Medical and Veterinary Entomology (2002) 16, 329-334
SHORT COMMUNICATION
Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species W. VAN BORTEI/, T. SOCHANTA^ R. E. HARBACH^, D. SOCHEAT^ P. ROELANTS^ T. BACKELJAU8 and M. COOSEMANS* ’Department of Parasitology, Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium, ^National Malaria Centre, Phnom Phen, Cambodia, Department of Entomology and Biomedical Sciences Theme, The Natural History Museum, London, U.K.. and Department of Invertebrates, Royal Belgian Institute of Natural Sciences, Brussels, and Evolutionary Biology Group, University of Antwerp (RUCA), Belgium Abstract. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north-eastern Cambodia. In addition to finding An. aconitus Donitz, An. minimus species A and An. pampanai Buttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR-RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR-RFLP assay can detect species not included in the initial set-up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non-vector anophelines in South-east Asia.
Key words. Anopheles culicifacies. An. minimus, Cellia, ITS2, Myzomyia, malaria vector, PCR-RFLP, species complex, species identification, Cambodia.
Mosquitoes of the Anopheles minimus group are widespread across South-east Asia, with malaria transmitted by at least two sibling species, A and C, of the An. minimus complex (Chen etal., 2002). In addition to specific isoenzyme characters (Green etal., 1990; Van Bortel etal., 1999), molecular methods have been developed for identifying An. minimus species A and C from the Asian mainland (Sharpe etal.,
Correspondence: Dr Wim Van Bortel, Department of Parasitology, Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Amwerpen, Belgium, E-mail:
[email protected] 2002 The Royal Entomological Society
1999; Van Bortel etal., 2000; Kengne etal., 2001). Moreover, these methods allow the identification of other species in the Myzomyia Series ofsubgenus Cellia (Table 1), which cannot be reliably distinguished from An. minimus sensu lato based on adult morphology. Thus, molecular identification may be useful for interpretation of malaria vector biology and control studies (Van Bortel etal., 2001). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) identification assay developed by Van Bortel etal. (2000) targets the internal transcribed spacer 2 (ITS2) of ribonuclear DNA (rDNA). Based on the sequence differences observed in this region, it 329
330 W. Van Bortel et al. Table 1. Overview of the molecular assays available for the identification of the Anopheles minimus complex and closely related species. ASA allele specific amplification; SSCP single-strand conformational polymorphism; SCAR sequence characterized amplified regions; PCR-RFLP polymerase chain reaction-restriction fragment length polymorphism.
An. m’.inimus comp]lex
Other species of
Other
An.
Myzomyia species
Identification Based an technique
A
C
aconitzis pampcawi varuna
ASA SSCP Multiplex PCR-RFLP
X X X X
X X X X
X X X
rDNA, D3 region in 2,8S rDNA, D3 region in 2;8S SCAR via RAPD rDNA, ITS2
minimus groiip
was suggested that this method has the potential to distinguish other species, closely related to An. minimus s.l., but not included in the initial assay (Van Bortel etal., 2000).
