Multiplex PCR assay for malaria vector Anophelesminimus and four related species in the Myzomyia Series from Southeast Asia H.. For identification of specimens from Indochina Cambodia, L
Trang 1Multiplex PCR assay for malaria vector Anopheles
minimus and four related species in the Myzomyia
Series from Southeast Asia
H K P H U C , A J B A L L , L S O N*, N V H A N H*, N D T U*, N G L I E N*,
A V E R A R D I and H T O W N S O N
Liverpool School of Tropical Medicine, U.K and *Hanoi University of Science, Vietnam
Series are important malaria vectors in Africa, India and Southeast Asia Among
10 named species of Myzomyia known from the Oriental Region, seven form the
An minimusgroup Even for expert taxonomists, the adults of these species remain difficult to identify morphologically For technical staff of malaria control pro-grammes, confusion may extend to misidentification of species that are not for-mally within the minimus group For identification of specimens from Indochina (Cambodia, Laos, Vietnam), we describe a multiplex polymerase chain reaction (PCR) assay, based on rDNA internal transcribed spacer 2 (ITS2) sequences, that employs a cocktail of primers to identify An minimus Theobald sibling species A and C (sensu; Green et al., 1990) and three other species in the An minimus group (An aconitus Do¨nitz, An pampanai Bu¨ttiker & Beales, An varuna Iyengar), as well
as An jeyporiensis James, also belonging to the Myzomyia Series As the test is DNA-based, it can be applied to all life stages of these mosquitoes for ecological investigations and vector incrimination studies This PCR assay is simpler, quicker, cheaper and more readily interpreted than previous assays
An varuna, Myzomyia, malaria vectors, polymerase chain reaction assay, rDNA ITS2, sibling species, species identification, Southeast Asia, Cambodia, Laos, Vietnam
Introduction
vector throughout the Oriental region (Reid, 1968; Harrison,
1980; Lien, 1991; Pholsena, 1992; Baimai et al., 1996; Dev,
1996; Sawabe et al., 1996; Khan et al., 1998; Kobayashi
et al., 2000; Chen et al., 2002) In Thailand, Green et al
(1990) considered An minimus to comprise at least two sib-ling species, A and C, based on the absence of heterozygotes for two alleles of octanol dehydrogenase (Odh 100 and 134) The work of Sharpe et al (2000) and Somboon et al (2001) confirms that An minimus (sensu Harrison, 1980) is a com-plex of several species, with important differences in vector roles and distributions (Chen et al., 2002)
For other species in the An minimus group (Harrison, 1980; Chen et al., 2003), evidence of a role in malaria trans-mission is scanty and difficult to evaluate In addition to the
of the An minimus group among 11 Oriental species
Correspondence: Professor H Townson, Liverpool School of
Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.
