185911

SYSTEMATICS

Mitochondrial DNA Differentiation Between Two Closely RelatedSpecies, Phlebotomus (Paraphlebotomus) chabaudi and Phlebotomus(Paraphlebotomus) riouxi (Diptera: Psychodidae), Based on DirectSequencing and Polymerase Chain Reaction-Restriction Fragment

Length Polymorphism

RAJA BOUDABOUS,l'e AZZEDINE BOUNAMOUS,a'4 DAMIEN JOUET,a JIROME DEPAQUIT,a

DENIS AUGOT,a HUBERT FERTI,3 StLIMA BERCHI,5 ARNAUD COULOUX,6

MICHEL VEUILLE,7 AND HAMOUDA BABBA

Ann. Entomol. Soc. Am. 102(3): 347-353 (2009)ABSTRACT Phlebotomus chabaudi Croset, Abonnenc & Rioux, 1970 and Phlebotomus riouxi Depa-quit, Killick-Kendrick & L6ger, 1998 (Diptera: Psychodidae) are closely related species of phleboto-mine sand flies, the females of which are suspected of transmitting Leishmania killicki Rioux, Lanotte,and Pratlong 1986 in Tunisia. Although males of these species are distinguishable by the number andsize of setae on the basal lobe of the coxite, morphologically differential characters between thefemales are blurred. A molecular study, based on 40 Algerian and Tunisian specimens, was conductedto distinguish females ofthese two species. Alignment ofthe cytochrome c oxidase I (COI) sequencesand their analysis by using neighbor-joining and maximum likelihood showed the separation of P.chabaudi and P. riouxi, irrespective of intraspecific variability. Both COI and cytochrome b have beentested as molecular identification tools. Single strand conformation polymorphism analysis did notdistinguish these two species, whereas restriction fragment length polymorphism can be applied toidentify P. chabaudi and P. riouxi for surveillance purposes.

KEYWORDS Phlebotominae, Phlebotomus chabaudi, Phlebotomus riouxi, mitochondrial DNA, DNAbarcoding

Phlebotomine sand flies are the exclusive vectors ofleishmaniasis through the bite of the hematophagousfemales. Within the genus Phlebotomus Rondani &Brete, 1840, the subgenus Paraphlebotomus Theodor,1948 includes 14 species, some ofwhich are proven (P.sergenti Parrot, 1917 [Guilvard et al. 1991] and P.saevus Parrot & Martin, 1939 [Gebre-Michael et al.2004]) or suspected (P. alexandri Sinton, 1928 [Guanet al., 1986] vectors of Leishmania tropica Wright,1903 and L. donovani Laveran and Mesnil, 1903, twocausative agents ofhuman leishmaniasis. Moreover, P.chabaudi Croset, Abonenc & Rioux, 1970 (Rioux et al.

Laboratoire de Parasitologie-Mycologie code 99UR/08-05, Fac-ult6 de Pharmacie, D6partement de Biologic Clinique B, Rue Avi-

cenne, 5000 Monastir Tunisie.Corresponding author, e-mail: rajaboudabous@yahoo.fr.USC AFSSA "VECPAR," Facult6 de Pharmacie, Universit6 de

Reims Champagne-Ardenne, Reims France.Laboratoire de pharmacologic et phytochimie, Facult6 des Sci-

ences de Jijel, Alg6rie.Laboratoire de Biosyst6matique et Ecologic des Arthropodes,

Facult6 des Sciences de la Nature et de la Vie, Universit6 Mentouri,25000 Constantine, Alg6rie.

Genoscope, 91000 Evry, France.Ecole pratique des hautes etudes, Unit6 de recherche 5202 CNRS-

MNHN, 16 Buffon, 75005 Paris, France.

