MORPHOLOGY, SYSTEMATICS, EVOLUTION

Polymerase Chain Reaction Identification of Three Members ofthe Anopheles sundaicus (Diptera: Culicidae) Complex,

Malaria Vectors in Southeast Asia

ISABELLE DUSFOUR,1,2 JOHANNA BLONDEAU,1 RALPH E. HARBACH,3 INDRA VYTHILINGHAM,4

VISUT BAIMAI,5 HO D. TRUNG,6 THO SOCHANTA,7 MICHAEL J. BANGS,8

AND SYLVIE MANGUIN1,9

J. Med. Entomol. 44(5): 723Ð731 (2007)

ABSTRACT Anopheles sundaicus s.l., a major malaria vector taxon, occurs primarily along coastalareas and on islands in Southeast Asia. Our previous studies using cytochrome oxidase I, cytochrome-b,and internal transcribed spacer 2 markers discriminated three allopatric species: An. sundaicus s.s. innorthern Borneo,An. epiroticus in Southeast Asia, andAn. sundaicusE on Sumatra and Java, Indonesia.Morphological comparisons of three developmental stages did not reveal unique diagnostic charactersthat could reliably distinguish the three species. Therefore, we developed a multiplex polymerasechain reaction (PCR) assay based on two mitochondrial DNA markers to unambiguously identifythem. This PCR was tested on 374 specimens from 24 different geographical populations, expandingour knowledge of the distribution of these species.

KEY WORDS Anopheles sundaicus complex, morphology, multiplex polymerase chain reaction,mitochondrial DNA, Southeast Asia

Most of the important malaria vectors in SoutheastAsia are members of complexes in which the speciesare morphologically very similar or indistinguishable(Subbarao 1998). The difÞculties of distinguishing sib-ling species stimulated the development of moleculartools for precisely and reliably identifying cryptic spe-cies (Scott et al. 1993; Collins and Paskewitz 1996;Walton et al. 1999; Kengne et al. 2001; Manguin et al.2002; Garros et al. 2004a, 2004b). Among major vectorsin Southeast Asia, Anopheles (Cellia) sundaicus Ro-denwaldt occurs primarily along coastal areas and is-lands extending from India to Vietnam, and southwardto Malaysia and Indonesia (Dusfour et al. 2004a) (Fig.1). It is considered as either a major or secondary

vector of malaria, depending on locality and epide-miological factors inßuencing transmission (Schaeferand Kirnowardoyo 1983, Meek 1995). For example,Kirnowardoya and Yoga (1987) observed that malariatransmission in Cilacap on the southern coast of west-ern Java was mainly due to An. sundaicus but thattransmission ßuctuated widely from year to year andfrom different localities during the same year, whichimplied complex and poorly understood epidemiologyof transmission and the difÞculties with developingeffective control strategies in this area.

The wide geographical distribution and ecologicaland behavioral diversity ofAn. sundaicus s.l. suggestedthe presence of a species complex (Reid 1970). Anotable indication was the presence of immaturestages both in brackish and freshwater. Cytogeneticstudies and isozyme analyses showed the presence ofthree distinct species, A, B, and C, in Indonesia andThailand (Sukowati et al. 1996, 1999). Because thetype series described by Rodenwaldt (1925) was non-extant (Bonne-Webster and Swellengrebel 1953, Reid1968), the identity of An. sundaicus s.s. was Þxed by aneotype designated from material collected at PandanBeach, Lundu District in the state of Sarawak on theisland of Borneo (Linton et al. 2001). A fourth cyto-type, D, including freshwater and brackish water pop-ulations, also was recently identiÞed on the Andamanand Nicobar Archipelagos (Nanda et al. 2004, Alam etal. 2006). In addition, the analysis of two partial mi-tochondrial DNA (mtDNA) markers, cytochrome ox-idase I (COI) and cytochrome-b (Cyt-b), and an

1 Institut de Recherche pour le Developpement (IRD), UMR22Biologie et Gestion des Populations (CBGP), Campus de Baillarguet,CS30016, Montferrier sur Lez 34988, France.

