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Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276

Mem. Inst. Oswaldo Cruz vol.109 no.4 Rio de Janeiro July 2014  Epub June 06, 2014

http://dx.doi.org/10.1590/0074-0276130584 

Articles

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

Azzedine Bounamous 1   2  

Véronique Lehrter 1  

Leila Hadj-Henni 1  

Jean-Claude Delecolle 3  

Jérôme Depaquit 1   +  

1Transmission Vectorielle et Épidémiosurveillance de Maladies Parasitaires EA 4688-USC, Agence Nationale de la Sécurité Sanitaire de l’Alimentation, de l’Enviromment et du Travail, Faculté de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France

2Laboratoire des Sciences Naturelles et Matériaux, Institut des Sciences et de la Technologie, Centre Universitaire de Mila, Mila, Algeria

33Université de Strasbourg, Strasbourg, France

ABSTRACT

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

Key words: Algeria; mtDNA; PCR-RFLP; Phlebotomus sergenti

Algeria is a country where four leishmaniases are endemic. The leishmaniasis due to Leishmania infantum is transmitted by phlebotomine sandflies belonging to the subgenus Larroussius: Phlebotomus perniciosus, Phlebotomus perfiliewi [proven vectors according to Killick-Kendrick (1990)] and possibly Phlebotomus longicuspis (suspected vector) (Izri et al. 1990, Izri & Belazzoug 1993, Harrat et al. 1996, Berdjane-Brouk et al. 2012). Leishmania major is transmitted by the proven vector Phlebotomus papatasi (Izri et al. 1992). Leishmania tropica and Leishmania killicki are transmitted by the proven vectors Phlebotomus sergenti (Guilvard et al. 1991, Boubidi et al. 2011, Jaouadi et al. 2012).

The phlebotomine sandfly fauna of Algeria has been studied in the past (Parrot 1917, 1935, 1942, Rioux et al. 1970a, b, Dedet et al. 1973, 1984, Dedet & Addadi 1977, Belazzoug & Mahzoul 1980, 1986, Belazzoug et al. 1986, Berchi et al. 1986, Belazzoug 1991, Russo et al. 1991). Recently, two molecular studies characterised two closely related species (Phlebotomus chabaudi and Phlebotomus riouxi) having undistinguishable or very difficultly distinguishable females from North Africa (Bounamous et al. 2008, Boudabous et al. 2009) and a new species for the country (and for Africa) has been recorded: Phlebotomus mascittii Grassi (Berdjane-Brouk et al. 2011).

In a recent paper, Latrofa et al. (2012) suggested to use mitochondrial DNA (mtDNA) cytochrome b (cyt b) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as a rapid molecular identification of the common phlebotomine sandflies in the Mediterranean Region. The goal of this paper is to check the proposed method on a sampling of 17 Algerian species (131 specimens) belonging to three genera: Phlebotomus, Sergentomyia and Grassomyia. We coupled cyt b and cytochrome C oxidase I (COI) of the mtDNA. The first marker is considered as the “gold standard” for phlebotomine sandflies systematic. The second one serves as a DNA barcode for the identification of animal species (Hebert et al. 2003). However, it was used in a few studies carried out on phlebotomine sandflies to study taxa from the Americas (Arrivillaga et al. 2002, Azpurua et al. 2010), from North Africa (Boudabous et al. 2009) and in India (Kumar et al. 2012).

MATERIALS AND METHODS

Sandflies collection - Sandflies were collected from different provinces of Algeria coupling three methods in order to increase the diversity (Rioux et al. 2013): sticky traps, CDC and ultraviolet miniature light traps and aspirators (Fig. 1). They were stored in 96% ethanol. One hundred thirty-one males and females selected for this study are indicated in the Supplementary data.

Fig. 1 : sampling realised for this study in different areas of Algeria. Aïn-Touta is the type locality of Phlebotomus sergenti. Circles: main cities; stars: locations where sandflies have been caught. 

Sandflies mounting and identification - The head and genitalia of individual male sandflies were cut off within a drop of ethanol, cleared in boiling Marc-André solution and mounted between slide and cover slide for species identification. The body related to the specimen was stored dried in a vial at -20ºC before DNA extraction.

The specimens have been identified by observation of the head and genitalia under a BX50 microscope. The identification keys and characters used for the identification of specimens are those of Abonnenc (1972), Dedet et al. (1984), Depaquit et al. (1998a), Pesson et al. (2004) and Bounamous et al. (2008). Measures and photos have been performed using the Perfect Image software (Aries Company, Chatillon, France) and a video camera connected to the microscope.