1986) and P. riouxi Depaquit, Killick-Kendrick &L6ger, 1998 are suspected vectors for the transmissionof L. killicki Rioux, Lanotte and Pratlong, 1986 inseveral foci in Tunisia. The detection of new foci ofleishmaniasis due to L. killicki in Tunisia (Bouratbineet al. 2005, Haouas et al. 2005, 2007), Libya (Aoun etal. 2006), and Algeria (Harrat et al. 2006) offers theopportunity for further study of the involvement ofthese species.Within the genus Phlebotomus, a specific diagnosis

is often difficult or impossible between closely relatedspecies belonging to the subgenera Adlerius, Larrous-sius (e.g., P. neglectus Tonnoir 1921 and P. syriacusAdler and Theodor 1931 ), or Paraphlebotomus (e.g., P.caucasicus Marzinowsky 1917, P. mongolensis Stintoni928, and P. andrejevi Shakirzyanocva 1953) (Depa-quit et al. 2000). Among the latter subgenus, males ofP. chabaudi and P. riouxi are distinguishable by thenumber and size of setae on the basal lobe of thecoxite, but females are considered to be morpholog-ically indistinguishable (awaiting validation of a char-acter recently proposed by Bounamous et al. (2008).Consequently, a rapid molecular typing for the sureidentification of these two species is needed.

0013-8746 09 0347-0353504.00 0 (C) 2009 Entomological Society of America

348 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 3

Table 1. Algerian and Tunisian sand fly specimens

Species Country Region Locality Traps Capture date Sex Specimen

P. chabaudi Algeria Aurs Arris ST 30 Aug. 2006 4` CB1Menaa ST 31 Aug. 2006 4` CB2

CDC 26 Sept. 2007 4` CB3CDC 26 Sept. 2007 4` CB572

Ain-Zaatout CDC 20 Sept. 2007 4` CB573CDC 20 Sept. 2007 CBZAT583

Tunisia Monastir Sayada ST 4 Oct. 2005 9 SMO419ST 4 Oct. 2005 4` SMO430CDC 4 Oct. 2005 4` SMO436CDC 4 Oct. 2005 $ SMO270

Khenis ST 30 Sept. 2005 9 SMO615ST 30 Sept. 2005 $ SMO617

Teboulba ST 4 Oct. 2005 4` SMO560ST 4 Oct. 2005 4` SMO554ST 04 Oct. 2005 4` SMO557ST 4 Oct. 2005 4` SMO562

Moknine CDC 30 Sept. 2005 9 SMOll2Skanes ST 28 Sept. 2005 ? SMO310Sidi Ameur CDC 29 Sept. 2005 4` SMO631

Mahdia E1 Bradaa ST 19 Oct. 2005 4' SMA34ST 19 Oct. 2005 4` SMA19ST 19 Oct. 2005 4` SMA27

M. Hached ST 21 Oct. 2005 4` SMA39ST 21 Oct. 2005 4` SMA38

Ksour Essaf ST 19 Oct. 2005 4` SMA5ST 19 Oct. 2005 4` SMA1

Echabba ST 23 Oct. 2005 4` SMA161Melloulech ST 24 Oct. 2005 4` SMA124Sidi Alouan ST 19 Oct. 2005 4` SMA60

P. riouxi Algeria Ghardaa Ghardaa ST 20 Sept. 2006 4` RX1ST 20 Sept. 2006 4` RX3

Metlili ST 19 Sept. 2006 4` RX4ST 19 Sept. 2006 4` RX7ST 19 Sept. 2006 4` RX8ST 19 Sept. 2006 4` RX9

Tunisia Tatahouine Ghomrassen: Bokhtit CDC 15 Oct. 2007 9 TAT24Ghomrassen: Om Sefri ST 15 Oct. 2007 4` TAT74Ghomrassen: Om Sefri ST 15 Oct. 2007 4` TAT63Ghomrassen: Ksar Hadada CDC 16 Oct. 2007 $ TAT23Ghomrassen: Ksar Hadada CDC 16 Oct. 2007 TAT186

CDC, miniature light trap; ST, sticky trap.

In a preliminary study, Bounamous et al. (2008)proposed the sequencing of mitochondrial cyto-chrome b (mtDNA cyt B) for the separation andidentification of P. chabaudi and P. riouxi. Here, we

propose an additional molecular marker for this group:the cytochrome c oxidase I (COI) gene of mitochon-drial DNA. Despite that this marker is commonly se-quenced for the "barcode of life" program (Hebert et

TAT24-74TAT23TAT63TAT186RXl

RX4RX7X8RX9SMA1-60SM0430SM0557-562SM0617SI0631SM0310

22222222222333333333333333344444444444444445555555555555666666666334455556668892455779993455666723455778899344455566778034566777899912345677577369258365385558565226273825583576947969497362583936856928627925873612665435

AACATTCTGGAATATTAAACAAGAATGGTATCTATAAAAAATCATACCGGTGTAGTGAGATATATGTACTGTATT T C

A A

CT G..C T C C

.G.GC.T. . G AAGG.A TCG. .G.GTG.A.C. ,TA AA C.G A.G..A. .TCA T.ACG.