2 Current address: Uniformed Services University of the HealthSciences (USUHS), Dept. PMB, 4301 Jones Bridge Rd., Bethesda, MD20814.

3 Department of Entomology, The Natural History Museum, Crom-well Rd., London SW7 5BD, United Kingdom.

4 Institute for Medical Research, Jalan Pahang, 50588 Kuala Lum-pur, Malaysia.

5 Department of Biology, Mahidol University, Rama IV RD,Bangkok 10400, Thailand.

6 National Institute of Malariology, Parasitology and Entomology,Luong the Vinh St., BC 10.200 Tu Liem, Hanoi, Vietnam.

7 National Center for Malaria Control, Parasitology and Entomol-ogy, 372 Monivong Blvd. Corner St. 322, Phnom Penh, Cambodia.

8 Preventive Medicine Department, Navy Region Northwest, 2850Thresher Ave., Silverdale, WA 98315.

9 Corresponding author, e-mail: manguin@mpl.ird.fr.

0022-2585/07/0723Ð0731$04.00/0 � 2007 Entomological Society of America

rDNA internal transcribed spacer 2 (ITS2) (Dusfouret al. 2007), did not match the three cytogenetic spe-cies recognized by Sukowati et al. (1996, 1999). There-fore, currently there are four cytogenetic species, A,B, C, and D (Sukowati et al. 1996, 1999; Nanda et al.2004), and three molecularly isolated entities. Two ofthe three taxa are formally recognized as species,namely An. sundaicus s.s. (Linton et al. 2001) and An.epiroticus (Linton et al. 2005), based on distinct ITS2and mtDNA sequences. The third taxon is distinctfrom bothAn. sundaicus s.s. andAn. epiroticusbased onmtDNA (Dusfour et al. 2007). The analysis of ITS2 ofrDNA showed that this new genetic entity was distinctfrom An. sundaicus s.s. but that it shared variants withAn. epiroticus and exhibited intragenomic variation.These results based on the ITS2 suggest a possibleintrogression event (Tang et al. 1996), not revealed bymtDNA. This observation might be explained by thecombination of genetic homogenization at nuclearloci while retaining the original mtDNA (Garcia-Pariset al. 2003), along with loss of diversity caused bycyclical climate and sea level ßuctuation during thePleistocene (Gorog et al. 2004). We referred to theIndonesian lineage as An. sundaicus species E. More-over, the three taxa have an allopatric distribution.An.sundaicus s.s. includes freshwater (concentrations�0.5 g salt/liter) and brackish (concentrations �0.5and �30 g salt/liter) water populations in northernBorneo.An. epiroticus (formerlyAn. sundaicus speciesA), which is characterized by brackish water popula-tions, only occurs in coastal areas from southern Viet-nam, Cambodia, and Thailand southward to peninsu-lar Malaysia (Linton et al. 2005). An. sundaicus E isknown to from brackish and freshwater sites on theislands of Sumatra and Java. The three species are

currently isolated by geographic barriers such as theSouth China Sea, the Malacca Strait, and the Java Sea.An. sundaicus D is also geographically isolated on theNicobar Island and this species has been found in bothbrackish and freshwater (Nanda et al. 2004, Alam et al.2006).

Morphological studies on populations of An. sun-daicus s.l. have revealed differences in the size ofcostal spots of the wings of specimens from Malaysia(Reid 1968) and in the preapical pale spots on thepalpus of specimens from the Andaman Islands (Nag-pal andSharma1983).However,neithercharacterwascorrelated with species status. Recently, the compar-ison of two members of the complex,An. epiroticus andAn. sundaicus s.s., revealed no morphological differ-ences that could reliably distinguish them (Linton etal. 2005). However, the morphology of An. sundaicusspecies E has not been compared with that of eitherAn. epiroticus or An. sundaicus s.s. SpeciÞc ecological,behavioral, and distributional data are currently un-available for the individual species of the SundaicusComplex.

