J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 462 http://jad.tums.ac.ir Published Online: October 04, 2016 Original Article ITS2-rDNA Sequence Variation of Phlebotomus sergenti s.l. (Dip: Psychodidae) Populations in Iran Vahideh Moin-Vaziri 1, *Mohammad Ali Oshaghi 2, Mohammad Reza Yaghoobi-Ershadi 2, Pupak Derakhshandeh-Peykar 3, Mohammad Reza Abaei 2, Fatemeh Mohtarami 2, Ali Reza Zahraei-Ramezani 2, Aboulhassan Nadim 4 1Department of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2Department of Medical Entomology and Vector Control, School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran 3Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 4Department of Epidemiology and Biostatistics, School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran (Received 26 Nov 2014; accepted 17 Feb 2015) Abstract Background: Phlebotomus sergenti s.l. is considered the most likely vector of Leishmania tropica in Iran. Although two morphotypes- P. sergenti sergenti (A) and P. sergenti similis (B)-have been formally described, further morphological and a molecular analysis of mitochondrial cytochrome oxidase I (mtDNA-COI) gene revealed inconsistencies and suggests that the variation between the morphotypes is intra-specific and the morphotypes might be identical species. Methods: We examined the sequence of the ITS2-rDNA of Iranian specimens of P. sergenti s.l., comprising P. cf sergenti, P. cf similis, and intermediate morphotypes, together with available data in Genbank. Results: Sequence analysis showed 5.2% variation among P. sergenti s.l. morphotypes. Almost half of the variation was due to the number of an AT microsatellite repeats in the center of the spacer. Nine haplotypes were found in the species constructing three main lineages corresponding to the origin of the colonies located in southwest (SW), northeast (NE), and northwest-center-southeast (NCS). Lineages NCS and NE included both typical P. cf sergenti and P. cf similis and intermediate morphotypes. Conclusion: Phylogenetic sequence analysis revealed that, except for one Iranian sample, which was close to the European samples, other Iranian haplotypes were associated with the northeastern Mediterranean populations in- cluding Turkey, Cyprus, Syria, and Pakistan. Similar to the sequences of mtDNA COI gene, ITS2 sequences could not resolve P. sergenti from P. similis and did not support the possible existence of sibling species or subspecies within P. sergenti s.l.. Keywords: Phlebotomus sergenti, P. similis, Leishmania tropica, ITS2-rDNA, Iran Introduction “The Phlebotomine sand fly Phlebotomus sergenti s.l. Parrot, 1917 originally described from Algeria in 1917, has a broad range of distribution which covers areas of the south- ern Mediterranean (Morocco, Algeria, Tunisia), the western Mediterranean” (Portugal, Spain, Sicily), Middle East, Arabia, Iran, Afghani- stan, Pakistan, and northern parts of India. Flies of this species have been incriminat- ed as the main vector of cutaneous leish- maniasis due to Leishmania tropica (CLT) throughout Iran and other CLT foci in the world (Nadim et al. 1971, Al-zahrani et al. 1988, Guilvard et al. 1991, Yaghoobi-Ershadi et al. 2002, Oshaghi et al. 2010). It has an extensive geographical distribution, wider than *Corresponding author: Dr Mohammad Ali Oshaghi, E-mail: moshaghi@sina.tums.ac.ir J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 463 http://jad.tums.ac.ir Published Online: October 04, 2016 that of the parasite. The presence of this sand fly in L. tropica free areas and the differ- ences in the transmission patterns of the CLT could be related to the existence of in- traspecific variability or even cryptic vector species (Depaquit et al. 2002, Yahyia et al. 2004). Intraspecific variability has been shown in some morphological and molecular charac- teristics of various populations from different countries. Depaquit et al. (2002) studied the intraspecific variability of the internal tran- scribed spacer 2 (ITS2) of 12 populations of P. sergenti s.l. from ten different countries. Accordingly, two branches could be identi- fied: one