E-mail: htownson@liverpool.ac.uk
Trang 2described in the Myzomyia*Series (sensu Edwards, 1932) of
over-lapping morphological characters lead to confusion for field
entomologists seeking to implicate the species in malaria
transmission, particularly where two or more species are
in sympatry For example, the appearance of pale spots on
the costal wing vein is used in diagnosis, yet in some parts of
southeast Asia, An minimus has highly variable wing
spot-ting that may lead to it being confused with other
Myzo-myia species (Van Bortel et al., 2000, 2001, 2002) Anopheles
but usually zoophilic Rahman et al (1995) found
cattle in Pensinsula Malaysia, whereas Hassan et al (2001)
collected it biting humans indoors in Malaysia Anopheles
(Webster, 2000) and was the primary inland vector in Java
considered a malaria vector in the hilly and forested parts
of east-central India (Rao, 1984), but its overall distribution
and importance as a malaria vector in the Oriental region is
uncertain because it has been, and continues to be, much
confused with An minimus (Reid, 1968; Van Bortel et al.,
2001) In the case of An pampanai there are few records to
indicate its true vector status
Regarding other Oriental Myzomyia, An filipinae and
and E) that may all contribute to malaria transmission in the
Indian subcontinent (Subbarao, 1998; Kar et al., 1999), of
which only species B has been recorded from southeast Asia
(Van Bortel et al., 2002) Anopheles jeyporiensis is the
com-monest anopheline in the western Ghats and has been
recorded as a vector of malaria in many parts of India,
particu-larly in the Jeypore Hills (Rao, 1984) from where it was
described, although Reid (1968) regarded it as only a minor
vector of malaria in the Orient In Vietnam, hundreds of
specimens of An jeyporiensis have been examined using a
sporozite enzyme-linked immunosorbent assay and not a
sin-gle positive has been found (Binh Nguyen Thi Huong, personal
communication) Likewise, in Assam the sporozoite rate of
0.02–0.7% in An varuna, where the major vector is An minimus
with much higher sporozoite rates (Rao, 1984; Dev, 1996)
Sharpe et al (1999) used allele-specific amplification of the D3 variable region of the 28S rDNA gene to distinguish
polymorphism (SSCP) of the D3 amplified region to dis-criminate four species, An varuna, An aconitus, An minimus
A and C Unfortunately, this method is more time consuming and the results less easy to interpret than those from conventional polymerase chain reaction (PCR) An alternative approach was adopted by Van Bortel et al (2000) who used PCR amplification of the rDNA rDNA internal transcriber spacer 2 (ITS2) region followed by BsiZI restriction enzyme digestion to distinguish An aconitus,
An jeyporiensis, An minimus A and C, An pampanai,
et al., 2002) Compared to SSCP, this is simpler, but it requires local medical entomologists to employ both PCR and restriction fragment length polymorphism (RFLP) analy-sis with consequent increased costs and methodological complexity Furthermore, RFLP fragments of very similar size are produced in An minimus C and An varuna, so that these species may easily be confused
Most recently, Kengne et al (2001) described the use of random amplified polymorphic DNA (RAPD) markers for the identification of species in the An minimus group RAPD fragments specific for species A and C were cloned and sequenced From these sequences, specific primer pairs were designed to create a multiplex PCR able to identify five species:
In this assay, the PCR products from hybrids of minimus
A and C were different from both parents These so-called
‘hybrizymes’ add to the confusion of using such a method in routine, large-scale sampling of mosquito populations
In this study, we describe the development and use of a method, based on a single multiplex PCR, for identifying
related species, An aconitus, An jeyporiensis, An varuna and
Cambodia, Laos and Vietnam (Indochina) This method has proved both simple and reliable in our hands, and when employed by others (C Walton, personal communication)
Materials and methods Mosquitoes
The specimens examined in this study were collected from
36 study sites in Vietnam, Laos and Cambodia The geo-graphical location and ecological character of the sites are shown in Fig 1 and Table 1 Female mosquitoes were also collected using landing collections on human bait A pro-portion were allowed to feed (either on the senior author or
a graduate volunteer, with informed consent) and their eggs collected and larvae reared Specimens were identified to species using morphological characters and the key of Harrison (1980) Because the molecular characteristics of these species are unambiguously different (see below), any specimens that initially were of doubtful identity, were
*
According to Harbach (1994) the Myzomyia Series includes
more than 60 recognized species of Anopheles (Cellia), mostly
endemic to the Afrotropical Region, with 11 named taxa in the
Oriental Region: aconitus Do¨nitz, filipinae Manalang, culicifacies
Giles, flavirostris (Ludlow), fluviatilis James, jeyporiensis James,
majidi (Young & Majid), mangyanus (Banks), minimus Theobald,
pampanai Bu¨ttiker & Beales and varuna Iyengar Those in bold
comprise the An minimus group Several of these nominal
species-group taxa are complexes of multiple sibling species with
non-Linnean terminology Chen et al (2002) reviewed the An minimus
group in China, where Hong Kong is the type-locality of minimus.