An initial polymerase chain reaction (PCR)method of species identiÞcation was developedbased on the recognition of the sequence charac-teristic ampliÞed region from a study of randomampliÞed polymorphism DNA that could differen-tiate An. sundaicus s.s. and An. epiroticus (Dusfouret al. 2004b). The aim of the current study was tocompare morphological characters and DNA se-quences of the three sibling species concomitantwith the development of a simple and reliable iden-tiÞcation method to foster studies of their bionomicsand medical/epidemiological importance.

Fig. 1. Map of Southeast Asia showing the known distribution of An. sundaicus s.l. (in gray) and the populations of An.sundaicus s.s., An. epiroticus, and An. sundaicus species E collected, represented by a triangle, circle, and star, respectively.Enlargement illustrates nine study sites in southern Vietnam (see Table 1 for details on population codes).

724 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 5

Materials and Methods

Mosquito Collections.All Þeld-collected specimenswere Þrst identiÞed as An. sundaicus s.l. by using themorphological keys of Reid (1968) and IMPE (1987).Morphological Samples. Fourth instars, pupae, and

adults reared from progeny broods of females col-lected in the Asahan and South Tapanuli Districts ofnorthern Sumatra, Indonesia, were compared with thetype specimens of An. sundaicus s.s. and An. epiroticusfrom Pandan Beach, Lundu District, Sarawak, Malay-sia, and Can Gio District, Ho Chi Minh Province,southern Vietnam, respectively (Fig. 1; Table 1). TheIndonesian specimens were sampled within the batchof sister-females of the 27 An. sundaicus species Especimens identiÞed by sequence analysis (Dusfour etal. 2007).Molecular Samples. Specimens used for the devel-

opment of the PCR identiÞcation method were col-lected in 15 localities in Þve countries, across theknown distribution of An. sundaicus s.l. (Fig. 1; Table1). In total, 374 specimens from 24 populations col-lected from Þve countries, including Cambodia (25),Indonesia (41), Malaysia (47), Thailand (47), andVietnam (214) were used to validate the PCR (Fig. 1;Table 1). Larval habitats of populations (Table 1, inbold), from Sarawak, Malaysia in northwesternBorneo (MAM), and South Tapanuli on the island ofSumatra (INS) were collected from freshwater sites(Table 1, F in bold), whereas all other sites containedbrackish water.Morphological Study. The recent molecular iden-

tiÞcations deÞned the presence of three sibling spe-

cies that have never been morphologically compared,and because these species are allopatric, we compared89 pupal and 160 larval setae, by using the descriptionsand illustrations of An. sundaicus s.s. (Linton et al.2001) and An. epiroticus (Linton et al. 2005) for guid-ance. Eighty-nine characters were compared among12 females, and Þve wing types were identiÞed basedon variation of costal spots described by Linton et al.(2001). Additionally, the Palpus Ratio (PR) [(apicalpale spot size/(apical dark spot size � apical pale spotsize)] was calculated from 12 specimens of each spe-cies and compared with Andaman specimens (Nagpaland Sharma 1983). The Costal Ratio (CR) [(preapicaldark spot size/(subcostal pale spot size � preapicaldark spot size � preapical pale spot)] suggested byReid (1968) also was estimated. The taxonomic ter-minology used is the same as that in Nagpal andSharma (1983) for easier comparison of the resultsbetween both studies.DNA Extraction. DNA from 462 females was indi-

vidually extracted following the protocol of Linton etal. (2001) based on a phenol-chloroform procedure.Amplification and Sequencing.A 524-bp segment of

theCOIgenewasampliÞed from86 femalesbyusing theuniversal primers C1-J-1718 and C1-N-2191 (Simon et al.1994)andthereactionconditionsdescribedbyLintonetal. (2001). A 575-bp segment of mitochondrial Cyt-bwasampliÞed from 88 females by using the primers CBsunAand CBsunB (Table 2) and the reaction conditions de-scribed by Dusfour et al. (2004b). Sequencing was donein both directions. The sequences, available in GenBankunder the accession numbers AY243788ÐAY243799,

