5-Hashareh Final Rassi New case Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 22 Original Article Identification of Sand flies of the Subgenus Larroussius based on Molecular and Morphological Characters in North Western Iran A Absavaran1, *Y Rassi1, P Parvizi2, MA Oshaghi1, MR Abaie1, S Rafizadeh3, M Mohebali4, Z Zarea5, E Javadian1 1Departement of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Iran 2Department of Parasitology, Institute of Pasteur, Tehran, Iran 3Center of Disease Control, Tehran, Iran 4Department of Parasitology, School of Public Health, Tehran University of Medical Sciences, Iran 5Institute of Public Health Research, Meshkinshahr Health Research Station, Iran (Received 1 Jul 2009; accepted 12 Dec 2009) Abstract Background: The adult female sand flies (Diptera: Psychodidae) of the subgenus Larroussius are important vectors of Leishmania infantum (Kinetoplastida: Tripanosomatidae) in Meshkinshahr district, Northwest of Iran. Four Phle- botomus (Larroussius) species are present in this area, i.e. Phlebotomus (Larroussius) kandelakii, P. (La.) major, P. (La.) perfiliewi and P. (La.) tobbi. The objective of the present study was to identify and distinguish the females of P. perfiliewi, P. major and P. tobbi, in this district. Methods: Adult sand flies were collected with sticky papers, CDC light traps, and aspirator in 2006. Individual sand flies of this four species from thirty different locations were characterized morphologically and by comparative DNA sequences analyses of a fragment of mitochondrial gene Cytochrome b (Cyt b) and nuclear gene Elongation Factor 1- alpha (EF-1α). PCR amplification was carried out for all three species P. major, P. perfiliewi and P. tobbi in the sub- genus Larroussius. Results: Phylogenetic analyses of P. major populations in this study displayed two different populations and genetic diversity. Spermathecal segment number, pharyngeal armature and other morphological characters of these three spe- cies were examined and found to present consistent interspecific differences. Conclusion: According to our findings, the phylogeny of Cyt b and EF-1α haplotypes confirms the relationships be- tween P. major, P. tobbi and P. perfiliewi as already defined by their morphological similarities. Keywords: Phlebotomus, Larroussius, Cytochrome b, Elongation Factor-1α, Morphology, Iran Introduction The blood-feeding female of phleboto- mine sand flies (Diptera: Psychodidae, Phle- botominae) include natural vectors of pro- tozoa of the genus Leishmania (Kinetoplas- tida: Trypanosomatidae), which are the para- sitic causative agents of mammalian leish- maniasis (Killick-kendrick 1990). There are approximately 700 species of phlebotominae sand flies divided among 6 genera, of which only two, i.e. Phlebotomus in the old world (OW) and Lutzomyia in the new world (NW) are medically importance (Lane 1987, Lewis 1982, Lane 1993, Sharma and Singh 2008). Only 10% of these Phlebotominae sand flies act as disease vector. Further, only 30 species of these are important from public health point (Sharma and Singh 2008). A to- tal of about 21 Leishmania spp. have been identified to be pathogenic to human (Singh *Corresponding author: Dr Yavar Rassi, E-mail: rassiy@sina.tums.ac.ir Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 23 2006). However, recent studies based on molecular data have provided evidence that the genus Phlebotomus is not monophyletic (Depaquit et al. 1998). Furthermore, Rispail and Leger (1998a) have recently revised the definition of morphological characters states. In the Mediterranean Region, species of the subgenus Larroussius are the main vectors of Leishmania infantum, which cause Visceral Leishmaniasis (VL) in humans and dogs (Kil- lick-kenrick 1990, Guernaoui et al. 2005). The subgenus is readily identified by the devel- opment long extension of the spermathecal neck in females (Rispail 1990). Description of the subgenus Larrous- sius created by Nitzulescu (1931) with P. ma- jor Annandale, 1910 as the type species (Per- filiev 1968, Lewis 1982). Among 27 species of Phlebotominae sand flies in the OW (Artemiev and Neronov 1984, Rispail and Leger 1998a, Rispail and Leger 1998b, Secombe et al. 1993), at least 12 species of the subgenus Larroussius are proven or probable vectors of leishmaniasis (Killick-kendrick 1990). The subgenus Lar- roussius is closely related to the subgenera Transphlebotomus and Adlerius, and the pro- liferation of species within Larroussius and Adlerius is probably recent (Rispail and Leger 1998b, Rispail and Leger 1991). Although, identification