.G.G..T. .AG G AAGG.A TCG, .GoGTG.A,C...A AA C. ,C.G C..A.G TCA T.A.G.

.G. GC. T..AG G AAGG.A TCG, G. GTG.A.C...A AA C, C. G A. G TCA T .ACG.,G.GC.T, . G. .AAGG.A. .TCG. ,G.GTG.A.C. ,A. .AA. .C .C.G A,G. ,T.A. .T.ACG..G.GC.T..AG G AAGG.A TCG..G.GTG.A.CC. .A AA C .C .G G TCA T.ACG..G.CC.T..AG G AAGG.A TCGC.G.GTG.A.C...A AA C..C.G GA TCA T.A.G.G. TG.. TAAAC,CC. CG. GGTT .A. GGTCG .AA... GGG.T. GAGC. T ,AA.7,G ,A.AGG.. G. CAGAGAG. GCTAAT.A.AAG..A, GC .AT... GG, TG. TAAAGCC. CG. GGTT .A. GGTCG ,AA... GGG.T. GAGC...AXIAGG .A.AGG.. G. CAGAGAG. GCTAAT.A.A.CA. GC .AT. G

G.TG. TAA/t,CC.CG.GGTT.A.GGTTG.AA. ,GGG,T.GAGC.T.AA.AGG.A.AGG. G.CAGAGAG.GCTAAT.A,AAG.CA.GC.AT. .GG. TG.. TAAAGTC. CG. GGTT .A. GGTCG .AA... GGG.T. GAGC. T.AA.AGG. A .AGG..G. CAGAGAG, GCTAAT.A,AAG. CA. GC .AT., GG.TG. TAAAGCC.CG.GGTT.A,GGTCG,AA. .GGG.T..T,AA.AAG,A.AGG..G.,GCTAAT.A.A;.CA.GC.AT.G. TG. TAAAGCC CG. GGTT .A. GGTCG .AA. GGG. T. G.GC, T.AA.AGG.A,AGG.. G. CAGAGAGAGCTAAT A .AAG. CA. GC ,AT... G

other specimens SMA/SMO (n=15) G,TG..TC.CG.GGTT.A.GGTCG,AA...GGG.T.GAC.T.AA.AGG.A.AGG..G..GCTAAT.A.AAG.CA.GC.AT., .GCBI-2-3-573 ,TG..T.AA.TCG. ,G.GTT.A. GGA.G,A GG.T.GA.CGT.A..AGG.AAAGGG... CAGAGAG. G.T.AT.AGAAGT.AAG, GAT. ,G

to.. r..reG....a..a.a ...e.... .a. .at......a

Fig. 1. Position of 97 segregating sites obtNned ter alignment of sequences of COI from 40 specimens of P. chabaudiand P. ouxi.

May 2009 Botroous gr hI.: MoIcutaa DETERMINATION OF Phlebotomus SPECIES 349

100

soc-cox "SMO430CB.COX

SMO436CB-COX

SMA34CB-COX

SMO615CB-COX

SMAt61CBCOX

SMO270CB-COX

SMO560CB-COX17 SMO112CB-COX

SMA19CB-COX

SaA2,CB-COXTunisia

SMO554CB-COX

SMA39CB-COX

SMA38CB-COX

SMASCB-COX

29 SMA27CB-COX

SMO310CB-COX

SMO631CB.-COX

SMOgf7CB-COX

1006 SMO562CB-COX

SMO617CB*COX

SMAICB-COX

t SMA60CBCOX

CBZTf83-COX

CB573-C,OX

I iCB'COX Algeria711 CB3-COXL 22&x

75J TAT24-COX7 TAl"74RX-COX_t.cox Tunisia

RX3-COXlixx

Algeria

51E;LRCOX

SE*COX P. sergenti.i-cox P. alexandri

100

P. chabaudi

P. riouxi

Fig. 2. Neighbor-Joining tree inferred from 689 bp ofthe COI gene ofP. chabaudi and P. riouxi from Tunisia and Algeria.A similar topology was obtained by maximum parsimony method with P. sergenti and P. alexandri used as outgroups. Thebootstrap values obtained after 1,000 replicates are shown on the nodes.

