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Anopheles pseudowillmori is the predominant malaria vector in Motuo County, Tibet Autonomous Region
© Song et al; licensee BioMed Central Ltd. 2009
Received: 09 October 2008
Accepted: 16 March 2009
Published: 16 March 2009
Malaria is endemic in Linzhi Prefecture in the Tibet Autonomous Region (TAR), but the vector for malaria transmission had never been identified.
Adult Anopheles spp. were collected in Motuo County, Linzhi Prefecture on the Sino-Indian border in July and August, 2007. Multiplex PCR was adopted for species identification, and a nested PCR approach was used to detect sporozoites in the salivary glands of the mosquitoes.
3,675 mosquitoes of the Anopheles maculatus group were collected and processed for species identification. Among them, 3,602 (98.0%) were Anopheles pseudowillmori and 73 (2.0%) were Anopheles willmori. The Plasmodium vivax SSUrDNA fragment was amplified in two of 360 pooled An. pseudowillmori samples.
The local An. maculatus group comprises the species An. pseudowillmori and An. willmori. Anopheles pseudowillmori is considered the sole malaria vector in Motuo County in Linzhi Prefecture.
Linzhi Prefecture is located in the south-eastern part of the Tibet Autonomous Region (TAR) of China. Motuo County in south of Linzhi Prefecture had a total population of 10,019 in 2006 and shares borders with both India and Myanmar. A total of 2,459 malaria cases were reported from Linzhi Prefecture between 1986 and 2004. Most of these infections were attributed to Plasmodium vivax, and 2,441 (99.3%) of the cases originated from Motuo County . In 2005 and 2006, the annual malaria incidence rates (IR) in Motuo County were 56.8 and 69.4 cases per 10,000 persons, respectively. Also in 2005, a malaria outbreak was reported from Bayi Town out of Motuo County, indicating an increasing malaria threat.
Up to the present day, Motuo County is inaccessible by car, and most villages can only be reached by foot. Although malaria appears to be a considerable public health problem in Motuo County, only few studies focused on this area, and the local malaria vector(s) remained elusive. This can be attributed both to the lack of local health workers, and to the challenging geographical conditions.
Studies conducted in 2006 established that Anopheles maculatus s.l. is the dominant Anopheles taxon in Motuo County (unpublished). The An. maculatus Theobald group belongs to the Neocellia series of the subgenus Cellia and is distributed throughout the Oriental region . Globally, nine members of the An. maculatus group have been described [3, 4]. Five among them occur in China. These are An. maculatus s.s., An. willmori, An. pseudowillmori, Anopheles sawadwongporni and Anopheles dravidicus [5, 6]. Anopheles maculatus s.l. and related species have long been recognized as important malaria vectors in Malaysia , Thailand  and the Philippines . Anopheles maculatus s.l. infected with Plasmodium were also detected in Yunnan Province, south-west China .
The aim of the present study was to elucidate the significance of An. maculatus s.l. for malaria transmission in Motuo County, thus providing the basis for the development and implementation of a locally adapted integrated malaria control strategy.
Study sites and period
Motuo County stretches across the lower reaches of the Brahmaputra River between 27°36' and 29°50' N latitude and 93°42' and 96°36' E longitude. The altitude of the mountainous county ranges between 700 and 2,100 m above sea level (mean: 1,200 m) and the average annual temperature is 16.1°C. Almost all malaria-endemic villages are scattered along the Brahmaputra River. These villages are mostly inhabited by members of the Zang, Menba and Luoba nationalities.
Cow-baited traps (CBT), human-baited net traps (HBNT) and CDC light traps (New Standard Miniature Light Traps 512 6 V 150 mm) were set up to collect adult mosquitoes between 21:00 and 01:00, or between 21:00 to 06:00. Each morning, the trapped mosquitoes were counted and identified according to morphological criteria using the key developed by Lu BL . According to Manguin et al. , the morphological identification of adult members of the An. maculatus group is error-prone due to overlapping characteristics; therefore, the collected samples were morphologically classified by species complex rather than species. Following the classification, the mosquitoes were killed by chloroform and dried on silica-gel, and subsequently transported to the laboratory where they were stored at -20°C pending DNA extraction.
All An. maculatus s.l. adults were individually registered. Subsequently, about 70% An. maculatus s.l. DNA was extracted from the legs of each individual for species identification by multiplex PCR, and pooled samples each comprising 10 thoraxes of the same species were homogenized to extract DNA for sporozoites identification as described by Li et al.  and Sun et al. . DNA extraction followed the protocol presented by Collins et al. , and the quality of the isolated DNA was assessed by measuring the proportional optical density (OD) at 260/280 nm.