Table 1. List of collection sites, type of larval habitat (LH) with brackish water (B) and freshwater (F), population code (PC), yearof collection (Yr), number of specimens used for each marker (N), number of specimens tested for PCR validation (n), and speciesidentification results

Country, province Latitude/longitude LH PC YrN

n IdentiÞcationCyt-b COI

Cambodia, Koah Kong 11� 27� N, 103� 04� E B KKKA 2003 5 5 25 An. epiroticusIndonesia, Jakarta, Java 06� 01� S, 106� 41� E B INJ 2001 1 1 0 An. sundaicus EIndonesia, Patuk, Java 08� 04� S, 110� 23� E B INP 2001 5 5 7 An. sundaicus EIndonesia, Asahan, Sumatra 03� 16� N, 99� 32� E B INA 2001 7 6 19 An. sundaicus EIndonesia, Lampung, Sumatra 05� 49� S, 105� 10� E B INL 2001 7 7 3 An. sundaicus EIndonesia, South Tapanuli, Sumatra 06� 11� S, 106� 50� E F INS 2001 7 6 12 An. sundaicus EMalaysia, Johore 01� 44� N, 103� 53� E B MAJ 2003 0 0 2 An. epiroticusMalaysia, Sabah, Pulau Gaya 06� 00� N 116� 04� E B MAG 2003 4 6 4 An. sundaicus s.s.Malaysia, Sarawak (Lundu) 01� 40� N, 109� 50� E B MAL 1999 7 7 10 An. sundaicus s.s.Malaysia, Sarawak (Miri) 04� 22� N 113� 58� E F MAM 1999 6 6 21 An. sundaicus s.s.Malaysia, Terrenganu 04� 45� N, 103� 05� E B MAT 2003 6 4 10 An. epiroticusThailand, Pattani 06� 55� N, 101� 20� E B TPA 2003 8 8 10 An. epiroticusThailand, Phangnga 08� 54� N, 98� 16� E B TPG 1999 5 5 15 An. epiroticusThailand, Suratthani 09� 13� N, 98� 56� E B TSU 2004 0 0 7 An. epiroticusThailand, Trat 12� 02� N, 102� 45� E B TTR 1999 6 6 15 An. epiroticusVietnam, Bac Lieu 09� 11� N, 105� 18� E B VBLB 1998 7 7 30 An. epiroticusVietnam, Ben Tre 09� 51� N, 106� 38� E B VBTB 2003 0 0 15 An. epiroticusVietnam, Ca Mau 09� 01� N, 105� 18� E B VCMD 2003 0 0 20 An. epiroticusVietnam, Ho Chi Minh 10� 34� N, 106� 48� E B VHCA 1998/2003 7/- 7/- 50a An. epiroticusVietnam, Ho Chi Minh 10� 37� N, 106� 51� E B VHCB 2003 0 0 20 An. epiroticusVietnam, Tien Giang 10� 16� N, 106� 46� E B VTGA 2003 0 0 19 An. epiroticusVietnam, Tra Vinh 09� 42� N, 106� 31� E B VTVA 2003 0 0 21 An. epiroticusVietnam, Kien Giang 09� 36� N, 105� 02� E B VKGA 2003 0 0 19 An. epiroticusVietnam, Kien Giang 09� 45� N, 104� 54� E B VKGS 2003 0 0 20 An. epiroticus

Total 88 86 374

a Fifty including 30 for 1998 and 20 for 2003.

