JOllrnal of Ihe American Mmqllito COlllrol As.wdtlliOlI. 16(3): 189-198. 2000Copyright ([;) 2000 by the American Mosquito COlllrol Association. Inc.

A NEW SPECIES OF THE HYRCANUS GROUP OF ANOPHELES,SUBGENUS ANOPHELES, A SECONDARY VECTOR OF MALARIA IN

COASTAL AREAS OF SOUTHERN VIETNAM

NGUYEN DUC MANH.' TRAN DUC HINH.' RALPH E. HARBACH.' JOANNA ELPHICK' AND Y.-M. LINTON'

ABSTRACf. Anopheles (Annpllf!le.r) nimpe. a new species of the Hyrcanus Group from lhe coa.~tal areas ofsouthern Vietnam. is described and iIIustraled in Ihe adult. pupal. and larval stages. Partial DNA sequence datafrom paratypes are included for the mitochondrial cytochrome c oxidase 1 and 165 rRNA genes. The species isdistinguished from other members of the group occurring in Southeast Asia.

KEY WORDS Anopheles nill/fle. Culicidae. mosquitoes. new species. Vietnam

•

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INTRODUCTION

As currently defined, the Hyrcanus Group in­cludes nearly three quarters of the species that com­prise the Myzorhynchus Series of Anopheles sub­genus Anopheles. With the addition of the newspecies described in this paper. the group includes28 formally recognized species in the Oriental andsouthern Palaearctic regions. Ten of these species.8 in China. I in Japan. and I in Vietnam. wererecognized after the Southeast Asian members ofthe group were described in detail by Reid (1968)and Harrison and Scanlon (1975). Whether thesespecies occur outside of the countries where theywere discovered is unknown. and their affinitieswith other members of the group are likewise un­known. In fact. relationships. based on morphologicsimilarity. have been hypothesized for only 9 spe­cies of the group. those that comprise the Lesteri(5 species) and Nigerrimus (4 species) Subgroups(Harrison 1972). The other species of the group.including the new one described here. are unplacedwithin the Hyrcanus Group because they cannot besegregated into supraspecific taxa on the basis ofavailable information. As pointed out by Reid(1968) and Harrison (1972). much additional tax­onomic work needs to be done on the group to dif­ferentiate the species, determine their distributions.and establish their relationships to one another. Thecomplexity of the taxonomic problem lends itselfto an integrated approach, using morphologic. mo­lecular. and bionomic data. The description of thenew species below is a contribution toward thisgoal.

MATERIALS AND METHODSThis study is based on specimens collected in

coastal areas of southern Vietnam. Wild-caught lar-

, Department of Entomology. National Institute of Ma­lariology. Parasitology and Entomology. Luong TM VinhStreet. Be 10.200 1'0 Liem, Hanoi. Vietnam.

, Depanment of Entomology and Biomedical SciencesTheme. The Natuntl History Museum. Cromwell Road.London SW7 58D. United Kingdom.

vae and the progeny of wild-caught adults were in­dividually reared to provide adults with associatedlarval and pupal exuviae. Observations of theadults were made under simulated natural light.Larval and pupal chaetotaxy were studied using acombination of bright-field and differential inter­ference contrast microscopy. Measurements andcounts were made from available specimens. Num­bers in parentheses represent modes of the reponedranges. Except for wing spot nomenclature (Wilk­erson and Peyton 1990). the morphologic terminol­ogy used in the species description follows Harbachand Knight (1980. 1982). The new species is rec­ognized on the basis of correlated anatomical fea­tures in associated life stages. Diagnostic and dif­ferential characters were confirmed in all availablespecimens. Type specimens are deposited in TheNatural History Museum (BMNH). London. andthe National Institute of Malariology. Parasitologyand Entomology (NIMPE). Hanoi.