Species identification by multiplex PCR
3,675 An. maculatus s.l. specimens underwent species identification by multiplex PCR. A protocol capable of distinguishing five members of the An. maculatus s.l. was adapted from Ma et al. , with primers designed based on sequence variations in the second internal transcribed spacer (ITS2) of the ribosomal DNA (rDNA). Anopheles pseudowillmori, An. maculatus s.s., An. willmori, An. dravidicus and An. sawadwongporni are characterized by 119 bp, 186 bp, 231 bp, 327 bp and 406 bp fragments, respectively. Well-characterized DNA samples were used as positive controls. The amplified products were spread by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and visualized using the Gel Imaging System (Alpha Imager HP, USA). A subsample of the PCR products was cloned and sequenced for confirmation.
Detection of P. vivax by nested PCR
The DNA of 3,600 An. pseudowillmori (= 360 pooled samples) and 70 An. willmori (= 7 pooled samples) thoraxes was extracted according to the Collins method . For the nested PCR, the following primers described by Snounou et al.  were used: the Plasmodium specific SSUrDNA primers rPLU5 (5'-CCTGTTGTTGCCTTAAACTTC-3') and rPLU6 (5'-TTAAAATTGTTGCAGTTAAAACG-3'), and the Plasmodium vivax species-specific SSUrDNA primers rVIV1 (5'-CGCTTCTAGCTTAATCCACATAACTGATAC-3') and rVIV2 (5'-ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA-3'). DNA amplification followed the protocol of Tassanakajon . Specifically, the first amplification cycle was performed using the primers rPLU5 and rPLU6 in 50 μl reaction mixture containing 1 μl DNA template. For the second cycle, 1 μl of the first-round product was used as template for the primers rVIV1 and rVIV2. Pooled samples were considered positive if a 121 bp fragment was obtained in the second PCR cycle. All products were cloned, sequenced and blasted in NCBI BLAST http://blast.ncbi.nlm.nih.gov/Blast.cgi.
Anopheline species complex and species identification
This is the first investigation aiming at identifying the malaria vector(s) in Motuo County of Linzhi Prefecture, TAR. According to the present results, the Anopheles spp. population in Motuo County almost exclusively consists of An. maculatus s.l. with Anopheles peditaeniatus accounting for the remainder. A previous study  reported that Anopheles sinensis, one of the major malaria vectors in central China, also occurred in the TAR. However, the present study failed to consolidate this claim. Although an experimental study  had confirmed that the sporozoites of Plasmodium cynomolgi could develop in An. peditaeniatus and that this mosquito could also transmit them to monkeys, there currently is no direct evidence for the transmission of human malaria by this species. Thus, An. maculatus s.l. can be regarded as the sole local malaria vector.
The study further showed that the An. maculatus group in Motuo County comprises the two species An. willmori and An. pseudowillmori. Anopheles willmori is one of the primary malaria vectors in Nepal  and has previously been reported to occur in the TAR . However, its local relevance for malaria transmission might be limited regarding its smaller abundance compared to An. pseudowillmori. The latter has been established as a malaria vector in northwest Thailand along the border with Myanmar [19, 20] but has not been recorded in the TAR before. Dong et al. , however, reported a clear relationship between the seasonal abundance variation of An. pseudowillmori and malaria IR in a mountainous area of Yunnan Province in the south of the TAR.
The detection of sporozoites in the salivary glands of Anopheles spp. conclusively identifies them as malaria vectors. Traditionally, sporozoite infection rates were determined by dissection and examination of salivary glands of individual mosquitoes under a light microscope, a time consuming and labor intensive approach. Therefore, alternative sporozoite detection methods had been developed in recent years: enzyme-linked immunosorbent assay (ELISA) [7, 9, 22–24] detecting the circumsporozoite protein (CSP), and different approaches based on the PCR technique [25, 26] aiming at amplifying specific DNA sequences. It appears that the detection of Plasmodium falciparum and P. vivax specific DNA sequences in mosquitoes by PCR has a higher sensitivity compared to ELISA tests .
In this study, a nested PCR approach aiming at the identification of the SSUrDNA of P. vivax sporozoites had been selected to identify malaria vectors. A conventional PCR test can detect as few as 10 sporozoites per salivary gland, making it a useful tool for screening small numbers of Anopheles spp. . The sensitivity of the employed nested PCR test is as low as three sporozoites , and the pooling of samples allows screening of large samples . In our study, pooling samples [11, 12] and screening them by nested PCR [14, 15] resulted in the identification of P. vivax SSUrDNA in salivary glands of two pooled An. pseudowillmori samples, thus establishing that An. pseudowillmori is the main malaria vector in this area.