September 2007 DUSFOUR ET AL.: IDENTIFICATION METHOD FOR SUNDAICUS COMPLEX 725

AY245283, AY245284, AY256954ÐAY256957, AY253150ÐAY253155, AY299094ÐAY299120, AY299339ÐAY299346,and AY672287ÐAY672404, also were used in anotherstudy of the Sundaicus Complex (Dusfour et al. 2007).Sequence Analysis.The 86 and 88 sequences of COI

and Cyt-b, respectively, were aligned within BioEdit(Hall 1999) by Clustal W (Thompson et al. 1994).Distribution of variation was evaluated among speciesby analysis of molecular variance (ARLEQUIN 2.0 bySchneider et al. 2000). Fixation indices evaluating theinterbreeding level among groups (Fst) and withingroups (Fsc) also were estimated. Polymorphic sitesand parsimony informative mutations were evaluatedusing DNAsp 4.0 (Rozas et al. 2003). Species-speciÞcmutations were subsequently identiÞed.Allele-Specific PCR. Allele-speciÞc primers were

designed from the mtDNA sequences. Each species-speciÞc PCR was tested independently before devel-oping the multiplex assay. In a Þnal volume of 20 �l,concentrations within the PCR reaction mix were 1�reaction buffer (QIAGEN, Valencia, CA), 0.4 mMdNTPs, 1 mM of MgCl2, 0.5 �M each primer, 0.5 U ofTaqpolymerase(QIAGEN), and2 �l ofDNAtemplatediluted 1/10. The PCR conditions were 94�C for 5 min,followed by 35 cycles at 94�C for 30 s, 50�C for 30 s, and72�C for 30 s. An additional autoextension at 72�C for10 min was included at the end of the reaction. ThePCR products were subjected to electrophoresis on a2.5% agarose gel stained with ethidium bromide.Multiplex PCR. To detect simultaneously eachAnopheles species, all primers were used in combina-tion to develop a one-step reaction. Because combin-ing more than two primers can result in unwantedinteractions between them during ampliÞcation, thisphenomenon was limited by using gradients of an-nealing temperature, MgCl2, and primer concentra-tions for optimizing the PCR reaction. We assessedtemperatures from 46 to 54�C, MgCl2 concentrationsfrom 0.5 to 1 mM, and primer concentrations from 0.2to 1 �M. This PCR multiplex was developed using 86mosquitoes identiÞed as An. sundaicus s.s., An. epiroti-cus, or An. sundaicus species E from a previous study(Dusfour et al. 2007).

Results

Morphological Comparison. No diagnostic or dif-ferential morphological characters or combinationof characters involving the branching and positionsof setae were found to distinguish the immaturestages of An. sundaicus s.s., An. epiroticus, and An.

sundaicus species E (data not shown). Among 324adults, Linton et al. (2001) described Þve differentwing types for An. sundaicus s.s. (AÐE). Of 12 spec-imens of An. epiroticus, four specimens exhibitedpattern A and eight specimens exhibited pattern B.Of the 12 specimens of An. sundaicus species Eexamined, two individuals had pattern A, seven hadpattern B, one had pattern C, and two had patternD. The Palpus Ratio (PR) ranged from 0.59 to 0.66for An. sundaicus s.s., from 0.66 to 0.75 for An. epi-roticus, and from 0.33 to 0.76 forAn. sundaicusE. Thedata for the last species are similar to those of Nagpaland Sharma (1983), i.e., PR ranging from 0.35 to 0.80.The Costal Ratio (CR) ranged from 0.33 to 0.56 forAn. sundaicus s.s., from 0.32 to 0.50 forAn. epiroticus,and from 0.43 to 0.60 for An. sundaicus E. Reid(1968) showed a CR difference between specimensfrom Java/Singapore and Borneo of 0.56 and 0.47,respectively. These data are within the range of ourobservations, but no diagnostic differences wereevident. Therefore, no individual character or com-bination of characters was found to distinguish thethree species.Sequence Analysis, Determination of NucleotideSpecificity andPrimerDesign.The observed variationwas highest among the species (58.15%), and reached13.84% among populations within species and 28.01%within a population. Fixation indices revealed thatgene ßow occurs within species (Fct � 0.330; P �0.000), whereas a limited gene ßow was detected be-tween species (Fst � 0.719; P � 0.000).

The 524-bp fragment of the COI gene ampliÞedfrom 86 specimens showed 60 variable sites, includ-ing 31 informative sites that represent 51.7% of thepolymorphic sites. In the 485 bp of Cyt-b, 31 variablesites were detected, of which 21 were parsimonyinformative, representing 67.7% of the polymorphicsites.