was related to the northeastern Med- iterranean area (Cyprus, Syria, and Turkey) and Pakistan, and the second related to southwestern of the first one including North Africa, Egypt, and Morocco. These branches are in accordance with postulated migration routes of P. sergenti s.l. along the Tethys Sea during the Miocene era. The sand flies belonging to these two different branches seem to differ in ecology, host preferences, and possibly also in vectorial capacity (Depaquit et al. 2002). Morphological and mtDNA characters of P. sergenti s.l. were also studied on 28 Ira- nian populations and a few samples from Greece, Morocco, Lebanon, Turkey, Paki- stan, and Syria (Moin-Vaziri et al. 2007). Ac- cording to this study, based on the number of setae and the width of basal lobe of coxite, three morphotypes were identified as A, B and C, with some intermediate forms. Se- quence analysis of mtDNA revealed a 6–7% genetic variation among the populations studied. However, there was no consistency between the morphotypes and the genotypes. Morphotype A was considered as P. sergenti sergenti, morphotype B, was identified as P. sergenti similis, and morphotype C had an elongated style in comparison with P. ser- genti sergenti. In another molecular study on P. sergenti s.l. populations of Spain and Mo- rocco using rDNA ITS2 and mtDNA Cytb sequences (Baron et al. 2008), a high genetic diversity including five ribosomal and 16 mitochondrial haplotypes was found within 25 specimens. The authors suggested testing the vectorial capacity of those haplotypes. Based on these studies, it is judicious to consider the potential existence of sibling species within this taxon. If sibling species within P. sergenti s.l. were proven, it would have important implications in epidemiology as well as in experimental studies. However, having found several intermediate mor- photypes as well as sympatric ecological niche of morphotypes A, B and C, postulated us to test the gene flow between the sympat- ric morphotypes of this taxon using sequence analysis of the ITS2 region of rDNA gene. This multicopy gene involves homogeneiza- tion processes usually called molecular drive (Dover 1982) and has provided resolution in several studies at the taxonomic level for the Larroussius and Paraphlebotomus subgen- era (Dover 1982, Depaquit et al. 2002). The use of ITS2 region has many advantages in- cluding, high and low mutation rhythm at interspecies and intraspecies level respectively, speed and ease of use, multiple target sites, predefined marker systems, known PCR pri- mers, pre-existing knowledge of them in some sand fly species (Di Muccio 2000, Depaquit et al. 2002). This study was conducted to verify the molecular variation of Iranian P. sergenti pop- ulations, and to compare them with available data in GenBank. Materials and Methods Sand fly collections and morphological identification The geographical locations from which P. sergenti s.l. was sampled are shown in Fig. 1. Sand flies were collected from different provinces using sticky traps and aspirators. Specimens caught by sticky papers were J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 464 http://jad.tums.ac.ir Published Online: October 04, 2016 washed in a bath of acetone before being stored. They were stored in 96% ethanol at 4 °C until morphological and molecular identi- fication. Only male specimens were selected for the study of morphological and molecu- lar variability since their characters tends to be more reliable. The head and genitalia of individual male sand flies were cut off within a drop of etha- nol, cleared in boiling Marc-André solution, and mounted between slide and cover slide in Berlese fluid and morphologically identi- fied based on external and internal characters of the head and genitalia according to the known identification keys (Theodor and Mesghali 1964). Morphometric measurements were performed to determine morphotypes of specimens as explained by Moin-Vaziri et al. (2007). The body related to the specimen was stored dried in a vial at -20 °C before DNA extraction. DNA extraction Based on ecological