al. 2003), it has been used only once for phlebotominesand flies; Arrivillaga et al. (2002) characterized sev-eral cryptic species within the Neotropical complexLutzomyia longipalpis Young & Duncan 1994.

In the current study, the use of COI and cyt B wereevaluated as reference markers for the identificationof P. chabaudi and P. riouxi by restriction fragmentlength polymorphism (RFLP) analysis and singlestrand conformation polymorphism analysis (SSCP).

Materials and Methods

Insect Collection. Sand fly specimens from Tunisiaand Algeria were collected on sticky papers or in CDC

miniature light traps. Specific localities, dates, and num-bers of specimens are given in Table 1. Forty A]gerianand Tunisian specimens of P. chabaudi and P. riouxi areselected and stored in analytical grade 96% ethanolat -20C waiting molecular processing and slide-mount-ing in Berlese fiuid (Lewis 1982) after dissection withsterile forceps and microneedles (Testa et al., 21302). Headand genitalia were mounted and stored in the collectionsofResearch unit JEgN3%USC AFSSA WECPAR" (Jeune6quipe 2533-Unit6 sous contrat de 1' Agence Franaisede Scurit6 Sanitaire des Aliments-Universit de ReimsChampagne Ardenne "transmission vectorielle et pi-dmiosurveillance de maladies parasitaires"). Male fliesused in this study were identified to species according to

P. chabaudi P. riouxi specimens

Fig, 3. SSCP analysis of the COl gene of some samples of P. ctutbaudi and P. riouxi. (Online figure in color.)

morphological characters (Croset et al. 1970,1978; Depa-quit et at. 1998), Females

were procegsed similarly tomales, identified

as P. dmbaudi/P, riouxi on the basis ofspermathecae shalx, and finally assigaaed to P chabaudi

or P. 7"/oux according to the molecuka" analyses.Polymerase Chain Reaction (PC'R) Amplification.

Genomic DNA wza extracted fi'om the thorax, wings,legs, and abdomen ofindividual sand flies by using theQIAmpDNA Mini kit (QIAGEN GnlbH, Hilden, Ger-many). To amplify the eyt B fragment,

weused the

protocol of Esseghir et at, (2000). To amplify thebrocade fragment of the CO1 gene, the primers LepF(5'-ATTCAACCAATCATAAAGATA'VrGG-3) andLepR (5'-TAAACqCTGGATGTCCAAAAAATCA-3') (Hajibabael et at. 2006) were

used under the fol-lowing thermal profile (Costa et at, (2007): an initialdenaturation step at 94C for 3 min, followed by fivecycles of (denaturation at 94C for 30 s, annealing at45C for 90 s, and extension at 86C for 60 s), and then,35 cycles of (denaturation at 94C for 30 s, annealingat 51C for 90 s, and extenston at 86C for 60 s) and

a

final extension at 68C for 10 min by using Taq poly-merase (5', QIAGEN GmbH).

Sequencing. Direct sequencingin both directionswas

perfomaed using thesame primers as

for DNA ampfifl-cation, The correction ofsequances was doneumgpre-gap and gap software (Bonfield and Staden 1996).

Phylogenetic Atmlyses. COI sequences wereused

for these analyses and P. sergiti Parrot 1917 md P.alexandrl Stlnton 1928 were selected

as outgroupsaccording to their phylogenetie position (Depaquit etal. 2000). Sequence alignment was performed usingthe ClustalW routine included in the MEGA version3.1 sotware (Kumar et al. 2004), and checked by eye.Neighbor-joiling (NJ) analyses were performed usingMEGA 3.1 software. Mttrdmum likelihood (ML) ara]-ysis was perlbrined i, PHYML onhne (Cuindon et al.2005). Models and parameters were chosen using thehierarchical likelihood ratio test implemented in Mod-eltest 3.7 (Posada and Crandall 2001) mad node sup-port was assessed by bootstrapping (1,000 replieates).