COI and Cyt-b showed two (Fig. 2) and six spe-cies-speciÞc nucleotides, respectively (Fig. 3).Cyt-b sequences exhibited three species-speciÞcnucleotides for An. sundaicus s.s. (positions 95, 267,and 297) in all 17 sequences from three populations,two for An. epiroticus (positions 90 and 552) in 44sequences from seven populations, and one for An.sundaicusE (position 480) in 27 sequences from Þvepopulations. COI exhibited two speciÞc mutations,one forAn. epiroticus (position 300) in 42 sequencesfrom seven populations and another (position 384)where three different bases occurred, each speciÞc

Table 2. List of primers and characteristics: code, original gene, specificity, primer sequences, primer length (PL), T°ann, and lengthof specific fragment (FL)

Code Gene SpeciÞcity Sequence (5�33�) PL (bp) T�ann FL (bp)

CBsunA Cyt-b Universal AATGTTACAAGAATTCA 17 42CBsunB Cyt-b Universal TTAGCTATACATTATGC 17 47 575SunSS Cyt-b An. sundaicus s.s. TATCATTCTGAGGAGCC 17 50 313SunE Cyt-b An. sundaicus E ATGATTTTTACGAATTTGC 19 48 498SpCO COI Universal GAACGGTTTATCCTCCT 17 48Epi COI An. epiroticus TATTCGATCTAAAGTAATC 19 48 167

726 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 5

to one of the three species. No population-speciÞcmutations were found.

These combined results allowed us to design threeuniversal primers and three species-speciÞc primers(Table 2). An. sundaicus s.s. and An. sundaicus speciesE speciÞc-primers: SunSS (positions 297Ð313) andSunE (positions 480Ð498), respectively (Fig. 2; Table2). Both primers amplify with the universal primerCBsunA for speciÞc-gene fragments. A pair of primers,SpCO (positions 152Ð168) and Epi (positions 300Ð

318), was designed on the COI gene for the identiÞ-cation of An. epiroticus (Fig. 3; Table 2). The oligo-nucleotide sequence for each primer and therespective annealing temperature (T�ann) are shownin Table 2. The expected lengths of speciÞc ampliÞ-cations are 313 bp for An. sundaicus s.s., 167 bp for An.epiroticus, and 498 bp for An. sundaicus species E(Table 2).Multiplex PCR. All Þve primers designed for identi-

fying each species were combined into a one-shot PCR

Fig. 2. Alignment of the consensus sequences of the 44 An. epiroticus, 17 An. sundaicus s.s., and 27 An. sundaicus speciesE sequences for Cyt-bmarkers. Four primers on the 575-bp Cyt-b consensus sequence alignment, including universal primersCbsunA and CbsunB and two species-speciÞc primers SunSS and SunE, are indicated. Additionally, species-speciÞc nucle-otides are indicated in bold and with a star.

September 2007 DUSFOUR ET AL.: IDENTIFICATION METHOD FOR SUNDAICUS COMPLEX 727

reaction (Fig. 4). The aim was to maximize the yield ofthe desired product while retaining sufÞcient speciÞcity.After testing different conditions, we selected the best

conditions for a Þnal volume of 20 �l as 0.5 mM MgCl2,0.25 �M of each primer, 0.5 U of Taq polymerase, and53�C for 30 s for the annealing temperature.

Fig. 3. Alignment of the consensus sequences of the 42 An. epiroticus, 19 An. sundaicus s.s., and 25 An. sundaicus speciesE sequences for COI markers. Two primers on the 524-bp COI consensus sequence alignment, including the universal SpCOand the species-speciÞc Epi, are indicated. Additionally, species-speciÞc nucleotides are indicated in bold and with a star.

Fig. 4. Gels of multiplex PCR for identiÞcation of members of the Sundaicus Complex. IdentiÞcation bands of An.epiroticus (lane 2), An. sundaicus species E (lane 3), and An. sundaicus s.s. (lane 4), including a positive control, are shown.Lane 1, bp ladder. Size of the band and primer pairs are indicated in parentheses for each band.