conditions of the lo- cation where specimens were collected and on the morphological differences noted by means of morphometric analysis, a few spec- imens from each morphotype (Moin-Vaziri et al. 2007) and ecological condition were chosen for molecular investigation. Genomic DNA was extracted from the thorax, wings, legs and abdomen of either individual sand flies using the QIAmp DNA Mini Kit (Qi- agen, Germany) (Depaquit et al. 2002) or ISH-Horowicz with small modification as described by Rassi et al. (2011). PCR amplification PCR was used to amplify a fragment of 480–516 bp containing the ITS2 of sand fly rDNA (Depaquit et al. 2002). PCR were per- formed in a 50 µ l volume using 5 µ l of ex- tracted DNA solution and 50 pmol of each of the two primers of C1a: 5'-CCT GGT TAG TTT CTT TTC CTC CGC T-3' and JTS3 : 5'-CGC AGC TAA CTG TGT GAA ATC-3'. The PCR mix contained (final concentra- tions) 10 mM Tris HCl, pH 8.3, 1.5 mM MgCl2 , KCl 50 mM, Triton X 100 0.01%, 200 µ M dNTP each, and 0.25 µ l (1.25 units) of Taq DNA polymerase (Eurobio). Initial denaturation at 94 °C for 5 min was fol- lowed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 62 °C for 1 min. and extension at 72 °C for 1 min with a final elon- gation time of 10 min at 72 °C. Amplicons were analysed by electrophoresis in 1.5% agarose gel containing ethidium bromide. Sequencing and comparative sequence analysis Purification of the PCR product was made by agarose-gel fractionation, using the Per- fect Prep Gel Cleanup (Eppendorf, Germa- ny). Direct sequencing of both DNA strands was performed by Qiagen (Hilden, Germa- ny) and the Department of Parasitology (University of Valencia, Spain) using the primers used for DNA amplification. Se- quences were edited and aligned to identify haplotypes (=unique sequences) by means of the CLUSTALW software package (www. ebi.ac.uk/clustalw) and manually adjusted, if necessary. They were analyzed using the Neighbor-Joining (NJ) method provided in the MUST software package (Philippe 1993). Available sequences of P. sergenti and P. similis were retrieved from GenBank and used for phylogenetic analysis (Table 1). Results Morphometric analysis Morphological investigation on specimens of 28 populations of P. sergenti sl collected from 11 provinces of Iran revealed three main morphotypes (A, B and C) and a few intermediate forms (Moin-Vaziri et al. 2007). Morphotype A was similar to P. sergenti Parrot (1917). Morphotype B was related to P. cf similis, according to Perfiliev (1968) and morphotype C represents specimens with J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 465 http://jad.tums.ac.ir Published Online: October 04, 2016 a curved basal lobe of coxite without a style as globulous as that of P. sergenti. In addi- tion to the three main morphotypes, some intermediate forms were identified among the collected samples. These morphotypes were found sympatric in several provinces of the country. However, the proportion of each morphotype varied within each region. Mor- photype A was the most frequent in all col- lection sites of the country. Molecular analysis The size of rDNA-ITS2 fragments ampli- fied was about 480 bp, from which 278 nu- cleotides were attributed to ITS2, 130 bp to 5.8S and 72 bp to 28S genes. However, in addition to the main PCR product, there was an additional product, close to the main am- plicon, in all amplification. Despite of gel purification before sequencing, these unwant- ed products affected the results of sequenc- ing. By the way, we could obtain a total of 249–253 bp length, including most part (218–222 bp) of ITS2 and 31 bp of 28S, from 11 specimens comprising 5, 4, and 2 individuals of morphotype A, B and inter- mediate form A/C respectively. The sequenc- es were submitted to Genbank (Accession Numbers: EF434818-EF434828). Compara- tive ITS2 sequence analysis of 11 P. sergenti s.l. individuals showed 13 (5.2%) pol- ymorphic sites, from which 46% was due to indel (insertion/deletion) and 54% was due to substitutions. All polymorphic sites were located in ITS2 region and 28S region was identical in all specimens. The region was