of male speci- mens of Larroussius are not very difficult, but determination of some females of this sub- genus has been considered impossible based on morphological characters. Leger et al. (1983) showed that three sympatric species of Larroussius in Greece could readily be separated by the morphol- ogy of base of the spermathecal ducts. Killick-Kendrick et al. (1991) separated 13 species of Larroussius by the morphol- ogy of the base of the spermathecal ducts. Recent studies showed that molecular tools could help resolve phylogenetic relationships between species of subgenus Larroussius (Es- seghir et al. 1997, Esseghir et al. 2000, Muc- cio et al. 2000, Pesson et al. 2004, Parvizi and Assmar 2007). There are four species of subgenus Larroussius including P. kandelakii, P. per- filiewi, P. major and P. tobbi in Meshkin- shahr district, north western Iran. The first species is a proven vector and the second is the probable vector of viscerotropic Leish- mania spp. in this area (Rassi and Javadian, 1998, Rassi and Javadian 1999, Rassi et al. 2001a, Rassi et al. 2001b, Rassi et al. 2005). The objective of the present study was to identify and distinguish the females of P. perfiliewi, P. major and P. tobbi. They are similar in morphological characters and indis- tinguishable. We analyzed the sequences of cytochrome b (mtDNA) and elongation fac- tor-1α (nDNA) genes of these three species and compared with morphological charac- teristics. Complete phylogenetic information is available for these genes and they are use- ful for the study of molecular systematic and speciation. Material and methods Study area Meshkinshahr District (48° 17′ N, 38° 15′ E) is located at 1890 masl in Ardebil Province, Iran. The district occupies the north- ern foothills of the Sabalan Mountains, which rise to an altitude 4881 masl. Temperature varies from -27 °C in winter to 41 °C in sum- mer. The human population was 156141 in 2006 and the principal economic activity is sheep farming. Sand fly sampling Sand fly sampling was carried out from Jun-October 2006 (during the period of peak activity), in 30 villages distributed through- out the Meshkinshahr District. Sand flies were collected once every 15 d from indoor habitats (bedroom, stable, toilet, bath room, hen nest, hay loft and store room) and outdoor habitats (yard, rodent burrow, stone and wall Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 24 crevices, fox burrows and dog kennel, rubble and riverbanks) using sticky papers, CDC light trap and Aspirator. Sand fly specimens were stored in 96% ethanol. Dissection, Mounting and Morphological identification After recording the sampling data and locations, sand fly specimens were washed in 1% detergent then twice in sterile distilled water. Each specimen was then dissected in fresh drop of sterile normal saline by cutting off the head and abdominal terminalia with sterilized forceps and single used mounted needles. The remainder of the body was stored in the sterile eppendorf microtubes. Specimens were mounted in Puri’s or Berlese’s medium and identified using the identification keys of Theodor and Mesghali (1964), Perfiliev (1968), and Lewis (1982). Morphological characters, which used in this study, included pharyngeal armature, sper- mathecal segments number, length of sperma- thecal neck, palpal and ascoids formula. First and second are more important characters. DNA extraction DNA was extracted from the dissected thorax and attached anterior abdomen of in- dividual sand flies using the method of Ish- Horowiz (Ready et al. 1991). In the 1.5 ml microtubes, the thorax plus anterior abdo- men of each sand fly was frozen and defro- sted twice to break up tissue using a sampler tips or pestel, with grinding mix. Then SDS mix was used to denature proteins associated with the DNA, then ice cold 8M KOAc was added to remove effectively the SDS pro- teins from solution. Cell debris and proteins were separated from the DNA by centrifuga- tion then the DNA in the supernatant was precipitated over night at -20 °C in 96% etha- nol. Following ethanol precipitation, the DNA was dissolved in 15µl 1X TE (10mM Tris- HCl, 1mM EDTA pH= 8.0) and stored at -20 °C. PCR amplification of Cyt b and EF-1α For Cyt b one pair primers were used. CB3-FC (forward) (5′-CA(C/T) ATTCAA- CC (A/T)GAATGATA-3′) with CB-R06 (re- verse) (5′-TATCTAATGGTTTCAAAACA ATTGC-3′) to amplify an overlapping 3′ frag- ment of 499 bp without primers (CB3 frag- ment). Also for EF-1α one pair primers were used, EF-F05 (forward) (5′-CCTGG ACAT- CGTGATTTCAT-3′) with EF-F08 (reverse) (5′-CCACCAATCTTGTAGACATCCTG-3′) to amplify of 454 bp without primers. The PCR reaction conditions were iden- tical for both Cyt b and EF-1α. 2µl 10X PCR buffer, 1.2 µl MgCl2, 0.15µl primers (F and R), 2µl dNTPs, 1.5µl DNA with the reaction volume completed to 20 µl by distilled water, followed by initial denaturation of Cyt b at 94 °C for 3 min. PCR consisted of 35 cycles of denaturation at 94 °C for 30 sec, anneal- ing1 at 40 °C for 30 sec, annealing 2 at 44 °C for 30 sec, extension at 72 °C for 90 sec and then final extension at 72 °C for 10 min. For EF-1α, initial denaturation at 94 °C for 3 min. PCR consisted of 35 cycles of denatu- ration at 94 °C for 30 sec, annealing at 48°C for 30 sec, extension at 72 °C for 30 sec and then final extension at 72 °C for 10 min. Direct sequencing of PCR products One hundred nanograms of each puri- fied DNA sample was cycle-sequenced us- ing an ABI Parsim® Big Dye™ Terminator cycle sequencing Ready Reaction Kit (version 2.0) and ABI 373/377 sequencing systems (ABI, PE Applied Biosystems), with 3.2 Pmol of the same primers that were used for PCR. Aligning and phylogenetic analysis of DNA sequences DNA sequences from both strands were aligned and edited using Sequencher Demo 4.7 and BioEdit softwares. Multiple or pair- wise sequence alignments of DNA were used with CLUSTAL W PPC: Clustalw version 1.7. Phylogenetic analyses were done using Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 25 Parsimony PAUP. Relationships were inferred based on genetic distances using the Neighbor Joining (NJ) option with default settings. Results Species composition of subgenus Larroussius Out of the 1743 sand fly specimens col- lected, 660 specimens (37.9%) belonged to the subgenus Larroussius, including: Phleboto- mus (La.) kandelakii (31%), P. (La.) major (1.5%), P. (La.) tobbi (1.5%), P. (La.) per- filiewi (1.7%) and P. (La.) major group (2.1%). Aligning and phylogenetic analysis of Lar- roussius DNA sequences PCR amplification of Cyt b and EF-1α was successfully achieved for all 3 species of the subgenus Larroussius. Seven sequences for Cyt b and four for EF-1α were compared for P. major. Com- parison of pairwise genetic similarity or score of sequences showed 87%-100% similarity for Cyt b and 96%-100% similarity for EF- 1α. Also 2.7% genetic diversity for Cyt b and 2.8% for EF-1α was observed. There were two haplotypes for Cyt b from seven sequences (Table 1) and for EF- 1α, there were two haplotypes within four sequences (Table 2). The Neighbor-joining (NJ) phylogram for Cyt b showed two line- ages, and each of these had subgroups with short branches. One of the lineages had one haplotype from sand flies from the same habitat (Stone crevices) but different locations (Ur kandi, Mueel, Agh daragh). The second line- age had one haplotype from sand flies from different habitats (Rodent burrow and Fox burrows) and locations (Ghurt tappeh, Alni, Niaz suee) (Fig. 1). In addition, phylogenetic tree for EF-1α showed two lineages and only one haplotype had subgroups. One of the line- ages had one haplotype from sand flies from the same location (Ur kandi) but different habi- tats (Stone crevices, bedroom, and hen nest). The second lineage had one haplotype from one specimen in rodent burrow (Fig. 2). Seven sequences for Cyt b and three for EF-1α were compared for P. tobbi. Com- parison of pairwise genetic similarity of se- quences indicated the 100% similarity for both Cyt b and EF-1α sequences. Genetic diver- sity and unique haplotype for Cyt b and EF- 1α were not observed. For Cyt b and EF-1α one haplotype was obtained (Table 1 and 2). The Neighbor-joining (NJ) phylogram for both Cyt b and EF-1α showed one lineage for all specimens in the same location and habitat (Fig. 1 and 2). Six sequences for Cyt b and three for EF-1α were compared for P. perfiliewi. Com- parison of pair wise genetic similarity of se- quences indicated 100% similarity for both Cyt b and EF-1α sequences. In this species genetic diversity for Cyt b and EF-1α were not observed (Table 1 and 2). Phylogenetic tree for both Cyt b and EF-1α showed one line- age for all specimens in the different location and habitats (Fig. 1 and 2). The Neighbor- joining (NJ) phylogram in combination of Cyt b and EF-1α for seven specimens showed two lineages for P. perfiliewi and P. tobbi and one lineage for P. major (Fig. 3). Identification of the female Larroussius spe- cies using morphological characters In the present study, the morphological characteristics of the three species female of Larroussius were described as follows: Phlebotomus major and Phlebotomus ne- glectus Palpal formula: 1, 4, 2, 3, 5 and the for- mula of ascoids: 2/3-8, 1/9-5. Pharyngeal ar- mature has occupied ⅓ of pharynx space and punctiform, arranged in several rows and ante- rior elements have serrated margin (Fig .4). Spermatheca with 14-16 segments and length of spermatheca neck ⅔ of spermatheca cap- sule (Fig. 5). Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 26 Phlebotomus tobbi Palpal formula: 1, 4, 2, 3, 5 or 1, (2, 4), 3, 5 and the formula of ascoids: 2/3-8, 1/9- 15. Pharyngeal armature has occupied over ½ of pharynx space and punctiform, arranged in several concave irregular rows of large dots and anterior part of pharynx have a fine dots (Fig. 6). Spermatheca with 11-13 seg- ments. Length of spermatheca neck as long as spermatheca capsule (Fig. 7). Phlebotomus perfiliewi Palpal formula: 1, 4, 2, 3, 5 and the formula of ascoids: 2/3-9, 1/10-15. Pharyngeal armature has occupied over ½ of pharynx space and punctiform, arranged in several con- cave regular rows of large dots (Fig. 8). Sper- matheca with 17-20 segments. Length of sper- matheca neck ½ of spermatheca capsule (Fig. 9). Identification of the male P. major and P. neglectus using morphological characters Among 26 male specimens in this spe- cies 17 specimens of P. major (65.4%) with 20-30 ventrally directed and long hairs stand densely on coxite and Palpal formula 1, 4, (2, 3), 5 (Fig. 10 and 11). In 9 specimens of P.neglectus (34.6%) with less than 20 ven- trally directed hairs, widely spaced and sparser and Paplal formula 1, 4, 2, 3, 5 (Fig. 12 and 13). Discussion According to the results of previous studies, the vectors of viscerotropic Leishmania spp. in OW mainly belong to the subgenera Larroussius, Adlerius, Euphlebotomus, Syn- phlebotomus and Paraphlebotomus. In the subgenus Larroussius, P.perniciosus in Al- geria, France, Italy, Malta, Spain, P.ariasi in France, Spain, Italy, P. perfiliewi in Italy, East Mediterranean, North of Africa, Greece, Azer- baydzhan, Tunisia, P. tobbi in Cyprus, East Mediterranean, Sicil, P. kandelakii in Afghani- stan, Russia, and P. neglectus in Greece, Al- bania, Portugal (Killick-kendrick 1990). In North West of Iran P. kandelakii and P. per- filiewi transcaucasicus are proven and prob- able vector of viscerotropic Leishmania spp. (Rassi et al. 2005). Table 1. Sequences comparison of Cyt b-mtDNA gene in the subgenus Larroussius Species Total Sequences Pairwise genetic similarity (%) Total aplotype Unique haplotypes Genetic diversity (%) P. major 7 87 – 100 2 0 2.7 P. tobbi 7 100 1 0 0.3 P. perfiliewi 6 100 1 0 0 Table 2. Sequences comparison of EF-1α-nDNA gene in the subgenus Larroussius Species Total Sequences Pairwise genetic similarity (%) Total aplotype Unique haplotypes Genetic diversity (%) P. major 4 96-100 2 0 2.8 P. tobbi 3 100 1 0 0 P. perfiliewi 3 100 1 0 0 Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 27 Table 3. Collected sand flies of the subgenus Larrossius in different location and habitats in Meshkinshahr District, Iran Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 28 Fig. 1. Neighbor-joining phylogenetic tree for DNA haplotypes of Cyt b (mtDNA) of the subgenus Larroussius sand flies species 0.1 tobbiAF161210 tobbiAF161212 MSH899tobbi MSH1657tobbi MSH1481tobbi MSH1292tobbi MSH903tobbi MSH551tobbi MSH480tobbi MSH721perfiliewi MSH738perfiliewi MSH756perfiliewi MSH795perfiliewi MSH812perfiliewi MSH836perfiliewi perfiliewiAF161201 MSH1201major MSH297major MSH619major MSH1397major neglectusAF161191 neglectusAF161193 MSH861major MSH007major MSH52major P.caucasicusFJ217390 P.sergentiEU980367 Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 29 Fig. 2. Neighbor-joining phylogenetic tree for DNA haplotypes of EF-1α (nDNA) of the subgenus Larroussius sand flies species 0.01 MSH297major MSH312major MSH317major majorEF416834 neglectusAF160802 neglectusAF160801 MSH522major MSH721perfiliewi MSH738perfiliewi MSH836perfiliewi perfiliewiAF160805 MSH903tobbi MSH1292tobbi tobbiAF160810 P. caucasicusEF416836 Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 30 Fig. 3. Neighbor-joining phylogenetic tree for DNA haplotypes of combination of Cyt b and EF-1α of the subge- nus Larroussius sand flies species. 