Molecular phylogeny of Oriental Myzomyia was interpreted by
Chen et al (2003).
Trang 3subsequently assigned readily to species on the basis of
the PCR test described below For An minimus s.l., several
offspring from each brood were then examined for their
octanol dehydrogenase electromorphs (Green et al., 1990)
to determine whether they were An minimus A or C The
remainder of the brood was preserved in absolute alcohol
for subsequent DNA studies Likewise, specimens collected
by CDC light-trap were identified morphologically and
then placed directly in absolute alcohol for assays later
Amplification and sequencing
extracted following the method of Ballinger-Crabtree et al
(1992), the final DNA pellet being re-suspended in 50 mL of
TE buffer The rDNA ITS2 region was then amplified
using conserved 5.8S and 28S primers modified from those
used by Paskewitz & Collins (1990) Each PCR reaction was
carried out in 0.5-mL microtubes in an Omni-E thermal
cycler (Hybaid: www.thermohybaid.com) using a 25 mL
DNA polymerase (Sigma: www.sigmaaldrich.com) and
20–50 ng DNA template The thermal cycle profile was as
Spin column (Qiagen: www.qiagen.com) and the products checked by running on agarose gels again before sequencing
(www.appliedbio-systems.com)
Species diagnostic PCR The assay employed six species-specific reverse primers and one universal forward primer derived from the highly conserved region of the 5.8S coding region (Tables 2 and 3) The PCR reaction was run in a 25 mL volume containing:
0.625 U Taq, and 10–20 ng DNA template This diagnostic PCR requires very little DNA and results were obtained with c 1/800 of the total DNA from a mosquito or 1/50 of the DNA extracted from a single leg The thermal cycle
5–10 min Ten microlitres of reaction products were run on 1.2% agarose gels containing ethidium bromide and pro-duced fragments whose sizes were diagnostic for the species (see Fig 2)
Results Specific ITS2 primers The complete ITS2 region was amplified from 13 speci-mens of An minimus A (373 bp) collected from Laos, Cambodia, Vietnam and on the border of Vietnam and China and from nine specimens of An minimus C (375 bp) from northern Laos, Vietnam and the border of Vietnam and China (see Table 2) There are a total of 23 bp differences between An minimus A and C, comprising two indels, 15 transversions and six transitions, and these differences were consistent throughout the sampling area In comparison, there are few similarities between the ITS2 sequences of
An aconitus, An pampanai and An varuna, whose ITS2 regions are 278, 255 and 227 bp, respectively These differ-ences confound alignment of their ITS2 sequdiffer-ences Based on species-specific differences, six primers were designed and using as reverse primers These reverse primers were mixed with 5.8S forward primer to amplify the whole ITS2 region (Manonmani et al., 2001) This diagnos-tic cocktail gives a 184 bp PCR product with template DNA from An minimus A, a 252 bp band for An varuna, a 306 bp band for An aconitus, a 346 bp band for An jeyporiensis,
a 452 bp band for An pampanai and a 509 bp band for
32 LAOS
CHINA
THAILAND
CAMBODIA
6 5
4 2
7 8
9 13 14 16 15 12 11 17 18 19
20 21 33 34
23 24 22
25
28 26 27 29
30 31 36
35
Fig 1 Map showing the mosquito collection sites in Cambodia,
Laos and Vietnam Details of sites are given in Table 1.