DNA was extracted from pinned male paratypes(En-I. E3) following a phenol-ehloroform proto­col (adapted from W. Tabachnick. personal com­munication) and resuspended in 100 JLlof 10 mMTris-HCI. pH 8.5. Partial DNA sequence data forthe mitochondrial genes cytochrome c oxidase I(COl) (472 bp) and 16S rRNA (504 bp) were ob­tained from the paratype EP2-I. and can be ob­tained from GenBank under the accession numbersAF216285 and AF216284. respectively. The COlgene fragment was amplified using the universalinsect primers CI-J-1718 and CI-N-2191. and theprimers LR-N-13398 and LR-J-12888 were used toamplify the 16S rRNA amplicon (Simon et aI.1994). In both ca.o;es. the last five digits indicate theposition of the 3' end of the primers with respectto the Drosophila yakuba (X03240) mtDNA ge­nome (Clary and Wolstenholme 1985). PurifiedDNA was amplified by polymerase chain reaction(PeR) using the following reaction mix (50 JLI): 2JLI DNA, 25.5 JLI double-distilled H20, 2.5 f.L1 2.5mM MgCI2• 0.1 f.LI Taq polymerase (BioLine. Lon­don. England), and 5 JLI each of primers at 5 JLM.deoxynucleotide phosphates (dNTPs). and NH.

189

190 JOURNAL OF TIm AMERICAN MosQUITO CONTROL ASSOCIATION VOl.. 16. No.3

buffer (BioLine). The PCR thennocycler programconsisted of a 2-min denaturation at 94°C; 34 cyclesof 94°C, 53°C, and noc for 30 sec each; followedby a 5-min extension at 72°C. Sequence data wereobtained after PCR purification using a commer­cially available PeR purification kit (QIAgen Ltd.Sussex. England) and cycle sequencing reactionswere read by an ASI 377 automated sequencer(Applied Biosystems. Perkin-Elmer Corporation.Wellesley. MA). Sequence data were edited andaligned on Macintosh@ computers using Sequench­ers 3.0 (Genes Codes Corporation. Ann Arbor.MI).

TAXONOMIC TREATMENT

Anopheles (Anopheles) nimpe Nguyen, Trantand Harbach

Anopheles (Anopheles) sp 1 of IMPE 1987: 13(adult key); Nguyen Due Manh et al. 1991 :54,55 (taxonomy); Nguyen Tang Am 1993:495.496(bionomics, control); Nguyen Tang Am et al.1993:465-468. 470. 472 (bionomics); BoanHanh Nhiin 1993:38 (vector incrimination); VuThi Phan 1998:49 (medical importance).

Anopheles (Anopheles) sp. I of Nguyen Duc Manhet al. 1993:215 (mention).

Diagnosis. Adults of An. nimpe are distin­guished from other members of the HyrcanusGroup in having narrow apical tarsal bands, apexof wing with long pale fringe spot. preapical palespot of costa and vein R. absent or weakly devel­oped, humeral crossvein with dense patch of darkscales, preapical dark spot of vein R. without palescales. and veins CuA and lA largely dark-scaledwith long basal dark spot on CuA (usually not welldefined) ending at nearly same level as base of up­per dark spot on IA. Pupae have the trumpet rimthin and unifonn. sela 5-ill-VII inserted on inter­segmental membrane. seta 8-VII inserted lateral tofold line. sela 9-Vill with well-developed branches.paddle with marginal serrations rarely reaching0.75 its length, and seta I-Pa with 2-7 branchesarising at base. Larvae are not so clearly distin­guished: seta I-A usually with fewer than 6 branch­es. seta 8-C has fewer than 12 branches (3-8, mode6), seta 9-C with 3-6(4) branches, and seta 14-Prelatively short and with 5 or fewer branches.