DNA extraction - Genomic DNA was extracted from the thorax, wings, legs and abdomen of individual sandflies using the QIAmp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions, modified by crushing the sandfly tissues with a piston pellet (Treff, Switzerland) and using an elution volume of 200 µL, as detailed in Depaquit et al. (2004).

PCR amplification and sequencing - All the mtDNA amplifications were performed in a 50 µL volume using 5 µL of extracted DNA solution and 50 pmol of each of the primers. The PCR mix contained (final concentrations) 10 mM Tris HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01% Triton X 100, 200 µM deoxynucleotide triphosphate each base and 1.25 units of 5 prime Taq polymerase (Eppendorf, Germany).

The cycle profiles were marker dependent. Each PCR begins by an initial denaturation step at 94ºC for 3 min and finishes by a final extension at 68ºC for 1 min. Amplification of a fragment of cyt b gene has been done by using the primers N1N-PDR and C3B-PDR, following the method previously published by Esseghir et al. (1997): five cycles (denaturation at 94ºC for 30 s, annealing at 40ºC for 60 s and extension at 68ºC for 60 s) followed by 35 cycles (denaturation at 94ºC for 60 s, annealing at 44ºC for 60 s and extension at 68ºC for 60 s). Their COI domain was amplified using the primers used by Hajibabaei et al. (2006): LepF and LepR, under the following thermal profile (Costa et al. 2007): five cycles (denaturation at 94ºC for 30 s, annealing at 45ºC for 90 s and extension at 68ºC for 60 s), then 35 cycles (denaturation at 94ºC for 30 s, annealing at 51ºC for 90 s and extension at 68ºC for 60 s).

Amplicons were analysed by electrophoresis in 1.5% agarose gel containing ethidium bromide. Direct sequencing in both directions was performed by Sanger’s method using the primers used for DNA amplification. Reagents for PCR cleanup were Agencourt® AMPure XP-PCR Purification and those for sequencing reaction were Big Dye Sequencing Buffer (Applied Biosystems Foster City, California, USA); Agencourt® CleanSEQ kit: CleanSeq® beads. The instruments used for the sequencing were Biomek NXp BiomekFXP (Bekman Coulter); Sequencer 3730 XLT (Applied Biosystems). The correction of sequences was done using Pregap and Gap softwares included in the Staden Package (Bonfield & Staden 1996).

Molecular analyses - They are based on the two datasets of sequences. Analyses were performed using haplotypes obtained from this study and sequences of P. chabaudi and P. riouxi available in GenBank (Supplementary data). Sequence alignment was performed using the CLUSTALW routine included in the MEGA v.4 software (Tamura et al. 2007) and checked by eye. The objective of this study is to provide a database for sandflies identification, not to do any phylogenetical analysis. Consequently, a neighbour-joining (NJ) analysis was performed using MEGA 4 software, with the Kimura-2 parameters model and using uniform rates among sites. Gaps were treated as missing data.

RFLP - The diagnostic endonuclease restriction sites on cyt b and COI mtDNA sequences were predicted for each specimen using CLC workbench 5.2 software (clc-genomics-workbench.com-about.com). A panel of restriction enzymes was tested including AseI enzyme proposed by Latrofa et al. (2012) for cyt b digestion. Because one restriction enzyme cannot provide an original digestion pattern per species, we selected double digestion for both molecular markers.

PCR-RFLP assays were performed in a 50-μL total volume reaction mix, containing 15 μL of PCR product (from PCR vials), 0.1 μL of AseI, 0.4 µL of MnlI for cytb and 0.05 µL of MspI, 0.05 µL of TaqI for COI, 5 μL of NEB buffer 3 for cyt b and 5 μL of NEB buffer 4 for COI containing bovine serum albumin (New England Biolabs, France).

For cyt b PCR-RFLP, we selected a double digestion coupling AseI with MnlI. PCR products were digested during 2 h at 37ºC.

For COI PCR-RFLP, we selected a double digestion coupling MspI with TaqI. According to their different temperature of activity (37ºC and 65ºC, respectively), PCR products were digested during 1 h at 37ºC then 1 h at 65ºC.

The digested samples were separated by electrophoresis in a 3% agarose gel to produce DNA fragments and sized by comparison with markers 50 bp ladder, 100 bp ladder and 20 bp ladder (Cliniscience, France).

According to the sequencing of all the specimens processed in the present study, we did not use any positive control, but we checked that each restriction enzyme had functioned properly in each reaction by comparison to the predicted digestion.