Trang 4PCR products that are significantly different in size to
distinguish them readily by examination of an ethidium
bromide-stained gel (Fig 2) A natural hybrid of An
as was a laboratory-produced hybrid; each gave two bands,
one from each parent
These primers were used to identify 661 adult female
mosquitoes, comprising 25 An aconitus, 65 An jeyporiensis,
280 An minimus A, 150 An minimus C, 31 An pampanai
and 110 An varuna All results were unambiguous and
agreed completely with isoenzyme or mitotic chromosome
determinations Smaller numbers of larvae and adult males
were also identified using this assay
Discussion From the area sampled, no intraspecific variation was detected in the sequence of the ITS2 region in mosquitoes
of the An minimus complex Samples of An minimus A from the extreme north of Vietnam (bordering southern China), northern Laos to central Vietnam and Laos, and southern Vietnam and north-west Cambodia have identical ITS2 sequence with An minimus A from Thailand, as described
by Sharpe et al (2000) Similarly, An minimus C from the Vietnam/China border, through middle Vietnam and Laos all have identical ITS2 sequences Studies of mitochondrial DNA sequences and of allelic variation at
Table 1 Details of sampling sites of Anopheles minimus A and C and An aconitus (Aco), An jeyporiensis (Je), An pampanai (Pa) and
An varuna (Va) Localities are those of a village or hamlet and the relevant province HBC, human bait catches; BBC, bovid bait catches; MIC, early morning indoor collections; LT, CDC light-trap collections; ORC, outdoor-resting collections; LS, larval sampling
Species Locality name/province Terrain
Date of collection
Geographical coordinates Collection method Vietnam
A, C, Je, Aco 1 Phuong Do/Ha Giang Mountain and foothills March 25, 1999 22 49 0 N 105 3 0 E BBC*, HBC*, MIC
A, Je, Aco 2 Quang Ngan Foothills March 26, 1999 22 31 0 N 104 54 0 E BBC*, HBC*, MIC
A, Je, Aco 3 Vuoc/Lao Cai Foothills February 10, 1999 22 31 0 N 104 6 0 E BBC*, HBC*, MIC
A, Je 4 Qua Foothills February 10, 1999 22 31 0 N 104 5 0 E BBC*, MIC*
A, C, Je 5 Thon/Son La Savanna October 9, 1999 21 4 0 N 104 5 0 E BBC*, MIC
A, C, Je 6 Huoi Oi High mountains October 11, 1999 20 55 0 N 104 3 0 E BBC*
A, C, Je, Aco 7 Ca Lieng/Cao Bang Mountains November 5, 1999 22 29 0 N 106 34 0 E BBC*, HBC
A, C, Je, Aco 8 Na Phuong Mountains November 4, 1999 22 31 0 N 106 33 0 E BBC*, HBC
A, C, Je, Aco 9 Ang Mo/Lang Son Mountains April 8, 2000 22 15 0 N 106 28 0 E BBC*, HBC, MIC
A, C, Je, Aco 10 Phu Cuong/Hoa Binh Mountains Three trips, 1999 20 37 0 N 105 16 0 E BBC*, HBC*, MIC*
A, C, Je, Aco 11 Quyet Chien High mountains Five trips, 1999 20 30 0 N 105 11 0 E BBC*, HBC, MIC*
A, Aco 12 Hoa Son Foothills Laboratory colony 20550N 105400E MIC*
A 13 Giang Bien/Hanoi Plain Tens of trips 21 3 0 N 105 55 0 E LS*, MIC*, ORC*
Aco, Je 16 Cat Ba/HaiPhong Island August 12, 1999 20 44 0 N 107 2 0 E BBC*
A, C, Je, Aco 17 Thach Thanh/Thanh Hoa Primitive forest November 12, 1999 20 14 0 N 105 36 0 E BBC*, HBC
A, Je, Aco 18 Quynh Thang/Nghe An Savanna and foothills November 23, 1999 19 19 0 N 105 25 0 E BBC*, HBC*
A, C, Aco 19 Cam Lam High mountains November 21, 1999 18 52 0 N 104 42 0 E BBC*, HBC*
A, C, Aco 20 Huong Khe/Ha Tinh Forest November 24, 1999 18 23 0 N 105 28 0 E BBC*
A, Aco 21 Ky Thinh Coast plain November 25, 1999 18 5 0 N 106 23 0 E BBC*
A 22 Van Canh Foothill Dry sample 13 37 0 N 108 59 0 E BBC*