Female. Head: Vertex with pale erect scalesbehind frontal tuft. dark erect scales posterolater­ally; frontal tuft with long pale setae. Clypeus withpatch of dark scales on either side. Antenna lengthabout 1.5 mm; pedicel with small pale scales onlateral surface; basal 2 or 3 flagellomeres with palescales on mesal surface. PrOboscis entirely dark­scaled, scales semierect proximally and appresseddistally; labella paler than prementum; lengthabout 2.0 mm. 1.25 X length of forefemur. slightlylonger than maxillary palpus (about 1.1 X). Max-

illary palpus (in dorsal view) with pale scales onapex of palpomere 5, narrow pale bands (incom­plete ventrally) across joints between palpomeres4-5, 3-4. and 2-3; palpomere 2 usually withoutpale scaling on mesal surface. occasionally withfew indistinct pale scales in this location. Thorax:Integument brown; scutum usually with darkermedian line extending buck to prescutellar area.often with darker lateral line on posterior dorso­central areas. and much darker spot just behindscutal fossa of either side; scutum sparsely cov­ered with fine golden pilifonn scales; anteriorpromontory with long pale setae and piliformscales mesally. dark setae and scales laterally;dark setae on acrostichal, dorsocentral. lateralprescutal. scutal fossal, antealar. and supraalar ar­eas. Scutellum with long dark setae and goldenpilifonn scales; integument dark medially. palelaterally. Mesopostnotum and postpronotum bare.Antepronotum with patch of dark erect scales ondorsoanterior margin. dark setae laterally. Pleurawith dark setae on upper proepisternum. prespi­racular area. prealar knob. upper and lower me­sokatepisternum. and upper mesepimeron. Willg(Fig. I): Length about 3.4 mm; mainly dark­scaled; small subcostal pale spot on costa. apex ofsubcosta. and R,; preapical pale spot on costa andR1 weakly developed or absent. especially on cos­ta; remigium dark-scaled, sometimes with few in­distinct pale scales; humeral crossvein with darkscales; vein R with scattered pale scales betweenbase and small sector pale spot; R. usually withfew scattered pale scales between sector and sub­costal pale spots. always entirely dark-scaled be­tween subcostal and preapical pale spots. apexwith dark scales; veins Rz, R~+5' MI' M2• and M,,~

usually with some pale scaling, as proximal spotson Rz• MI' and Mz• and speckling on R~'5 andMJ'~; CuA predominantly dark-scaled with darkscales more concentrated in "basal" and apicalspots. basal dark spot (generally not sharply de­limited) long, tenninating at or just before level ofbase of upper dark spot on vein IA; IA also usu­ally predominantly dark-scaled, dark scales con­centrated in "upper" and apical spots; apical palefringe spot long. extending from R. to R~+5 orslightly beyond (Figs. IA. IC), sometimes indef­inite and seemingly ending before R~, 5 (Fig. 1D);posterior margin of wing without pale fringe spotat apex of CuA. Halter: Pedicel mainly pale. sca·bellum and capitellum dark. capitellum dark­scaled. Legs: Coxae without pale scales. forecoxawith few dark scales at base; femora, tibiae. andtarsi mainly dark-scaled. posterior or ventropos­terior surfaces of femora, tibiae, and 1st tarsomer­es with dirty yellowish scaling. mid- and hindti­biae with few pale scales dorsally at apex;foretarsomeres 1-3 with apical pale bands approx­imately equal to width of tarsomeres; midtarso­meres 1-3 and hindtarsomeres 1-4 with narrowdorsoapical pale spots. AIJdomen: Integument dark

SEPTEMBER 2000 NEW SPECIES Or ANtI/'IIHts Il)l

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192 JOURNAl. OF THE AMERICAS MOSQuITO CONTROL ASSOCIATION VOL. 16. No.3

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SEPTEMBER 2000 NEW SPECIES OF ANOPHELES 193

Table I. Range of numbers of bnmches for pupal setae of Anopheles (Anopheles) nimpe. Mode in parentheses.