728 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 5

To incorporate a positive control to the PCR reac-tion, the universal primer CBsunB (Dusfour et al.2004b), complementary to CBsunA, was added. PCRconditions were improved with a CBsunA concentra-tion at 0.375 �M and CBsunB concentration at 0.25�M. The positive control was 575 bp (Table 2; Fig. 4).

In total, 374 specimens from 24 populations (Fig. 1)were tested to validate the multiplex PCR. These re-sults conÞrmed the distribution of each species, i.e.,An. sundaicus s.s. in northern Borneo (Malaysia); An.epiroticus in coastal areas of Vietnam, Cambodia, Thai-land, and peninsular Malaysia; and An. sundaicus spe-cies E on Sumatra and Java (Fig. 1).

Discussion

The present work aimed to elucidate diagnosticmorphological characters to easily and reliably iden-tify three members of the Sundaicus Complex. Nodiagnostic or differential characters or combination ofcharacters was found. Therefore, a multiplex PCR wasdeveloped to identify each of the three species in thecomplex.

For the most part, rDNA has been used to designspecies-speciÞc primers, especially ITS2 and inter-genic spacer sequences (Scott et al. 1993, Fettene andTemu 2003, Garros et al. 2004a). However, despite theunique ITS2 sequence of An. sundaicus s.s., the pres-ence of intragenomic variation and shared ITS2 typesbetween An. epiroticus and An. sundaicus species E(Dusfour et al. 2007) and An. sundaicus species E andAn. sundaicus species D (Alam et al. 2006) precludedthe use of this marker. To date, few PCR methods foridentifying sibling species of Anopheles have beenbased on mtDNA (Goswami et al. 2005) due to po-tential mitochondrial introgression. However, thelevel of genetic divergence, the low level of gene ßowamong species, and the presence of Þxed mutationsamong populations across a wide geographic scaleallowed us to base our PCR method on mtDNA mark-ers. In the Þrst instance, this PCR method will allow usto rapidly screen a large number of specimens beforefurther investigating populations in other geographicareas of interest.

Of the two markers, Cyt-b includes the largest num-ber of speciÞc nucleotides (six). Two species-speciÞcprimers, SunSS and SunE, and two universal primers,CBsunA and CbsunB, were designed based on varia-tion in this gene fragment. Resolution on agarose gelwas not optimum with An. epiroticus Cyt-b-speciÞcprimers. Consequently, we designed one An. epiroti-cus-speciÞc primer (Epi) from the COI fragment andone universal primer (SpCO) to achieve better reso-lution. All of the species-speciÞc primer designs werebased on a single nucleotide difference from the gen-eral sequences. This characteristic generated nonspe-ciÞc ampliÞcations during the development of themultiplex assays; however, improvement of PCR con-ditions by increasing annealing temperature and de-creasing MgCl2 concentration limited such ampliÞca-tions. Better resolution and intensity of each speciÞcband produced nonambiguity in the results.

The species of the Sundaicus Complex require fur-ther study. Therefore, the one-shot multiplex PCRassay will be crucial in future investigations for elu-cidating the biology, distribution, and disease relationsof each species. In future work, the presence of a PCRcontrol band will distinguish negative ampliÞcationfrom any additional species in the complex by theabsence of a speciÞcally recognized signal.An. sundaicus s.s. is distributed in northern Borneo,

and it is likely tooccurelsewhere incoastal areasof theisland. An. epiroticus occurs in coastal areas of conti-nental Southeast Asia from the 11th parallel in south-ern Vietnam, Cambodia, and Thailand to peninsularMalaysia.An. sundaicus species E has so far been foundonly on Java and Sumatra but may well occur in east-ern areas of the archipelago.