A, Je, Aco 23 Konthup/Gia Lai Highland June 10, 1999 13 44 0 N 107 51 0 E BBC*, HBC, LT*
A, Je, Aco 24 Alaba Highland June 15, 1999 14 12 0 N 107 55 0 E BBC*, HBC, LT* Aco, Je 25 Khanh Phu/Khanh Hoa Mountain June 2, 1999 12 14 0 N 108 58 0 E BBC*
A, Aco, Va 26 Binh Thanh/BinhThuan Coast plain Four trips, 1999 11 10 0 N 108 41 0 E BBC*
Aco, Pa, Va 27 Tanh Linh Forest Two trips, 1999 11 5 0 N 107 40 0 E BBC*, HBC Aco, Je 28 Lada Forest and mountain May 21, 1999 11 9 0 N 107 16 0 E BBC*, HBC
A, Aco 29 Dong Xoai/Dong Nai Plain July 20, 1999 11320N 106540E BBC*
A, Je, Aco 30 Dac U/Binh Phuoc Forest Three trips, 1999 12140N 107260E BBC*, HBC*
A, Aco 31 Thanh Tan/Tay Ninh Plain and foothills May 26, 1999 11 19 0 N 106 3 0 E BBC*, HBC
Laos
A, C, Je, Aco 32 Muong Son/Samneur High mountain Dec, 1999 20 53 0 N 104 2 0 E BBC*, HBC
A, Aco, Je 33 Voong Co/Bolikhamxay Forest February 26, 1999 18 12 0 N 104 46 0 E BBC*, HBC*, MIC*
A, Aco 34 Khamcot Mountainous town February 30, 1999 18 11 0 N 104 58 0 E BBC*, MIC*
A, Je 35 Phonxay Forest March 2, 1999 18 15 0 N 104 56 0 E BBC*
Cambodia
A, Aco, Pa,Va 36 Ta Trok/Battambang Forest and plain July 3, 1999 12 51 0 N 102 49 0 E BBC*, HBC
*Methods used successfully to collect An minimus.
Trang 5microsatellite loci (Phuc and Townson, unpublished data)
show evidence of significant genetic variation within
species A and C, but this is not reflected at the level of
the ITS2 sequences The significance of this variation and
its relevance to speciation in the minimus complex can only be assessed once these other data are complete and analysed
For An aconitus, one individual was sequenced from each collection site and identical ITS2 sequences were found in all specimens from northern and southern Vietnam The same is true of An jeyporiensis Samples of An varuna and
any ITS2 sequence variation being found For the six species studied, therefore, the apparent absence of intra-specific variation within the ITS2 region sequence between samples from Cambodia, Laos and Vietnam, together with the unambiguous differences in PCR product-size, make this assay very suitable for differentiation of An aconitus,
An jeyporiensis, An pampanai and An varuna, as well as distinguishing between species A and C of An minimus in Indochina and probably elsewhere
Acknowledgements
We thank the World Health Organization TDR pro-gramme, The Leverhulme Trust and the British Council for funding this research and associated field studies
Table 3 Sequence of the primers used for ITS2 amplification and species diagnosis within Myzomyia
Oligonucleotide sequence (5 0 3 0 ) Function
Size of the PCR product (bp) PCR primers used for ITS2 amplification
PCR primers used for species diagnosis
Table 2 DNA sequence of ITS2 in Anopheles minimus species A and C showing insertions/deletions (indels –), transitions (s) and transversions (v) The two species differ at 23 out of the 375 bases found in species C
Nucleotide position
4 1 2 9 3 4 8 0 2 4 5 8 0 3 7 1 2 5 pairs Percentage
An minimus A A C A C T – – T A G G C G G C A A A A A C A A 373
An minimus C T A T T A A A G C C A G A T A G G C C C T C C 375
184 252 306 346 452 509 bp
Fig 2 Products from the multiplex PCR run on a 1.2% agarose
gel Track 1, 100 bp molecular weight ladder (Sigma P1473); track
2, Anopheles minimus A; track 3, An varuna; track 4, An aconitus;
track 5, An jeyporiensis; track 6, An pampanai; track 7, An minimus
C; track 8, laboratory A/C hybrid; track 9, natural A/C hybrid.