Cephlllo- Abdominal segmentsSeta thorax Paddleno. CT II III IV V VI VII VIIJ Pa

0 1-3(2) 1-3(2) \-3(2) 1-3(2) 1-3(2) 1-3(2) \-3(1)I 1-3(1) 70-115 4-16(8) 10-33(18) 11-30(17) 2-17(6) 1-3( I) 1.2(1 ) 2-7(4)2 1.2(2) 2-6(3) 3-7 3-8(6) 2-5(4) 3-6(4) 2-7(4) 2-5(4) 1-3(1)3 1-3(2) 2-6(3) 1-3(2) 2-5(3) 3-7(5) 1-3(2) 1-3(2) 2-4(3)4 1-3(2) 2-5(3) 1-3(3) 1-3(2) 1-3(2) 1-4(2) 1-3(2) 1.2(2) 1-3(2)5 1-4(2) 1-4(2) 1-4(2) 9-38(14) 10-39 11-40(17) 4-31(13) 3-15(5)6 1-3(2) 1-3(1 ) 1.2(1 ) 1-4(2) I 1.2(1 ) 1-3(1) 1-3(2)7 1-4(2) 1-6(1 ) 2-4(3) 1-3(2) 1-4(2) 1-3(2) 1.2(1) 1-3(1)8 1.2(1) 1-3(2) 1-3(2) 1-3(2) 1-3(2) 1-4(3)9 1-3(2) 1-4(2) I I 1 I I 1 12-23

10 1-4(2) 0.1(0) 1.2(2) 1.2(1) I 1-3(1) 2.3(2)II 2-4(3) 1-4(2) 1-3(2) 1-3(2) 1-5(2) 1-3(2)12 2.3(2)14 1.2(1) 1.2(1) 1.2(\) 1.2(\)

with long golden-brown setae: without scales ex­cept sternum VII sometimes with few narrow darkerect scales near posterior margin (without con­spicuous tuft of dark scales that characterizes oth­er members of the Hyrcanus Group).

Male. Resembles the female except as follows.Head: Maxillary palpus with indistinct line of palescales on mesal surface of palpomere 2 and lateralpatches of pale scales at base of palpomere 4.across joint between palpomeres 4 and 5. and ondistal ponion of palpomere 5. Genitalia (Fig. 2):Gonocoxite with pale scales on lateral and dorsalsurfaces: inner and outer parabasal setae straight ornearly so. oUler seta slightly longer than inner seta:internal seta rather long. relatively stout. and slight­ly curved or bent apically; tergum IX with pair oflong slender caudally directed lobes with slightlyexpanded tips: aedeagus with 3 pairs of leaflets.leaflets without teeth or serrations; ventral lobe ofclaspctte with 2 long setae. outer seta nearly twiceas long as inner seta and noticeably curved or bent;dorsal lobe of claspelte with club formed of at least3 setae on inner-sternal side and 2 flat appressedsetae on outer-tergal side.

Pupa. (Fig. 2). Character and positions of setaeas figured; numbers of branches in Table 1. Ceph­alothorax: Lightly to moderately pigmented. all se­tae usually with ring of darker cuticle around al­veoli; mesothoracic wing with blurred latticepattern of darker spots; antenna usually with apexand most distal joint darkly pigmented. Trumpet:Without secondary cleft or tragus; rim thin and uni-

form. without thickened saw-toothed areas. Abdo­men: Lightly 10 moderately pigmentcd. terga III­VII without central dark spot. sterna II-IV darkeranteriorly, integument at bases of setae 2-4-II-V.2-VI. 2.3-VII. and 0-VW darkly pigmented; lengthabout 3.2 mm. Seta 10-11. or more often its alveo­lus. usually present; 5-W-VII insened on interseg­mental membrane posterior to tergum; 8-VII in­sened on lateral side of fold line; 9-vm withwell-developed branches (12-23). Paddle: Lightlypigmented; asymmetrical. outer part larger than in­ner part; refractile border about 0.6 paddle length;length about 0.70 mm. width about 0.53 rom. indexabout 1.3. Seta I-Pa shon. with 2-7(4) branchesarising at base.

