complex it seems likely that the specimen DA-70 belonged to the species X and the 2 differences found in the ITS2 sequences of DA70 and species X indicate some population structure in the species X. However, the distance gradient between the two sites of occurrence of species X – one in Yunnan province in the southwestern China and the other in Assam state in India – both separated by hundreds of km having no link or geographical continuity needs explanation. Closeness of species X to species A (An. dirus s.s.) in NJ tree topology is perhaps a reflection of the speciation process
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and the effect of various kinds of evolutionary forces which takes into account the balance between the gene flow and genetic drift. It is worth mentioning that this particular male individual (DA70) was collected as an immature from a small, partially shaded, semipermanent ground pool containing fresh water and emergent vegetation from a hilly area showing a different breeding ecology in comparison to An. baimaii which in Assam is known to breed in small, shaded/partly shaded muddy, transient ground pool with leaf litter on jungle path (Prakash et al., 1997). Identical ITS2 sequences of An. baimaii from as many as 27 places of north-east India and those from Thailand indicate a complete lack of intraspecific variation at this locus throughout the species range. This is in agreement with the low level of genetic population structure detected in this species using mitochondrial and microsatellite markers (Walton et al., 2001). The lack of intraspecific variation observed in the ITS2 sequences of An. baimaii populations over the wide geographical area may be due to sufficient gene flow, thus, homogenizing the sequences of r-DNA genes by concerted evolution (Elder and Turner, 1995). Given the fact that ITS2 variation is usually found among the sibling species we support the view expressed by Obsomer et al. (2007) that the intraspecific ITS2 variations between populations of An. baimaii from Thailand (as well as north-east India) and Yunnan province of China at one hand, and An. scanloni at Kanchanburi and Thung Song locations of Thailand (O’Loughlin et al., 2007) on the other hand indicate the possible existence of two new species in addition to the seven recognized species in the An. dirus complex in south-east Asia. More collections, morphological examination and molecular data are needed from different geographical areas of north-east India, especially the N.C. Hills area of Assam to further investigate the taxonomic status of species X, its biological attributes and role in malaria transmission. Acknowledgements This work was supported by a grant from Indian Council of Medical Research under the North-east initiative. We are thankful to the State Programme Officers of different north-eastern states, District Malaria Officers of different districts, Medical Officers and other staff members of the concerned Primary Health Centres for logistic help during the study. Technical help provided by Messrs. Ripunjoy Sonowal, Satyam Kumar Das, A.C. Rabha and Dipak Dutta is acknowledged. We express gratitude to Dr. Catherine Walton, University of Manchester and Dr. Samanta O’Loghulin Imperial College of London for useful discussions and help in several ways. References Baimai, V., Kijchalao, U., Sawadwongporn, P., Green, C.A., 1988. Geographic distribution and biting behaviour of four species of the An. dirus complex (Diptera: Culicidae) in Thailand. Southeast Asian J. Trop. Med. Pub. Health 19, 151–161. Baimai, V., 1989. Speciation and species complexes of the Anopheles malaria vectors in Thailand. In: Proceedings of the 3rd Conference on Malaria Research, Thailand, 18–20 October 1989. Chaing Mai University, Thailand, pp. 146–162. Beebe, N.W., Saul, A., 1995. Discrimination of all members of the Anopheles punctulatus complex by polymerase chain reaction–restriction fragment length polymorphism analysis. Am. J. Trop. Med. Hyg. 53, 478–481. Bhat, H.R., 1988. A note on Anopheles dirus Peyton and Harrison, 1979 [An. balabasensis (sensu lato) Baisas, 1936] in India. Indian J. Malariol. 25, 103–105. Das, B.P., Rajagopal, R., Akiyama, J., 1990. Pictorial key to the species of Indian anopheline mosquitoes. Zoology 2, 131–162. Elder Jr., J.F., Turner, B.J., 1995. Concerted evolution of repetitive DNA sequences in eukaryotes. Quart. Rev. Biol. 70, 297–320. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis programme for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and clustal X version 2. Bioinformatics 23, 2947–2948. Manguin, S., Kengne, P., Sonnier, L., Harbach, R.E., Baimai, V., Trung, H.D., Coosemans, M., 2002. SCAR markers and multiplex PCR-based identification of isomorphic species in the Anopheles dirus complex in South east Asia. Med. Vet. Entomol. 16, 46–54.
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