0.01 MSH-836-P. perfiliewi MSH-738-P. perfiliewi MSH-721-P. perfiliewi MSH-903-P. tobbi MSH-1292-P. tobbi MSH-297-P. major MSH-522-P. major P. caucasicus EF416836 Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 31 Fig. 4. Pharyngeal armature of P. major Fig. 5. Spermatheca of P. major Fig. 6. Pharyngeal armature of P. tobbi Fig. 7. Spermatheca of P. tobbi Fig. 8. Pharyngeal armature of P.perfiliewi Fig. 9. Spermatheca of P.perfiliewi Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 32 Fig. 10. Palpal formula of P.major Fig. 11. Coxite hairs of P.major Fig. 12. Palpal formula of P.neglectus Fig. 13. Coxite hairs of P.neglectus According to the finding of the present investigations, the phylogeny of Cyt b and EF-1α haplotypes confirms the morphologi- cal relationships among the three species P. perfiliewi, P. major and P. tobbi in the sub- genus Larroussius. The males of these three species have a several diagnostic morpho- logical characters, whereas the females of these species show very similarities in morphology of spermathecal segment number, pharyngeal ar- mature and other characteristics. Although morphological characteristics are the most practical methods for species dis- tinguishing, new molecular techniques are very useful to resolve problems of identification in the cases with morphologically similarities. Access on the genetic diversity and mo- lecular systematic of the Larroussius sand flies species not only useful to find the taxonomic status of them, but also indicates the ecologi- cal and geographical differences. In our analysis, all of the Cyt b and EF- 1α sequences, the monophyly of the subgenus Larroussius was confirmed, in concordance with the morphologically and molecularly based phylogenies of Rispail and Leger (1991, 1998b) and Esseghir et al. (1997). All of the branches of the parsimony and distances trees had strong support and showed identical re- lationships and indicated the validity of many of characters in inferring evolutionary rela- tionships. The trees were also topologically si- milar to the parsimony tree of Esseghir et al. (1997). On morphological characteristics, the present study confirmed the observations of Iranian J Arthropod-Borne Dis, (2009), 3(2): 22-35 A Absavaran et al: Identification of Sand flies… 33 Perfiliev (1968), Lewis (1982), Leger et al. (1983), and Killick-Kendrick et al. (1991). Phylogenetic analyses of P. major po- pulations showed 2 lineages in different lo- cations such as rodents burrow and stone crevices (Table 3). According to our molecular and morpho- logical finding, it seems that there are both P. major and P. neglectus in Meshkinshahr district. This is the first report of P. neglectus in this area as well as in Iran (Table 3) Further studies needs to resolve the ta- xonomic status of this species. P. major has many geographical variants the females of which have conventionally been distinguished by differences in pharyngeal armature. Adler (1933) stressed the tendency of P. major to form geographical races. He showed (1946) that they differ mainly in the form of the pha- ryngeal armature of the females. He also men- tioned ecological differences between the races and stresses that some races may have a dif- ferent importance in the epidemiology of VL. Our study on phylogenetic analyses of P. tobbi populations showed one lineage with single haplotype for either cytB or EF-1α gene. P. tobbi was first found in Iran and Is- rael, and was described as a variety of P. per- niciosus. Parrot (1934) reported that it is a single species with the name of P. tobbi. This was accepted also by Theodor (1948). Phlebotomus tobbi is mainly found in burrows and is rather common in Transcaucasia and it seems to be identical with the sand flies from Iran and southwest Asia. Therefore, P. tobbi is considered a separate species. Phlebotomus perfiliewi is the probable vector of VL in the north west of Iran. In the present study, this species had one lineage with a single haplotype. Phylogenetic tree retrieved from cytB or combination of cytB and EF-1α showed close relationship of P. perfiliewi with P. tobbi. Perfiliev (1966) recognized three sub- species of P. perfiliewi (perfiliewi, galilaeus and transcaucasicus) which were distingui- shed by minor morphological differences in the aedeagus of the male. Lewis (1982) listed the same three subspecies but commented that the distinction of the male galilaeus from transcaucasicus uncertain. Artemiev and Ner- onov (1984) raised galilaeus and transcau- casicus to level species. The spermatheca of galilaeus and perfiliewi are indistinguishable, suggesting that these allopatric sand flies are taxonomically very close and are perhaps two subspecies (Lewis, 1982). To find an exact distinction and identi- fication of the female species of Larroussius, needs more investigations in different parts of the world as well as Iran. 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