Larva, 4th-stage. (Fig. 3). Characler and posi­tions of setac as figured; numbers of branches inTable 2. Head: Slightly longer than wide. lengthabout 0.70 mm. width about 0.65 mm; unevenlypigmented. with mottled pattern of moderately darktanning. collar and dorsomentum darkly pigmented.Seta 2-C single. simple; 3-e dendritic. with 21­39(24) branches arising from distinct basal stem; 4­C small. double or triple; 5.6,8.9-C with relativelyfew branches. 5-C with 11-16(12). 6-C with 9­16(14), 8-C with 3-8(6). and 9-C with 3-6(4)branches. Antenna: Lightly pigmented; mesal andventral surfaces strongly spiculate; length about0.27 mm. Seta I-A moderately long. usually withfewer than 6 branches (1-7. mode 3). Thorax: In­tegument hyaline. smooth. Seta loP without setalsuppon plate. single or branched apically; 2-P on

Fig. 2. Pupa and male genitalia of Anopheles (Anopheles) IIimpe. Pupa: A. left side: of cephalothorax. dorsal toright; B. dorsal (left) and ventral (right) aspects of metathorax and abdomen. C. Male: genitalia. aspects as indicated.Ac. aedeagus; c. club on dorsal lobe of c1aspettc:; CI. c1aspeltc; CT. cephalothorax; Gc. gonocoxitc; Os. gcnostylus;InS. internal seta; is. inner seta on ventml lobe of claspelte; LAe. leanets of aedeagus; os. outer seta on ventral lobeof c1aspette; Pa. paddle; PBS. pambasal setae; I-VIH. abdominal segments I-VUI; IX-Te. tergum IX; 1-14. setalnumbers for specified areas. e.g.• seta 3-(. Scales in mm.

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SEPTEMBER 2CXX) NF.W SPECIES OF ANOf'Hf:U;S 195

setal support plate. with 7-17 branches; II-P sig­nificantly larger than II-M.T. with 3-5(4) branches;support plate of pleural setal group 9-12-P withSll'ong spine; 14-P with relatively few branches (2­5. usually 3); 4-M with 2-5(4) branches arisingfrom central stem. not sinuous; 3-T palmate withsimple lanceolate leaflets (not differentiated into ablade with apical filament). with 8-23(15) unpig­mented branches. Abdomen: Integument hyaline.smooth except for fine spicules ventrally on seg­ments II-VIU; tergal plate of segment VIII roughlyhexagonal. with anterior margin wider than poste­rior margin and greatest width about 1.7 length.Seta 1·1,11 similar to seta 3-1: palmate with simplelanceolate leaflets; I-III-VII fully palmate, leafletswith blades darkly pigmented proximal to shoul­ders, shoulders not well developed. filaments notpigmented; 6-1 most often with fewer than 21branches (14-28. mode (9). 6-11 most often withmore than 21 (18-27. mode 23); 5-1I usually withfewer than 12 branches (6-12. mode 9); 9-111 mostoften with more than 10 branches (6-13. mode II);13-IV imermediate in length between 13-111 andI3-V, with 2-11(5) branches and approximatcly 0.5length of seta IO-IV. Pecten plate lightly to mod­erately pigmented. with 7 or 8 long and 8-12(9)small spincs. Saddle moderately pigmented. lengthabout 0.65 mm. Seta I-X slightly longer than sad­dle.

Molecular characterization. The 472 bp partialsequence for the mtDNA COl fragment sequencecomprised the following nucleotide bases: 38.8% T,29.5% A. 16.5% C. and 15.2% G (see GenBankaccession AF2(6285). Higher insect mitochondrialDNA sequences are unique in their high AT content(Jenniin and Crozier (994). and the COl fragmentfor An. tJimpe (68.2% AT) falls within the range ofpreviously sequenced COl genes of Culicidae. forexample. Anopheles quodrimaculatus Say L04272(65.4%) and An. gambiae Giles L20934 (68.4%).A FASTA search of available sequences (Pearsonand Lipman 1988) revealed closest homology at90.4% with An. gambiae (Beard et al. 1993).

The 504 bp mtDNA 16s rRNA amplicon com­prised 40.7% 1: 33.7% A. 16.9% G, and 8.7% Cnucleotides, with an AT bias of 74.4% (seeGenBank accession AF216284). A FASTA searchrevealed closest homology with An. quadrimacu­latus (L04272) and An. gambiae (L20934). with97.5% and 96.0% shared bases. respectively.