The composition of the local anopheline population in Motuo County at the Sino-Indian border consists mostly of members of the An. maculatus group, with An. pseudowillmori being much more abundant than An. willmori. Anopheles pseudowillmori has been identified as the local malaria vector.
The authors thank Prof. Ma Ya-Jun from the Second Military Medical University of China in Shanghai for providing confirmed DNA samples of five species of the An. maculatus group. The smooth collaboration with the staff of the Linzhi Prefecture CDC and the Motuo County CDC is acknowledged, and we are grateful to Zhuoma Yang-jin, Hu Yong-Hong, Jiang Wei-Kang, Hu Song-Lin, Xu Hui-Mei, Ciren Quzh and Zhang Wei for their assistance in the field. We thank Peter Steinmann from Swiss Tropical Institute for assistance with the English.
- Luo S, Hu YH, Hu SL, Zhen N, Li CC: [An epidemiological analysis on malaria in Linzhi District of Tibet in 1986–2004]. Chin J Parasitol Dis. 2005, 12: 457-459.Google Scholar
- Manguin S, Garros C, Dusfour I, Harbach RE, Coosemans M: Bionomics, taxonomy, and distribution of the major malaria vector taxa of Anopheles subgenus Cellia in Southeast Asia: An updated review. Infect Genet Evol. 2007, 8: 489-503. 10.1016/j.meegid.2007.11.004.View ArticlePubMedGoogle Scholar
- Harbach RE: The classification of genus Anopheles (Diptera: Culicidae): a working hypothesis of phylogenetic relationships. Bull Entomol Res. 2004, 94: 537-553. 10.1079/BER2004321.View ArticlePubMedGoogle Scholar
- Walton C, Somboon P, O'Loughlin SM, Zhang S, Harbach RE, Linton YM, Chen B, Nolan K, Duong S, Fong MY, Vythilingum I, Mohammed ZD, Trung HD, Butlin RK: Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA. Infect Genet Evol. 2007, 7: 93-102. 10.1016/j.meegid.2006.05.001.View ArticlePubMedGoogle Scholar
- LU BL: Fauna Sinaca, Insecta, Diptera: Culicidae II. 1997, Beijing china, Science Press, 9:Google Scholar
- Ma YJ, Li SZ, Xu J: Molecular identification and phylogeny of the Maculatus group of Anopheles mosquitoes (Diptera: Culicidae) based on nuclear and mitochondrial DNA sequences. Acta Trop. 2006, 99: 272-280. 10.1016/j.actatropica.2006.09.005.View ArticlePubMedGoogle Scholar
- Reid JA: Anopheline mosquitoes of Malaya and Borneo. Stud Inst Med Res Malaysia. 1968, 31: 1-520.Google Scholar
- Rattanarithikul R, Konishi E, Linthicum KJ: Detection of Plasmodium vivax and Plasmodium falciparum circumsporozoite antigen in anopheline mosquitoes collected in southern Thailand. Am J Trop Med Hyg. 1996, 54 (2): 114-121.PubMedGoogle Scholar
- Torres EP, Foley DH, Saul A: Ribosomal DNA sequence markers differentiate two species of the Anopheles maculatus (Diptera: Culicidae) complex in the Philippines. J Med Entomol. 2000, 37: 933-937.View ArticlePubMedGoogle Scholar
- Zhou HN, Zhang ZX, Curtis C, Hill N, Li CF, Chao W: [Evaluation of the enzyme-linked immunosorbant assay in detecting circumsporozoite protein of anopheline vectors in Yunnan]. Chin J Parasitol Parasit Dis. 2004, 22: 227-230.Google Scholar
- Li FW, Niu C, Ye BH: Nested polymerase chain reaction in detection of Plasmodium vivax sporozoites in mosquitoes. Chinese Medical Journal. 2001, 114: 654-657.PubMedGoogle Scholar
- Sun QW, Zhu HM, Lu L, Gu ZC, Cheng X, Fang Y: [Restudy on a model for estimating the sporozoite infection rate in mosquitoes using pooled sampling]. Chin J Parasitol Parasit Dis. 2002, 20: 351-353.Google Scholar
- Collins FH, Mendez MA, Rasmussen MO, Mehaffey PC, Besansky NJ, Finnerty V: A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex. Am J Trop Med Hyg. 1987, 37 (1): 37-41.PubMedGoogle Scholar
- Snounou G, Pinheiro L, Goncalves A, Fonseca L, Dias F, Brown KN, do Rosario VE: The importance of sensitive detection of malaria parasites in the human and insect hosts in epidemiological studies, as shown by the analysis of field samples from Guinea Bissau. Trans R Soc Trop Med Hyg. 1993, 87: 649-653. 10.1016/0035-9203(93)90274-T.View ArticlePubMedGoogle Scholar