The assay has the advantage that PCR amplification should always produce a product, allowing anomalies to be more easily resolved, i.e. RFLP profiles differing from known diagnostic banding patterns can be detected. This is especially useful in South-east Asia, where the specific identification of anophelines belonging to Myzomyia Series (Harrison, 1980; Harbach, 1994) is unreliable on the basis of adult morphology, so their presence or absence and vector status remain uncertain in many areas. However, progress is now being made through application of molecular identification methods, e.g. Thailand (Sharpe etal., 1999), Japan (Somboon etal., 2001), Vietnam (Van Bortel etal., 2001), China (Chen etal., 2002) and now Cambodia. Adult mosquitoes were collected in north-eastern Cambodia at Char Ong Chan village (1345’N, 10656’E), Ratanakiri Province, using various methods of capture during March, July and November 1999. Larvae were also collected and reared to adults. Females were identified morphologically in the field using a modified key for the medically important anophelines of South-east Asia (adapted from IMPE, 1987). They were stored individually in Beem capsules (Agar Scientific Ltd, Stansted, Essex, U.K.) kept in plastic bags with silica gel for desiccation. Morphological identification of An. minimus s.l. was verified using the PCR-RFLP assay based on ITS2 sequences (Van Bortel etal., 2000). Progeny broods were obtained from blood-fed female Anopheles and reared individually to obtain adults with associated larval and pupal exuviae. Final identification of progeny was based on the correlated differential and diagnostic characters of the adult and immature stages (Harrison, 1980). The entire ITS2 rDNA region of three females (specimen nos. K211, K231 and K250) and the cytochrome oxidase I (COI) gene of mitochondrial DNA (mtDNA) of specimen K211 were sequenced. Specimens K211 and K250 are progeny from two different broods of which the mothers were collected by the indoor resting morning collection method. Specimen K231 was wild-collected on man outdoors. Additionally, the ITS2 region was sequenced from four reference specimens from the study in which Green & Miles (1980) first elucidated An. culicifacies
X X
X X X
culicifacies
X
jeyporiensis Reference
X
Sharpe et al. (1999) Sharpe etal. (1999) Kengne etal. (2001) Van Bortel etal. (2000), this paper
species A and B. These specimens, originally retained in the London School of Hygiene and Tropical Medicine and later transferred to The Natural History Museum, London, were as follows: species A, specimens L2/3 (S. J. Miles, Lahore, male) and L2/13 (S. J. Miles, Lahore, female); species B, specimens 44 A and 47 A (S. J. Miles, Delhi,
males). Protocols for DNA extraction, ITS2 rDNA PCR amplification and sequencing are described in Van Bortel etal. (2000). Because PCR amplification of the reference specimens failed after using the extraction method of Collins etal. (1987), the extracts were resuspended in 200 pL TE and the DNA was then carefully extracted once with 200 uL phenol/chloroiorm/isoamyi alcohol (25:24:1) at pH 8 and again with chloroform/isoamyi alcohol (24: 1) according to the method of Sambrook & Russell (2001). Amplification of the COI gene region was performed in a 50-uL reaction mixture containing 1.65mM Mgda, 10 mM Tris-HCl (pH8.4), 50mM KC1, 0.1% Triton X-100, 200 UM dNTPs, 125nM of each primer, 0.5 units Silverstar DNA polymerase (Eurogentec, Seraing, Belgium) and 1-3 pL of template. Cycling conditions were as follows: initial 94C denaturation for 3min, 35 cycles of min denaturation at 94C, min annealing at 50 C and 2 min extension at 72 C followed by a final 72C extension for 10 min. Amplification products were checked on a 1.5% agarose gel and visualized after ethidium bromide staining on the Image master VDS
(Amersham Pharmacia, Uppsala, Sweden). Forward primer UEA7 (5’-TAC-AGT-TGG-AAT-AGACGT-TGA-TAC-3’) (Lunt etal., 1996) and reverse primer UEA Obis (5’-TTC-ATT-GCA-CTA-ATC-TGC-CATATT-A-3’) were used. The latter is adapted from the UEA10 primer (Lunt etal., 1996) to the mtDNA sequence of An. gambiae (Beard etal., 1993). The amplified COI fragment was directly sequenced on an automated A.L.F.express DNA sequencer (Amersham Pharmacia) using the Thermo sequenase Cy5 dye terminator sequencing kit (Amersham Pharmacia). The following ITS2 rDNA sequences were deposited in GenBank: K211, AF440396; K250, AF479311; K231,
AF479312; 47A, AF479313; 44A, AF479314; L2/3, AF479315. The PCR amplification of reference specimen L2/13 failed. The COI mtDNA of K211 was deposited under accession number AF440397. Similarities ofnucleotide
2002 The Royal Entomological Society, Medical and Veterinary Entomology, 16, 329-334
PCR-RFLP of ITS2 shows Anopheles culicifacies B in Cambodia 331 sequences from Cambodian specimens were compared to those in GenBank by a FASTA-search (Pearson & Lipman,