Etymology. The species is named in recognitionof the National Institute of Malariology. Parasitol­ogy and Entomology (NIMPE-Hanoi) and its sup-

port of mosquito taxonomy in relation to malariacontrol. For purposes of nomenclature. the specificname nimpe is to be regarded as an arbitrary com­bination of leuers without gender.

Systematics. The Hyrcanus Group is an ex­tremely complex assemblage of species that occursin the southern Palaearctic and Oriental regions.with the majority of species known from China andSoutheast Asia. Despite recent studies of the groupin Thailand (Harrison and Scanlon 1975). China(Xu Jin-jiang and Feng Lan-chou 1975. Lu Baolinet al. 1997). and Vietnam (Nguyen Due Manh etal. 1993). present knowledge of the species in east­ern Asia is surprisingly inadequate and fragmen­tary. The group as a whole is distinct in the adultand immature stages. but individual species are of­ten difficult or impossible to distinguish in 1 ormore life stages. As currently interpreted. the groupincludes 28 species: 5 placed in the Lesteri Sub­group. 4 in the Nigerrimus Subgroup. and the other19. including An. nimpe. are unplaced within thegroup (Harbach 1994). Most species of the lastgroup were described in recent years by Chineseworkers without careful study of species that occuroutside of China. Needless to say. the taxonomicstatus and affinities of these nominal species areunclear and the entire group is in need of compre­hensive revision. To further complicate the situa­tion. the group undoubtedly includes a number ofunrecognized species; for example. we are awareof at least 2 additional new species in Vietnam thatrequire formal description. In order to aid identifi­cation. we intend to include keys to the species ofthe Hyrcanus Group in Vietnam in a later paper thatwill describe the other new species.

Anopheles nimpe seems to show closer affinitieswith An. crawfordi Reid. An. lesteri Baisas and Hu.An. puraliae Sandosham. and An. sinensis Wiede­mann than with other members of the HyreanusGroup. but it cannot be confused with any of thesespecies in the adult. larval. or pupal stages. Threeof these species. An. crawfordi. An. paraliae. andAn. sinensis. are known from southern Vietnam andoccur in various degrees of sympatry with An. nim­pe. Anoplleles lesteri. in addition to its presence inChina. the Philippines. Okinawa. and Japan (Har­rison and Scanlon 1975). is recognized in northernVietnam (IMPE 1987. unpublished records).

Adults of An. nimpe key to An. crawfordi inNguyen Thuong Hien (1968) and to An. sinensis inStojanovich and Scott (1965. 1966); larvae key toAn. sinensis in the former and to the combinationof Anopheles nigerrimus Giles and An. sinensis in

Fig. 3. Fourth-stage larva of Anopheles (AnolJheles) nimpe. reconstructed from exuviae. A, Head. dorsal (left) andventral (right) aspects of left side; B. thorax and abdominal segments I-VI. dorsal (left) and ventral (right) aspects ofleft side; C. abdominal segments VII-X. left side; D. palmate seta I-V showing pigmentalion of leaflets; E. abdominalsegments VII-X. left side. A. antenna; C. cranium; P, prothorox; M. mesothorax; S, spiracular lobe; T. meullhorax; 1­X. abdominal segments I-X: 1-15. selal numbers for specified areas. e.g.• seta 5-C. Scales in mOl.

196 JOURNAL OF THE AMERICAN MosQUITO CONTROL ASSOCIATION VOL. 16. No.3

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the latter. No life stage (females. males. larvae, orpupae) key to a terminal species in Harrison andScanlon (1975). Adults of An. ,limpe differ fromthese 3 species. as well as An. lesteri and An. par­aUae, in having the preapical pale spot of the costaand vein R, absent or weakly developed. Pupae areeasily distinguished from these species by theunique insenion of seta 5-IIJ-VII on the interseg­mental membrane. the insenion of seta 8-VII on thelateral side of the fold line, and the distinct devel­opment of seta I-Pa. The larval stage of An. nimpeis not as strongly differentiated as the adult andpupal stages. but larvae can be distinguished fromthose of the other species in having setae I-A. 8­C. 9-C. and 14-P with fewer branches.