RFLP. DNA from each amplicon was loaded anddigested for 2 h with restriction enzyme HttdIlI (forCOI amphcons) or HpyI88Ilq (for cyt B anaplicons).according to the manufacturer's recommendations.Digestion products were separated by submerged gelelectrophoresis at 1,30 V in 2% agarose gel for 2 h andstained with ethidium bromide.SSCP. Gel plates were cleaned with 95% ethanol and

double distilled water (ddIO). Aerylmnide was pre-pared before use, filtered, and stored at 4C. The acryl-amide solution consistedofthefollowing: 3 ml of5 TBE(0A,SM Tris base, 0.45 M boric acid, mad 10 nl EDTA,pH 8), 3 nd of56% acrylamlde-N24'-methylene bis-acryl-amide solution (99:1), 3 ml of50% glycerol mad 20 ml ofddH20. Before pouring into the gel plate assembly, 0.75ml of 1,6% aramoninm persalfate and 30 1 ofTEMEDwere 'added. Samples of purified polymerase chain

re-

action (PCR) product were diluted, 1:I in Ioadingbuffer(20 0,M EDTA, 0,05% bromphenol blue, ,05% formamlde,and 0.05% xylene cyanol), heated at 9C for 5 rain, mdhmnediately placed on wet ice. Aliquots are loaded on a

prerun gel (1 nan in thickness, 16 by/8 cm) and suh-jected to electxophoresis for 3.5 h at200V, 19C, by usingavertical slab gel unit and 0.5 TBE buffer. The gel

was

silver stained (SanKtinetti et at. 1994) to visualize thereannealed single-stranded PCIq. products. Initial fixationof the gel was for 10 rain (10% ethmiol mid 0.5% aceticacid), followed by staining ha silver nitrate (0.2% in fix-ative) for 5 rain and 1 min wash in deionized water. Thegel was placed in chilled developer (3% NaOH ead 0.1%formaldehyde) nntil bands became visible. After

afinal

fixation for 5 rain and 0-rain wash in deinnJzed water,the plate

wasah'-xlried at room temperature and photo-

graphed. All solutions were prepared in deionized waterto reduce background stalinng.

Results

Phylogenetlc Aaalyses. The frtgments resulting fromCO1 ampfification were 689 bp (GetBank tcctssJon nos.

1I196403 to FJ]96443). Their aliunent, without gaps,

p. P. fiouxi s

Fig. 4. SSCP analysis of the CYT B gene of samples of P. chahaudi and P. riouxi. (Online figure in color.)

shows a polymoghism of 14.1 mad a 5% sequl!rlcl diver-

gence between the two species. Froin 97 vm'iable sites.there were 34 pro-simony-ifformaLive md63 psimony-uninfomfiv sites. Twenty haplos were

obse'ed:l0 for P. ouxi (ibm- from Tisia zmd six fiom AlgeSa)and 10 ibr P. chabaudi (seven flom Tunisia and threeFom Mgefia). The agnment of vadable nucleofideposifio is sho in Fig. 1.The ML analysis

waspeTormed using tire Y+F

model (Discrete gamlna model with eighl categoriesand Gma shape pm'ameter 1.874/nucleotide fre-quencies: f(A) 0.3707; f(C) 0.i902: f(G)0.167; f(T) 0.29614). It shows, like N] analyses, thedivergence of the two species. High bootstrap valuessongly suppo dichotomy robustness (Fig. 2). Foreach species, there two-branch dichotomy thatre{leered Ihe countT of o]Sgin.

Differentiation beeen the Two Species by I'CR-SSCP. The maalysis by SSCP of each amplico (eyt Band COI) did not show a

clearly discrimfmtte profileeen the two species (Figs. 3 and 4).