- Tassanakajon A, Boonsaeng V, Wilairat P, Panyim S: Polymerase chain reaction detection of Plasmodium falciparum in mosquitoes. Trans R Soc Trop Med Hyg. 1993, 87: 273-275. 10.1016/0035-9203(93)90124-9.View ArticlePubMedGoogle Scholar
- Editorial Committee of malaria control and research in China: Malaria control and research in China. 1991, Beijing, People's Medical Publishing HouseGoogle Scholar
- Collins WE, Warren M, Galland GG: Studies on infections with the Berok strain of Plasmodium cynomolgi in monkeys and mosquitoes. J Parasitol. 1999, 85: 268-272. 10.2307/3285631.View ArticlePubMedGoogle Scholar
- Pradhan SP, Shrestha SL, Vaidya RG: Malaria transmission in high mountain valleys of west Nepal including the first record of Anopheles willmori (James) as a third vector of malaria. Nepal Med Assoc. 1970, 8: 89-97.Google Scholar
- Green CA, Rattanarithikul R, Pongparit S, Sawadwongporn P, Baimai V: A newly-recognized vector of human malarial parasites in the Oriental region, Anopheles (Cellia) pseudowillmori (Theobald, 1910). Trans R Soc Trop Med Hyg. 1991, 85: 35-36. 10.1016/0035-9203(91)90143-M.View ArticlePubMedGoogle Scholar
- Green CA, Rattanarithikul R, Charoensub A: Population genetic confirmation of species status of the malaria vectors Anopheles willmori and An. pseudowillmori in Thailand and chromosome phylogeny of the Maculatus group of mosquitoes. Med Vet Entomol. 1992, 6: 335-341. 10.1111/j.1365-2915.1992.tb00629.x.View ArticlePubMedGoogle Scholar
- Dong XS, Zhou HL, Bi Y, Dong LM: [The relationship between Anopheles maculatus geographic distribution, ecological features and malaria in Yunnan province]. Acta Parasitol Med Entomol Sin. 1996, 3: 100-105.Google Scholar
- Trung HD, Van Bortel W, Sochantha T, Keokenchanh K, Quang NT, Cong LD, Coosemans M: Malaria transmission and major malaria vectors in different geographical areas of Southeast Asia. Trop Med Int Health. 2004, 9: 230-237. 10.1046/j.1365-3156.2003.01179.x.View ArticlePubMedGoogle Scholar
- Benet A, Mai A, Bockarie F, Lagog M, Zimmerman P, Alpers MP, Reeder JC, Bockarie MJ: Polymerase chain reaction diagnosis and the changing pattern of vector ecology and malaria transmission dynamics in Papua New Guinea. The American journal of tropical medicine and hygiene. 2004, 71 (3): 277-284.PubMedGoogle Scholar
- Cuamba N, Choi KS, Townson H: Malaria vectors in Angola: distribution of species and molecular forms of the Anopheles gambiae complex, their pyrethroid insecticide knockdown resistance (kdr) status and Plasmodium falciparum sporozoite rates. Malar J. 2006, 5: 2-10.1186/1475-2875-5-2.PubMed CentralView ArticlePubMedGoogle Scholar
- Stoffels JA, Docters van Leeuwen WM, Post RJ: Detection of Plasmodium sporozoites in mosquitoes by polymerase chain reaction and oligonucleotide rDNA probe, without dissection of the salivary glands. Med Vet Entomol. 1995, 9: 433-437. 10.1111/j.1365-2915.1995.tb00020.x.View ArticlePubMedGoogle Scholar
- Moreno M, Cano J, Nzambo S, Bobuakasi L, Buatiche JN, Ondo M, Micha F, Benito A: Malaria Panel Assay versus PCR: detection of naturally infected Anopheles melas in a coastal village of Equatorial Guinea. Malar J. 2004, 3: 20-10.1186/1475-2875-3-20.PubMed CentralView ArticlePubMedGoogle Scholar
- Vythilingam I, Nitiavathy K, Yi P, Bakotee B, Hugo B, Singh B, Wirtz RA, Palmer K: A highly sensitive, nested polymerase chain reaction based method using simple dna extraction to detect malaria sporozoites in mosquitos. Southeast Asian J Trop Med Public Health. 1999, 30 (4): 631-635.PubMedGoogle Scholar
- Zhou HY, Zhu GD, Jin XL, Li JL, Wang WM, Cao J, Gu YP, Gao Q: [Detection of Plasmodium sporozoites in mosquitoes by nested-PCR]. Chin J Schisto Control. 2006, 18: 365-368.Google Scholar
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