RESULTS

PCR amplification was successful for all the specimens processed. The GenBank accessions for COI and cyt b are indicated in the Supplementary data. The length of the analysed markers is of 680 bp for COI. It varies from 510-525 bp for cyt b. Each gene from each haplotype had an open reading frame (ORF). The sequences labelled COI include exclusively this marker. The sequences labelled “cytB” included in fact an ORF of cytB (positions 1-321), the t RNA serine (positions 321-378), then the ORF for NADH subunit 1 (from 379 to the last position). These ORFs are translated in proteins, explaining the low probability they could be pseudogenes (Rogers & Griffiths-Jones 2012).

Global trees based on cyt b and COI sequences are presented in Figs 2, 3, respectively. According to Depaquit et al. (1998b), Phlebotomus bergeroti has been selected to root the tree. All the species morphologically recognised are well individualised. We note that P. sergenti included two populations without any morphological difference. The identification of P. perniciosus and P. longicuspis is not doubtful. We did not record any atypical specimen.

Fig. 2 : neighbour-joining tree based on mitochondrial DNA cytochrome b sequences rooted on Phlebotomus bergeroti. Bootstrap values after 1,000 replicates are indicated on the branches. 

Fig. 3 : neighbour-joining tree based on mitochondrial DNA cytochrome C oxidase I sequences rooted on Phlebotomus bergeroti. Bootstrap values after 1,000 replicates are indicated on the branches. 

The double digestion of each PCR product confirms the expected fragments for all haplotypes (Tables I, II). The resolution of DNA fragment size by the gel fractionation method used is about 10 bp. The double digestion of COI by restriction enzymes TaqI and MspI is very efficient, but cannot unequivocally distinguish Phlebotomus ariasi and Sergentomyia schwetzi.

TABLE I Species and populations examined in the present study - number and positions of cuts predicted for cytochrome C oxidase I mitochondrial DNA restriction fragment length polymorphism 

Species TaqI and Mspl (expected)
Cut sites
Cut (n) Fragments (n)
Phlebotomus bergeroti 4 5 85, 91, 101, 176, 254
Phlebotomus alexandri 5 6 22, 84, 85, 91, 156, 267
Phlebotomus longicuspis 4 5 22, 73, 91, 113, 408
Phlebotomus perniciosus 3 4 22, 91, 184, 408
Phlebotomus chabaudi 5 6 30, 85, 89, 91, 158, 256
Phlebotomus sergenti (group 1) 3 4 25, 62, 111, 507
Phlebotomus sergenti (group 2) 3 4 22, 85, 91, 507
Phlebotomus chadlii 6 7 14, 22, 85, 91, 101, 174, 224
Phlebotomus riouxi 6 7 21, 30, 85, 91, 141, 157, 186
Phlebotomus ariasi 2 3 85, 91, 527
Phlebotomus perfiliewi 2 3 11, 184, 408
Sergentomyia fallax 2 3 85, 265, 353
Sergentomyia schwetzi 2 3 85, 91, 527
Sergentomyia clydei ' 5 82, 91, 174, 176,184
Sergentomyia minuta 3 4 88, 90, 176, 350
Sergentomyia christophersi 5 6 32, 81, 85, 91, 101, 319
Sergentomyia dreyfussi 3 4 70, 91, 184, 360
Sergentomyia antennata 3 4 82, 110, 157, 356

TABLE II Species and populations examined in the present study - number and positions of cuts predicted for cytochrome b mitochondrial DNA restriction fragment length polymorphism 

Species AseI and MnlI (expected)
 
Cut (n) Fragments (n) Cut sites
Phlebotomus bergeroti 3 4 14, 20, 74, 434
Phlebotomus alexandri 5 6 14, 20, 62, 92, 108, 248
Phlebotomus longicuspis 5 6 14, 20, 62, 92, 103, 263
Phlebotomus perniciosus 4 5 14, 20, 62, 103, 353
Phlebotomus chabaudi 4 5 14,20, 62, 198,248
Phlebotomus sergenti (group 1) 4 5 14, 20, 96, 208,209
Phlebotomus sergenti (group 2) 3 4 14, 20, 62, 449
Phlebotomus chadlii 3 4 20, 62, 107, 356
Phlebotomus riouxi 3 4 14,20, 62, 445
Phlebotomus ariasi 3 4 14, 20, 62, 453
Phlebotomus perfiliewi 5 6 14, 20, 62, 92, 103, 263
Sergentomyia fallax 3 4 20, 62, 103, 353
Sergentomyia schwetzi 4 5 20, 52, 52, 62, 356
Sergentomyia clydei 5 6 10, 20, 62, 103, 134,212
Sergentomyia minuta 6 7 14, 19,20, 42,74, 93,296
Sergentomyia christophersi 6 7 20, 34, 46, 58, 62, 134, 184
Sergentomyia dreyfussi 7 8 14, 15, 20, 28, 62, 103, 134,171
Sergentomyia antennata 1 2 20, 515

The double digestion of cyt b by restriction enzymes AseI and MnlI provide restriction profiles indicated in Table II. On the one hand, it cannot individualise one population of P. sergenti, P. ariasi and P. riouxi. On the other hand, it cannot distinguish P. longicuspis and P. perfiliewi.