Differentiation be'een the Two Species by PCR-P. For COl amplicons, Him cut only at the417-bp site of P. o,xi specimens md generated two

fi'agments (417 and 272 bp). Thls ,-estriction site wasabsent in P. chabmtdi. Tbc electropboretic separationofdigestion products shoxved two types ofprofiles: thefirst group fi'om P. chabaudi with

one band and thes,ond group tom P. riouxi with two bands (Fig. 5).The species sep,'u'ations were

further co.firmed byrestriction analysis of the cyt B amplicons: Hpy188IHcut P. chabaudi specimens at position 216, generatingtwo fiagments for this spt!cies ('16 and 274 bp) (Fig,6). This restrictioll sitt, is absenl in P ciouxi.

Discussion

Tile COI seqllelee data set obtained in the currentstndy w-s eompmed ,4tb that fromeB (Bou.amonset al. '2008) (GenBmak accession nos. EU935791 toEU935827). The vmiability of CO1 ws similm" to thatofeyt B, de latter showing

apolymorphism of 11.7 and

a 4% sequence divergence, ]9 parsimony-informativeand 38 parsfinony-uninformative nueleotides (totallength, 490 bp), and 19 haplotypes: 12 for P. tiouxi(four from Tunisia and eight from Algeria) and

seven

for V. chabaIMi ([Ne fi'om Tunisia and two from Al-geria). No SSCP or restriction fi'agment length poly-

500b

P

Fig. 5. Elctrophoretic separation oi' the COl gene digested by HindlII of samples of P. chabaudi and P. riouxi,

P. chabaudi P. riouxi specimensspecimens

300bp

200bp

500bp

Fig. 6. Electrophoretic separation of the CYT B gene digested by Hpy188II1 o; samples of P. chabaudi md P

mo13hism diagnostic application sas proposed byBounamots et al. (2008).The analysis of COI and cyt B fi'agments by PCIL

SSCP did not provide any discminative profilesso it

cannot be informative for this group of phlebotominesand flies. PCR-restriction fiagment lenh po]ymor-phism wtcs

informative with both COI and eyt B for P.dmbaudi and P rioux. However, it cannot Ie used

aunivers method for the identification of North

Acan sand flies because many species have GOI

Hpy188 (data not shown), Consequently, the pro-posed method only be used during cpidemio-logical fieldwork, aher

apreliminary morphological

The sequences of COI showa

nueleofide diver-

similar to that obtned by the NJ method (F@ 2). It

poed by lfih bootsh'ap values ( and 1%). AnintrspeciGc divergmce (2-4%) observed pro-ducing 'o eounhTelades within each of these spe-cies. A large nuelcotide divergence 10.91-9.47%ohseed by Arfivillaga et al. (2003) among cladeswRhin populations of L. Ionipalpis. The tonomic

status of these elades remains unresolved. They may

species. We consider the country-clades detected

wilhin each branch of P. chaba,di md P. riouxi. Thisfinding allowed to sociate females to males in each

Aek.owledgments

We thank N. L6ger for help and relevant advice. Thesequencitlg done at Genoscope under grant BI4I (Barcoding insccts for identification) to M.V

Reirences Cited

Aoun, K., N. Bousslimi N. Haouas, H. Babba, A. EI-Buni, andA. Bouralbine. 2006. First report ofLeishmania (L,) killicki Rioux, Lanotte & Pratlong, 1986 in Libya. Parit [3:87- 88.

.rrivillaga, J. C., D. E. Norris M. D. Flleiangeli, and G. C.Lanzm'o. 2(XI2. Phylogeography of'the Neotropieal sand

May 2009 BOUDABOUS ET AL.: MOLECULArt DETERMINATION OF Phlebotomus SPECIES 353

Hypotheses ofdispersion and speciation. Insect Mol. Biol.9: 293-300.

Esseghir, S., P. D. Ready, and R. Ben-Ismail. 2000. Specia-tion ofPhlebotomus sandflies ofthe subgenus Larroussiuscoincided with the late Miocene-Pliocene aridification ofthe Mediterranean subregion. Biol. J. Linn. Soc. 70: 189-219.

Gebre-Michael, T., M. Balkew, A. Ali, A. Ludovisi, and M.Gramiccia. 2004. The isolation of Leishmania tropicaand L. aethiopica from Phlebotomus (Paraphlebotomus)species (Diptera: Psychodidae) in the Awash Valley,northeastern Ethiopia. Trans. R. Soc. Trop. Med. Hyg. 98:64 -70.