The next objective is to determine whether thethree species are sympatric or entirely allopatric. Par-ticular attention should be given to islands in theMalacca Strait that lie between the continent andSumatra, a possible convergence zone where bothAn.epiroticus and An. sundaicus species E may occur.What is known about the bionomics of each speciesmay need to be reÞned based on new Þndings; how-ever, if we consider the distribution of these threespecies as allopatric throughout the range of the com-plex (to also include species D from the Andaman andNicobar Islands, India), we can objectively retainmuch of the information already gathered about eachspecies based on locality.

The most commonly observed larval habitats for thewhole of this predominately euryhaline species com-plex are stagnant brackish water impoundments (e.g.,lagoons, blocked river outlets, Þsh ponds) containingsubmerged and ßoating aquatic vegetation (Þlamen-tous algae) (Ikemoto et al. 1986, Nguyen Tang Am etal. 1993, Nagpal and Kalra 1997, Chang et al. 2001).Only a few freshwater sites have been documentedacross its distribution (Sukowati et al. 1996, Chang etal. 2001). To date, An. epiroticus has only been col-lected in brackish water sites, whereas An sundaicuss.s. and An. sundaicus species E and D have beencollected in both brackish water, and, less frequently,from freshwater sites (Sukowati et al. 1996, Chang etal. 2001, Nanda et al. 2004). Characteristics of An.sundaicus s.s. in Lundu (neotype locality), northernBorneo, is that larvae occur in rock pools withoutvegetation (Chang et al. 2001), whereas vegetation isnormally present in all other habitats. Thus, the ap-parent ecological and behavioral plasticity of thistaxon does not seem to be linked to a particular speciesbut rather to an ability of each species to adapt toavailable aquatic habitats. In terms of adult feeding,resting and host preference, a wide range of behav-ioral patterns seem to occur in the complex, includingendo/exophagy, endo/exophily, with varying degreesand predilections for feeding on either humans orother animals (Dusfour et al. 2004a). Altered behav-ioral responses, most likely due to natural selection foravoidance (excito-repellency) of chemically-sprayedsurfaces, have resulted in an apparent shift from in-door to increased outdoor biting activity after the use

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of residual insecticides on Nicobar Island (Kalra 1978)and in Vietnam (Phan 1998). Most information re-garding patterns of insecticide resistance is outdatedand in need of reassessment.

SpeciÞc vector capacities for malaria transmissionremain poorly documented in many areas (Dusfour etal. 2004a). In Indonesia, Malaysia, and Vietnam, An.sundaicus s.l. is considered an important, if not prin-cipal, vector of malaria in most coastal areas (Rao 1984,Nguyen Tang Am et al. 1993, Miyagi et al. 1994, Barcuset al. 2002, Maguire et al. 2005); however, no recentmalaria cases have been documented where An. epi-roticus occurs in southern Vietnam (Erhart et al. 2004,Trung et al. 2004). In Cambodia, this species is con-sidered only a secondary vector (Meek 1995), and italso has been generally regarded as a secondary vectortoo in Thailand (Gould et al. 1966, Harinasuta et al.1974). An. epiroticus is considered a potential majorvector because of its occurrence near Thai tourist sites(Chowanadisai et al. 1989). Given the lack of morerecent data on vector capacity, the status of eachspecies needs further clariÞcation with regard to ma-laria transmission and possible differences in responseto control measures. The development of a rapid andreliable PCR assay is essential for investigating thedistribution, epidemiology, and ecology of the indi-vidual species of the complex. Better understanding ofAnopheles complexes is critical for incriminating andinvestigating the biology of malaria vector species, andimplementing appropriate vector control strategies.

Acknowledgments

We thank the staff of collaborating institutions in Cam-bodia, Indonesia, Malaysia, Thailand, and Vietnam for mak-ing mosquito collections, and Y. Linton for sharing specimensfrom Borneo, Malaysia, and Indonesia. We are especiallygrateful to Prof. M. Coosemans (ITM, Antwerp, Belgium),the coordinator of the EC grant “Malvecasia”, INCO-DCresearch project ERBIC 18CT970211, which Þnancially sup-ported this work.

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Received 14 April 2006; accepted 25 January 2007.

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