AT rich, with an average of 75%. There were two variable microsatellite regions in the ITS2 aligned part of the spec- imens, which showed variable repeats of a poly (AT) microsatellite. In the first variable microsatellite sites, two specimens contain 9, three specimens had 8, and six other spec- imens comprised 7 repeats of AT (Fig. 2). Other entries from Genbank also showed variable number of AT repeats in this site. In the second polymorphic microsatellite, vari- ations between the Iranian specimens were due to two single nucleotide insertions/dele- tions (indels) and one variable repeats of a poly (AT) microsatellite. Lower degrees of variation in the poly (AT) microsatellite also have been observed in non-Iranian entries (Fig. 2). Based on the sequence alignment, nine different haplotypes (nominated as haplotype I–IX) have been identified, three haplotypes (VII–IX) in the south west (SW), two haplo- types (I and VI) in the north east (NE) and 5 haplotypes (I–V) in the north-center-south (NCS) populations (Table 1, 2, Fig. 3). A sample of haplotype I was positioned in NE lineage. These haplotypes differed in 1–11 nucleotide positions. The length of the seg- ment sequenced varied between 249 to 253 bp. The ITS2 ribosomal haplotype I with three repeats was the most frequently hap- lotype among all haplotypes. Distance analysis of the ITS2 sequences indicated three main lineages (Fig. 3), which supports correlations between geographical distributions of those populations. These lin- eages were so-called SW, NE, NCS lineages which included haplotypes of South-West, North-East and a mix of haplotypes extended from Northwest to the Central to the South- east of the country, respectively. When we added the ITS2 sequence data of other P. sergenti and P. similis populations as repre- sentatives of other parts of the world for phylogenetic analysis, except for one Iranian sample which was close to the European samples, all of the Iranian haplotypes were associated with the north-eastern Mediterra- nean populations including Turkey, Cyprus, Syria, and also Pakistan (Fig. 4). The P. si- milis populations from Iran were associated with other P. sergenti populations, however, the European P. similis populations clustered separately. This might be due to independent accumulated mutations in distinct geograph- ical populations resulted in separate branch J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 466 http://jad.tums.ac.ir Published Online: October 04, 2016 in the tree. The phylogenetic tree deduced from combination of the ITS2 and the 381 bp of mtDNA CytB-NADH1 sequences ob- tained in our previous study (Moin-Vaziri et al. 2007) revealed similar tree and did not resolve P. segenti from P. similis (data are not shown). Table 1. Details of Phlebotomus sergenti sand flies and their sequence data used in this study Specimen code ( No. assigned in Fig. 1) Locality Morphotype Haplotype ITS2 Gen- Bank Ac. No CytB GenBank Ac. No Reference Iran-ser(A)-1 Tehran, Iran A I EF434822 DQ840367 Moin-Vaziri et al. 2007 Iran-sim(B)-1 Tehran, Iran B V EF434821 DQ840363 Moin-Vaziri et al. 2007 Iran-sim(B)-2 Mashhad, Iran B I EF434825 DQ840345 Moin-Vaziri et al. 2007 Iran-serI(A/C)-3 Neishabur, Iran A/C VI EF434820 DQ840352 Moin-Vaziri et al. 2007 Iran-serII(A/C)-3 Neishabur, Iran A/C I EF434819 DQ840359 Moin-Vaziri et al. 2007 Iran-ser(A)-4 Izeh, Iran A VIII EF434828 DQ840384 Moin-Vaziri et al. 2007 Iran-ser(A)-5 Bushehr, Iran A IX EF434826 DQ840391 Moin-Vaziri et al. 2007 Iran-sim(B)-6 Bandar-e- Abbas, Iran B VII EF434824 DQ840395 Moin-Vaziri et al. 2007 Iran-ser(A)-7 Bam, Iran A III EF434818 DQ840376 Moin-Vaziri et al. 2007 Iran-sim(B)-7 Bam, Iran B IV EF434827 DQ840375 Moin-Vaziri et al. 2007 Iran-ser(A)-8 Iranshahr, Iran A II EF434823 DQ840397 Moin-Vaziri et al. 2007 Malta-similis* Malta - - AF462334 Depaquit et al. 2002 Greece-similis1* Greece - - AF462333 - Depaquit et al. 2002 Greece-similis2* Greece - - AF218324 - Depaquit et al. 2000 Portugal-sergenti* Portugal - - AF462327 - Depaquit et al. 2002 Turkey-sergenti* Turkey - - AF462332 - Depaquit et al. 2002 Syria-sergenti* Syria - - AF462328 - Depaquit et al. 2002 Cyprus-sergenti* Cyprus - - AF462323 - Depaquit et al. 2002 Pakistan-sergenti* Pakistan - - AF218323 - Depaquit et al. 2000 Spain-sergenti1* Spain - S-C EU980387 - Baron et al. 2008 Spain-sergenti2* Spain - S-B EU980386 EU980375 Baron et al. 2008 Spain-sergenti3* Spain - S-A/M-B EU980385 EU980375 Baron et al. 2008 Spain-sergenti4* Spain - - AF462324 - Depaquit et al. 2002 Italy-sergenti* Italy - - AF462330 - Depaquit et al. 2002 Morocco-sergenti1* Morocco - M-C EU980384 EU980371 Baron et al. 2008 Morocco-sergenti2* Morocco - M-A EU960382 EU980370 Baron et al. 2008 Egypt-sergenti* Egypt - - AF462329 - Depaquit et al. 2002 Israel-sergenti* Israel - - AF462325 - Depaquit et al. 2002 P. papatasi Iran - - EF408801 - Depaquit et al. 2007 J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 467 http://jad.tums.ac.ir Published Online: October 04, 2016 Table 2. Comparison of nucleotide characters of Phlebotomus sergenti of Iran at polymorphic sites of the rDNA ITS2. Gap (indel) shows by – Specimen code 11 19 23 24 50 75 76 77 78 140 146 147 148 Iran-ser(A)-8 A T T A T T A T A G A - - Iran-ser(A)-7 A T T G T T A T A G A - - Iran-sim(B)-7 C T C G C T A - - G A - - Iran-sim(B)-1 A T T G T T A - - G A - - Iran- serII(A/C)-3 A T T G T - - - - G A - - Iran-ser(A)-1 A T T G T - - - - G A - - Iran-sim(B)-2 A T T G T - - - - G A - - Iran- serI(A/C)-3 A T T G T T A - - G C A T Iran-ser(A)-4 A C T G T - - - - G C A T Iran-ser(A)-5 A C T G T - - - - A C A T Iran-sim(B)-6 A T T T T - - - - A A - - Fig. 1. Collection sites of Phlebotomus sergenti s.l. for this study, (1) Tehran, (2) Mashhad, (3) Neishabur, (4) Izeh, (5) Bushehr, (6) Bandar-e-Abbas, (7) Bam, (8) Iranshahr. Dark spots represent CLT endemic foci in Iran J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 468 http://jad.tums.ac.ir Published Online: October 04, 2016 Fig. 2. Microsatellite region in ITS2-rDNA sequences of different populations of Phlebotomus sergenti s.l. from Iran and other parts of world retrieved from GenBank (Marked with *). A: Morphotype A, B: Morphotype B, A/C: Intermediate forms of morphotype A and C, ser, P. sergenti, sim, P. similis 468 http://jad.tums.ac.ir Published Online: October 04, 2016 J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 469 http://jad.tums.ac.ir Published Online: October 04, 2016 Fig. 3. Neighbor-Joining tree inferred from 253 bp of ITS2-rDNA sequences including 222bp of ITS2 and 31 bp of 28S of Phlebotomus sergenti s.l. populations from Iran. A, morphotype A, B, morphotype B, A/C, intermediate form of morphotype A and C, ser, P. sergenti, sim, P. similis J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 470 http://jad.tums.ac.ir Published Online: October 04, 2016 Fig. 4. Neighbor-Joining tree inferred from 253 bp of ITS2-rDNA sequences including 222 bp of ITS2 and 31 bp of 28S of Phlebotomus sergenti s.l. populations from Iran and other available data from Genbank originated from Europe, Africa, and Asia. A, morphotype A, B, morphotype B, A/C, intermediate form of morphotype A and C, ser, P. sergenti, sim, P. similis, P. papatasi (AN: EF408801) has been used as an outgroup Discussion Results of this study showed a high diver- sity between specimens of P. sergenti s.l. in Iran where we found nine ribosomal haplo- types. However, the rate of genetic variation (3%) within the ITS2 locus between the Ira- nian populations is half of the rate (6%) that was previously observed in the mitochondrial (CytB-NADH1) sequences (Moin-Vaziri et al. 2007). It seems that mtDNA sequences are more appropriate for the study of the intra- specific variability of P. sergenti s.l. in a more limited geographical environment. J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 471 http://jad.tums.ac.ir Published Online: October 04, 2016 The high diversity observed in ITS2 is in agreement with previous studies indicating different ecological, morphological, and mo- lecular variation among Iranian P. sergenti populations. Previous studies revealed at least three morphotypes (A, B and C). However, in this study like previous study, we