Trang 6Baimai, V., Kijchalao, U & Rattanarithikul, R (1996) Metaphase
karyotypes of Anopheles of Thailand and Southeast Asia: V, The
Myzomyia series, subgenus Cellia (Diptera: Culicidae) Journal
of the American Mosquito Control Association, 12, 97–105.
Ballinger-Crabtree, M.E., Black, W.C & Miller, B.R (1992) Use of
genetic polymorphisms detected by the random-amplified
polymorphic DNA polymerase chain reaction (RAPD-PCR)
for differentiation and identification of Aedes aegypti subspecies
and populations American Journal of Tropical Medicine and
Hygiene, 47, 893–901.
Chen, B., Harbach, R.E & Butlin, R.K (2002) Molecular and
morphological studies on the An minimus group of mosquitoes
in southern China: taxonomic review, distribution and malaria
vector status Medical and Veterinary Entomology, 16, 253–265.
Chen, B., Butlin, R.K & Harbach, R.E (2003) Molecular
phylo-genetics of the Oriental members of the Myzomyia Series of
Anopheles subgenus Cellia (Diptera: Culicidae) inferred from
nuclear and mitochondrial sequences Systematic Entomology, 28,
57–69.
Chow, C.Y., Ibnoe, M.R & Josopoero, S.T (1959) Bionomics of
anopheline mosquitoes in inland areas of Java, with special
reference to Anopheles aconitus Do¨n Bulletin of Entomological
Research, 50, 647–660.
Dev, V (1996) Anopheles minimus: its bionomics and role in the
transmission of malaria in Assam, India Bulletin of the World
Health Organization, 74, 61–66.
Edwards, F.W (1932) Genera Insectorum Diptera Fam
Culici-dae Fascicle, 194, 1–258.
Green, C.A., Gass, R.F., Munstermann, L.E & Baimai, V (1990)
Population genetic evidence for two species in Anopheles
minimus Thailand Medical and Veterinary Entomology, 4, 25–34.
Harbach, R.E (1994) Review of the internal classification of the
genus Anopheles (Diptera: Culicidae): the foundation for
comparative systematics and phylogenetic research Bulletin of
Entomological Research, 84, 331–342.
Harrison, B.A (1980) Medical entomology studies XIII The
Myzomyia series of Anopheles (Cellia) in Thailand, with
emphasis on intra-interspecific variations (Diptera: Culicidae).
Contributions of the American Entomological Institute, 17, 1–195.
Hassan, A.A., Rahman, W.A., Rashid, M.Z.A., Shahrem, M.R &
Adanan, C.R (2001) Composition and biting activity of
Anopheles (Diptera: Culicidae) attracted to human bait in a
malaria endemic village in peninsular Malaysia near the
Thailand border Journal of Vector Ecology, 26, 70–75.
Kar, I., Subbarao, S.K., Eapen, A et al (1999) Evidence for a new
malaria vector species, species E, within the Anopheles
culicifacies complex (Diptera: Culicidae) Journal of Medical
Entomology, 36, 595–600.
Kengne, P., Trung, H.D., Baimai, V., Coosemans, M & Manguin, S.
(2001) A multiplex PCR-based method derived from random
amplified polymorphic DNA (RAPD) markers for the
identifi-cation of species of the Anopheles minimus group in Southeast
Asia Insect Molecular Biology, 10, 427–435.
Khan, S.A., Handique, R., Tewari, S.C., Dutta, P., Narain, K &
Mahanta, J (1998) Larval ecology and mosquito fauna of upper
Brahmaputra valley, northeast India Indian Journal of
Malariology, 35, 131–145.