1988). The PCR-RFLP assay was applied to anophelines from Char Ong Chan village identified in the field as An. minimus on adult morphology. Among 106 mosquitoes assayed, 60 (56.6%) were identified as An. minimus species A, seven (6.6%) as An. aconitus and 26 (24.5%) as An. pampancd. The remaining 13 specimens (12.3%) showed a restriction pattern .that was clearly different from the previously known diagnostic PCR-RFLP profiles for members of the An. minimus complex and related species. Individually reared siblings of specimens K211 and K250 were subsequently identified as An. culicifacies based on correlated adult, larval and pupal morphology (Harrison, 1980). The ITS2 fragments, including partial sequences of the 5.8S and 28S genes, of the putative An. culicifacies specimens were 507 bp long. The sequences of specimens K211, K250 and K-231 were identical. The endonuclease BsiZI restriction pattern deduced from the sequences (378 bp and 129 bp) was in agreement with the banding pattern on agarose gel (Fig. 1). Results of the FASTAsearch showed that the ITS2 sequence shared closest similarity with An. culicifacies collected in Sichuan Province, China (GenBank accession no. AY007172, the exact species of the complex is not indicated). Three differences were found in this sequence of 489 bp (99.4% identity), all in the 28S region (Table 2). The approximate boundaries of the ITS2 were defined by comparison with previously determined 5.8S and 28S rDNA sequences of anophelines (Porter & Collins, 1991; Paskewitz etal., 1993). The second
1
2
3
closest match in FASTA-search was another An. culicifacies sequence from Sichuan Province, China, and the third most similar sequence was the ITS2 sequence for An. culicifacies A (AF402297), which shared 93.1% identity. No other An. culicifacies were detected in the FASTA-search. A partial sequence of the COI gene of 643 bp was obtained. Highest similarity (99.8%) was found with An. culicifacies B from India (GenBank accession no. AF117800: 605 bp with only one position different). The best score for An. culicifacies A was found for sequence AF117793, with 31 differences among 605 bp (94.9% identity) (Table 2). The ITS2 region of the Cambodian specimens had the same sequence as reference specimen 47A of An. culicifacies B (Table 2). Specimen 44A showed one difference at position 256. Specimens L2/3 and AF402297 of An. culicifacies A showed four differences, with one in the ITS2 region. Comparison of ITS2 restriction patterns from the An. culicifacies A and B reference specimens revealed that BsiZI restriction endonuclease does not differentiate the two species. This study revealed the presence of An. culicifacies B in Cambodia, which is not surprising because An. culicifacies s.l. was recently found to occur in Binh Thuan Province of south-central Vietnam (Van Bortel et al., 2001), i.e. ~300 km farther south than the Cambodian site. Prior to these records, the An. culicifacies complex had not been detected so far south in South-east Asia, having been recorded previously from more northerly parts of Thailand, Laos and Vietnam, and the extreme south of China (Harrison,
1980). Anopheles culicifacies is a complex of five species distinguishable by polytene chromosome banding patterns or metaphase karyotypes: species A and B (Green & Miles, 1980), species C (Subbarao etal., 1983), species D (Suguna etal., 1989; Vasantha etal., 1991) and species E (Kar etal., 1999). Species A and D are efficient malaria vectors, whereas species B is regarded as a poor vector of malaria in India (Subbarao, 1988; Subbarao etal., 1992). In Sri Lanka, however, species B has long been considered to be an important malaria vector. Likewise on Rameshwaram Island, India, where malaria incidence is high, only An. culicifacies B was found (Subbarao etal., 1993). On this island, Kar etal. (1999) found, on cytogenetic evidence, two reproductively isolated populations in An.
Fig.1. BsiZI restriction pattern of the ITS2 rDNA fragment on 1.5% agarose. Lane 1, specimen K211, An. culicifacies B; lane 2, An. minimus A; lane 3, ladder.
(C)
culicifacies
showing differences in sporozoite rates. Kar etal. (1999) designated the non-vector as species B and the vector as species E. Recently, Surendran etal. (2000) suggested that species E is present in Sri Lanka and that this species, and not species B, is responsible for malaria transmission. Species B is widespread in India, occurring in sympatry with one or more species of the complex, and it has the most easterly distribution of the five species (Subbarao etal., 1988; Subbarao, 1988). Anopheles culicifacies in Cambodia is undoubtedly species B of the complex, based on complete agreement of the ITS2 reference specimens of this taxon. This is supported by shared sequences of the COI region. Likewise, the specimen from Sichuan Province, China, can also be equated with species B. In 1987,
2002 The Royal Entomological Society, Medical and Veterinary Entomology, 16, 329-334
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W. Van Bortel et al. 8