have not found any correlation between genotypes, ecotypes, or morphotypes and the results ob- tained here do not support the presence of sibling species (P. sergenti and P. similis) with- in the taxon. However, results of this study are in conflict with a phylogenetic analysis of nuclear ribosomal DNA of Depaquit et al. (2002) that showed the monophyly both of P. sergenti s.l. and P. similis and they were not sister species. Their result confirmed a study previously carried out, using morpho- logical and morphometric approaches for ex- amining the status of the two species by the same investigators (Depaquit et al. 1998). Their worldwide attempt showed allopatric situation of the two taxa at that time. According to our findings, it seems that this group (morphotypes) cannot be consid- ered as two different species because firstly we have found many intermediate morpho- logical and genetically forms among the spec- imens, which indicate possible gene flow and lack of reproductive isolation between them. Secondly, finding identical genotypes (100%) among different morphotypes that are P. ser- genti and that P. similis in mtDNA (Moin- Vaziri et al. 2007) and ITS2 sequences sup- port our morphological findings. Considering the molecular drive characteristic of ITS2- rDNA strongly indicated that these two mor- photypes are not isolated reproductively. Moreover having found these two taxa sym- patric in most provinces of Iran differs from taxonomic conception of subspecies. Accord- ing to our data, we encountered with differ- ent morphologically populations of P. ser- genti s.l. in Iran. In light of our results, it is too early to come to a final decision on taxo- nomic status of the species. More molecular, morphological, and hybridization studies be- tween the two taxa, particularly between ge- ographically distinct populations is necessary. In Iran, CLT is endemic in 14 foci located in 8 provinces restricted to large and medium sized cities in different parts of the country (Yaghoobi-Ershadi 2012).We identified three ITS2 lineages of P. sergenti which is in agreement to the ones identified using the mtDNA loci (Moin-Vaziri et al. 2007). Lin- eage NE and NCS correspond to the main foci of CLT in the country which included both typical and intermediate morphotypes of P. cf sergenti and P. cf similis. In other hand SW lineage exist in clean area of CLT. These findings warrant studies to examine if CLT is due to differences in the vectorial capacity of the P. sergenti s.l. lineages (NE/ NCS versus SW) or other ecological and epidemiological factors are involved. Conclusion Phylogenetic sequence analysis revealed that most Iranian haplotypes were associated with the northeastern Mediterranean popula- tions. Similar to the sequences of mtDNA COI gene, ITS2 sequences could not resolve P. sergenti from P. similis and did not sup- port the possible existence of sibling species or subspecies within P. sergenti s.l.. More molecular studies on other genes or hybrid- ization should be done to clarify the status of different morphotypes of mentioned species in Iran. Moreover, with regard to the im- portance of the epidemiology of leishmania- sis, further studies need to be performed on the possible role of these three morphotypes in the transmission of L. tropica. Acknowledgements Authors are grateful to the staff of the leishmaniasis laboratory at Isfahan Training and Health Research Centre, and also AA J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 472 http://jad.tums.ac.ir Published Online: October 04, 2016 Akhavan, AA Hanafi-Bojd, K Akbarzadeh, M Aghasi, H Alipour, R Jafari, Z Zare, M Nazari, F Mohtarami, and F Yaghoobi dur- ing the field and laboratory studies. The au- thors are greatly also indebted to Dr B Hooshmand, M Boutry, C Grimplet, P Col- son, and F Muller for their kind assistance in this program. Our sincere thanks to Dr Sixte Blanchy for his help on various aspects, es- pecially for organising a close cooperation with our French colleagues at the University of Reims Champagne-Ardenne. This study was financially supported by Institute of Public Health