Kobayashi, J., Somboon, P., Koemanila, H et al (2000) Malaria
prevalence and a brief entomological survey in a village
surrounded by rice fields in Khammouan province, Lao PDR.
Tropical Medicine and International Health, 5, 17–21.
Lien, J.C (1991) Anopheline mosquitoes and malaria parasites in Taiwan Gaoxiong Yi Xue Ke Xue Za Zhi, 7, 207–223 Manonmani, A., Townson, H., Adeniran, T., Jambulingam, P., Sahu, S & Vijayakuma, T (2001) rDNA-ITS2 polymerase chain reaction assay for the sibling species of Anopheles fluviatilis Acta Tropica, 78, 3–9.
Paskewitz, S.M & Collins, F.H (1990) Use of the polymerase chain reaction to identify mosquito species of the Anopheles gambiae complex Medical and Veterinary Entomology, 4, 367–373 Pholsena, K (1992) The malaria situation and antimalaria program in Laos Southeast Asian Journal of Tropical Medicine and Public Health, 23, 39–42.
Rahman, W.A., Hassan, A.A., Adanan, C.R & Rashid, M.R (1995)
A report of Anopheles minimus (Diptera: Culicidae) attracted to cow bait in a malaria endemic village in peninsular Malaysia near the Thailand border Southeast Asian Journal of Tropical Medicine and Public Health, 26, 359–363.
Rao, T.R (1984) The Anophelines of India Malaria Research Centre, Indian Council of Medical Research, New Delhi Reid, J.A (1968) Anopheles mosquitoes of Malaya and Borneo Studies from the Institute for Medical Research Malaysia, 31, 1–520.
Sawabe, K., Takagi, M., Tsuda, Y et al (1996) Genetic differentiation among three populations of Anopheles minimus
of Guangxi and Yunnan Provinces in the People’s Republic of China Southeast Asian Journal of Tropical Medicine and Public Health, 27, 818–827.
Sharpe, R.G., Hims, M.M., Harbach, R.E & Butlin, R.K (1999) PCR-based methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand conformation polymorphism Medical and Veter-inary Entomology, 13, 265–273.
Sharpe, R.G., Harbach, R.E & Butlin, R.K (2000) Molecular variation and phylogeny of the Minimus group of Anopheles subgenus Cellia (Diptera: Culicidae) Systematic Entomology,
25, 263–272.
Somboon, P., Walton, C., Sharpe, R.G., Higa, Y., Tuno, N., Tsuda, Y.
& Takagi, M (2001) Evidence for a new sibling species of Anopheles minimus from the Ryuku Archipelago, Japan Journal
of the American Mosquito Control Association, 17, 98–113 Subbarao, S.K (1998) Anopheline Species Complexes in South-East Asia Technical Publication, SEARO No 18, pp 1–82 World Health Organization Regional Office for South-East Asia, New Delhi.
Van Bortel, W., Trung, H.D., Roelants, P., Harbach, R.E., Backeljau, T & Coosemans, M (2000) Molecular identification
of Anopheles minimus s.1 beyond distinguishing the members of the species complex Insect Molecular Biology, 9, 335–40 Van Bortel, W., Harbach, R.E., Trung, H.D., Roelants, P., Backeljau, T & Coosemans, M (2001) Confirmation of Anopheles varuna in Vietnam, previously misidentified and mistargeted as the malaria vector Anopheles minimus American Journal of Tropical Medicine and Hygiene, 65, 729–732.
Van Bortel, W., Sochanta, T., Harbach, R.E., Socheat, D., Roelants, P., Backeljau, T & Coosemans, M (2002) 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 Medical and Veterinary Entomology, 16, 329–334.
Webster, J (2000) Malaria control in complex emergencies Cambodia http://www.malariaconsortium.org/Cambodia.pdf Accepted 21 September 2003