Guan, L. R., Y. X. Xu, B. S. Li, and J. Dong. 1986. The roleof Phlebotomus alexandri Sinton 1928 in the transmissionof Kala-azar. Bull. OMS 64: 107-112.

Guilvard, E., J. A. Rioux, M. Gallego, F. Pratlong, J. Mahjour,E. Martinez-Ortega, J. Dereure, A. Saddiki, and A. Mar-tini. 1991. Leishmania tropica au Maroc. 111. Rrle vecteurde Phlebotomus sergenti. Ann. Parasitol. Hum. Comp. 66:96 -99.

Guindon, S., F. Lethiec, P. Duroux, and O. Gascuel. 2005.PHYML Online-a web server for fast maximum likeli-hood-based phylogenetic inference. Nucleic Acids Res.33: W557-W559.

Haouas, N., N. Chargui, E. Chaker, M. Ben Said, H. Babba,S. Belhadj, K. Kallel, F. Pratlong,J. P. Dedet, H. Mezhoud,et al. 2005. Anthroponotic cutaneous leishmaniasis inTunisia: presence of Leishmania killicki outside its orig-inal focus ofTataouine. Trans, R. Soc. Trop. Med. Hyg. 99:499-501.

Haouas, N., M. Gorcii, N. Chargui, K. Aoun, A. Bouratbine,F. Messaadi Akrout, A. Masmoudi, J. Zili, M. Ben Said, F.Pratlong, et al. 2007. Leishmaniasis in central and south-ern Tunisia: current geographical distribution of zymo-demes. Parasite 14: 239-246.

Hajibabaei, M., D. H. Janzen, J. M. Burns, W. Hallwachs, andP.D.N. Hebert. 2006. DNA barcodes distinguish species

of tropical Lepidoptera. Proc. Natl. Acad. Sci. U.S.A. 103:968 -971.

Harrat, Z., S. C. Boubidi, F. Pratlong, R. Benikhlef, J. P. Selt,Dedey, and M. Belkaid. 2006. Premiere description deLeishmania killicki en Algerie. Premieres Journees A1-gero-Francaises de Parasitologie-Myvologie, Alger, 15-16Novembre.

Hebert, P.D.N., S. Ratnasingham, and J. R. deWaard. 2003.Barcoding animal life: cytochrome c oxidase subunitdivergences among closely related species. Proc. R. Soc.Lond. B 270: $96-$99.

Kumar, S., K. Tamura, andM. Nei. 2004. MEGA3: integratedsoftware for molecular evolutionary genetics analysis andsequence alignment. Brief. Bioinform. 5: 150-163.

Lewis, D. J. 1982. A taxonomic review of the genus Phle-botomus (Diptera: Psychodidae). Bull. Br. Mus. (Nat.Hist.) (Entomol.) 45: 121-209.

Posada, D., and K. A. Crandall. 2001. Selecting the best-fitmodel of nucleotide substitution. Syst. Biol. 50: 580-601.

Rioux, J. A., G. Lanotte, F. Fetter, J. Dereure, O. Akalay, F.Pratlong, I. D. Velez, N. B. Fikri, R. Maazoun, M. Denial,et al. 1986. Les leishmanioses cutanres du bassin Mdi-terranren occidental. De l'identification enzymatique tl'analyse 6co-rpidrmiologique. Exemple de trois "foyers,"tunisien, marocain et frangais, pp. 365-395. In Leishmania.Taxonomie et phylogenise. Applications co-6pid6mi-ologiques (Coll. Int. CNRS INSERM), IMEEE, Montpel-lier, France.

Sanguinetti, C.J.E., D. Neto, and A.J.G. Simpson. 1994.Rapid silver staining and recovery of PCR products sep-arated on polyacrylamide gels. Biotechnology 17: 914-921.

Testa, J. M., J. Montoya-Lerma, H. Cadena, M. Oviedo, andP. D. Ready. 2002. Molecular identification ofvectors ofLeishmania in Colombia: mitochondrial introgression inthe Lutzomyia townsendi series. Acta Trop. 84: 205-218.

Received i July 2008; accepted 19 October 2008.