Research, Aca- demic Pivot for Education and Research, Tehran University of Medical Sciences: Project ID No: 241.82.72. The authors de- clare that there is no conflict of interests. References Al-Zahrani MA, Peters W, Evans PA, Ching- Chin I, Smith V, Lane RP (1988) Phlebotomus sergenti, a vector of Leish- mania tropica in Saudi Arabia. Trans R Soc Trop Med Hyg. 82: 416–419. Barón S, Martín-Sánchez J, Gállego M, Morales-Yuste M, Boussaa S, Morillas- Márquez F (2008) Intraspecific var- iability (rDNA ITS and mtDNA Cyt b) of Phlebotomus sergenti in Spain and Morocco. Acta Trop. 107: 259–267. Depaquit J, Ferte H, Leger N, Killick- kendrick R, Rioux JA (2000) Molecu- lar systematics of the Phlebotomine sandflies of the subgenus Paraphleboto- mus (Diptera, Psychodidae, Phleboto- mus) based on ITS2 rDNA sequences, Hypotheses of dispersion and specia- tion. Insect Mol Biol. 9: 293–300. Depaquit J, Ferté H, Léger N, Lefranc F, Alves-Pires C, Hanafi H, Maroli M, Morillas-Marquez F, Rioux JA, Svobodova M, Volf P (2002) ITS2 se- quences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Dip- tera, Psychodidae): Possible conse- quences in their ability to transmit Leishmania tropica. Int J Parasitol. 32: 1123–1131. Depaquit J, Leger N, Ferte H (1998) Le statut taxinomique de Phlebotomus sergenti Parrot, 1917, vecteur de Leishmania tropica (Wright, 1903) et Phlebotomus similis Perfiliev, 1963 (Diptera: Psychodidae). Approches mor- phologique et morphom´etrique. Corol- laires biog´eographiques et ´epid´ emi- ologiques. Bull Soc Path Exot. 91: 346–352. Di Muccio T, Marinucci M, Frusteri L, Maroli M, Pesson B, Gramiccia M (2000) Phylogenetic analysis of Phlebotomus species belonging to the subgenus Lar- roussius (Diptera: Psychodidae) by ITS2 rDNA sequences. Insect Bio- chem Mol Biol. 30: 387–393. Dover G (1982) Molecular drive: a cohesive mode of species evolution. Nature. 299: 111–117. Guilvard E, Rioux JA, Gallego M, Pratlong F, Mahjour J, Martinez-Ortega E, Dereure J, Saddiki A, Martini A (1991) Leish- mania tropica au Maroc. III. Rôle vecteur de Phlebotomus sergenti. Ann Parasitol Hum Comp. 66: 96–99. Moin-Vaziri V, Depaquit J, Yaghoobi-Ershadi MR, Oshaghi MA, Derakhshandeh- Peykar P, Ferté H, Kaltenbach M, Bargues MD, Léger N, Nadim A. Ferté H, Kaltenbach M, Bargues MD, Léger N, Nadim A (2007) Intraspecific varia- tion within Phlebotomus sergenti Par- rot (1917) (Diptera: Psychodidae) ba- sed on mtDNA sequences in Islamic Republic of Iran. Acta Trop. 102: 29–37. Nadim A, Seyedi-Rashti MA, Mesghali A (1971) Epidemiology of cutaneous leish- maniasis in Iran. B. Khorassan. Part IV. Distribution of sand flies. Bull Soc Path Exot. 64 (6): 865–870. J Arthropod-Borne Dis, December 2016, 10(4): 462–473 V Moin-Vaziri et al.: ITS2-rDNA Sequence … 473 http://jad.tums.ac.ir Published Online: October 04, 2016 Oshaghi MA, Rasolian M, Shirzadi MR, Mohtarami F, Doosti S (2010) First re- port on isolation of Leishmania tropica from sandflies of a classical urban Cu- taneous leishmaniasis focus in southern Iran. Exp Parasitol. 126(4): 445–450. Philippe H (1993) MUST, a computer pack- age of management utilities for se- quences and trees. Nucl Acids Res. 21: 5264–5272. Rassi Y, Oshaghi MA, Azani SM, Abaie MR, Rafizadeh S, Mohebai M, Moh- tarami F, Zeinali Mk (2011) Molecular detection of Leishmania infection due to Leishmania major and Leishmania turanica in the vectors and reservoir host in Iran. Vector Borne Zoonotic Dis. 11(2): 145–150. Theodor O, Mesghali A (1964) On the Phlebotomine of Iran. J Med Entomol. 1: 285–300. Yaghoobi-Ershadi MR (2012) Phlebotomine sand flies (Diptera: Psychodidae) in Iran and their role on Leishmania transmis- sion. J Arthropod-Borne Dis. 6(1): 1–17. Yaghoobi-Ershadi MR, Hanafi-Bojd AA, Javadian E, Jafari R, Zahraei-Rama- zani AR, Mohebali M (2002) A new cutaneous leishmaniasis caused by L. tropica. Saudi Med J. 23(3): 291–294. Yahia H, Ready PD, Hamdani A, Testa JM, Guessous-Idrissi N (2004) Regional genetic differentiation of Phlebotomus sergenti in three Moroccan foci of cu- taneous leishmaniasis caused by Leish- mania tropica. Parasite. 11: 189–199.