Sequence data for the 16S rRNA gene of An.nimpe revealed relatively high homology with dis­tantly related taxa within Anopheles, thus suggest­ing that this gene region has high levels of conser­vation due to functional or structural constraints.Results of recent studies on Culicoides (Linton etaJ., unpublished) indicated that the COl gene is use­ful in defining both intra- and interspecific relation­ships. The COl sequences are widely used and haveproven to be informative across a broad range ofdivergences in insects. for example, tetranychidmites (Navajas et al. 1996) and Coleoptera (How­land and Hewitt 1992; for review see Caterino etaJ. 2000). Brown et aJ. (1994) reponed high levelsof congruence between COl sequence data andmorphologic characters in Greya (Lepidoptera). Inaddition. the role of COl as a functional proteincoding region. adhering to structural (Saraste 1990)and amino acid constraints (Lunt et al. 1996), pro­vides a solid framework around which to investi­gate relationships between both closely related anddistant taxa. We advocate use of this gene regionin any future studies of the molecular systematicsof the Hyrcanus Group.

Bionomics. Larvae of An. nimpe are found inbodies of brackish water where Anopheles (Cellia)sUlldaicus (Rodenwaldt) and An. (Cel.) .fubpictusGrassi also occur. However. larvae of the last 2 spe­cies inhabit masses of floating plants (Ceratophyl­lum and Najas spp.) exposed to full or panial sun­light. whereas larvae of An. nimpe are found amongemergent grasses and plants shaded from the sun.Females of An. nimpe have been collected bitinghumans both indoors and outdoors. but they havenever been found resting indoors (Nguyen DueManh and colleagues, unpublished observations).The species has been incriminated as a secondaryvector of malaria by enzyme-linked immunosorbentassay detection of Plasmodium jalciparum and P.vivax in head-thoracic sections of specimens (BoanHanh NhSn 1993. Nguyen Tang Am 1993).

Distribution. Anopheles nimpe is only knownfrom coastal areas of southern Vietnam. from themost southerly province of Cll Mau nonhward to

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SEPTEMBER 2000 NEW SPECIES OF ANOPHEU.s 197

the Can Gio, Binh Chanh, and Duyen Hai districtsof Ho Chi Minh City.

Material examined. One hundred one speci­mens (17 ~, 150. 60 genitalia. 32 larval exuviae[LeJ, 31 pupal exuviae [Pe)) from 32 larval rear­ings. Holotype, ~ (EP I-I), with LePe on separatemicroscope slides. VIETNAM: Ca Mau Province,Blim D6i District. Tan Tien Commune, VillageTK97. 22.x.98 (U Xuiin Hoi) (BMNH). Paratypes,60 (EPI-2; En-l [used for DNA extraction]. -2,­5, -6, -II), 5~ (EPI-3, -4; EP2-8, -9. -10),6& gen­italia. IILe. lOPe (EP-ll without Pel, same data asholotype (BMNH); Ho Chi Minh City, Binh ChWlhDistrict. Phong Phu Commune. ii.1988 (Pham XuiinSinh et al.), 40 (012. El. E3 [used for DNA ex­traction], E6), 3 ~ (C12, C27. E2), 7LePe on sep­arate microscope slides (BMNH); 50 (B8. C2. C6,06, D7), 8~ (B2, Bll. C4, C5, C7, C29, 016,EIO), 14LePe on separate microscope slides (NIM­PE-Hanoi).

ACKNOWLEDGMENTS

We are grateful to La Xuan H6i, National Insti­tute of Malariology, Parasitology and Entomology(NIMPE), Hanoi, for collecting and rearing thespecimens from Ca Mau Province; to the staff ofNIMPE for help in the field and laboratory; to thePhotographic Unit of the The Natural History Mu­seum (NHM) for preparing Fig. I; to TheresaHoward (NHM) for drawing Figs. 2 and 3; and toBruce A. Harrison, North Carolina Department ofNatural Resources Public Health Pest Manage­ment, for critically reviewing the manuscript. Thiswork was carried out as part of the EuropeanCommission INCO-DC research project ER­BIC I8CT9702 I I.

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