No partial digests were recognised in the analysis.

DISCUSSION

The specimens identified by morphology as belonging to a species are branched together regarding independently cyt b or COI sequences (Figs 2, 3).

Cyt b sequences provide a NJ tree in agreement with the traditional morphological taxonomy of the phlebotomine sandflies (Fig. 2). Sergentomyia are grouped together including Grassomyia dreyfussi. Concerning Larroussius, all of them are grouped together and two branches are individualised: one including P. ariasi and Phlebotomus chadlii and another containing P. perniciosus, P. longicuspis (including 2 lineages) and P. perfiliewi. These data are in accordance with those obtained by Esseghir et al. (2000) on cyt b and Di Muccio et al. (2000) on rDNA internal transcribed spacer 2. Concerning the subgenus Paraphlebotomus, the species P. sergenti, P. chabaudi and P. riouxi are grouped together. The first one shows two lineages in its type-locality. Moreover, Phlebotomus alexandri is not included in this branch, as previously observed (Depaquit et al. 2000, Krüger et al. 2011).

The NJ tree based on COI sequences (Fig. 3) has a surprising topology: the species belonging to the subgenera Sergentomyia, Larroussius and Paraphlebotomus are not grouped together. Despite this curious branching, some results are congruent with cyt b: (i) the position of P. alexandri, (ii) the existence of two molecular lineages within P. sergenti topotypes, (iii) the individualisation of P. chabaudi and P. riouxi, (iv) the existence of two lineages within P. longicuspis and (v) a high variability within Sergentomyia minuta and Sergentomyia antennata.

Many lineages have been identified in P. sergenti in the literature from populations from different parts of the species distribution area and no study emphasise a link between the molecular variability and the morphology (Depaquit et al. 2002, Yahia et al. 2004, Moin-Vaziri et al. 2007, Barón et al. 2008, Dvorak et al. 2011). The two mitochondrial lineages (cyt b as well as COI) within Algerian specimens of P. sergenti coming from different localities, all located just around the type locality area (Fig. 1) called Ain Touta, formerly Mac Mahon (Parrot 1917). This locality has not been precisely designated by Parrot (1917). The specimens processed in the present study can be considered as being topotypes, clearly labelled and stored in the collection of the laboratory of Parasitology of the Faculty of Pharmacy of Reims. These two populations are strongly separated and are characterised by many variable nucleotidic positions: about 30 for cyt b and 40 for COI (Fig. 4). The mean pairwise distance between the two populations of P. sergenti (> 5%) is comparable to the pairwise distances individualising P. perniciosus from P. longicuspis or P. perfiliewi in the present study and question about the status of these populations.

Fig. 4 : variable positions observed within Phlebotomus sergenti for cytochrome b and cytochrome C oxidase I (COI) mitochondrial DNA. Stars indicate the sites characterising the two populations. 

The haplotypes obtained for P. perniciosus and P. longicuspis have been compared with those of cyt b available in the literature (Esseghir et al. 1997, Pesson et al. 2004, Perrotey et al. 2005) (Fig. 5). The main P. perniciosus Algerian haplotype is the same than the main Mediterranean haplotype (= pern01), but three new haplotypes are recorded from Algeria for this species. Concerning P. longicuspis, the most common haplotype is the lcus01. However, we record four new haplotypes including a couple (LC2 and LC637) strongly individualised from the other ones. The COI sequences also individualise the latter specimens from all other P. longicuspis (Fig. 3).

Fig. 5 : alignment of cytochrome b haplotypes concerning Phlebotomus perniciosus and Phlebotomus longicuspis and showing exclusively the variable sites reported by Esseghir et al. (1997), Pesson et al (2004) and Perrotey et al. (2005). The Algerian haplotypes are written in bold. The variations observed in Algerian specimens are underlined. The stars indicate the sites individualising the haplotypes LC2 and LC637. 

Recently, the single digestion based on cyt b sequences by Latrofa et al. (2012) as a rapid molecular identification method for the common phlebotomine sandflies in the Mediterranean Region does not apply in Algeria. In fact, these authors focused on five species only, commonly caught in Italy: P. papatasi, P. perniciosus, P. perfiliewi, Phlebotomus neglectus and S. minuta. We tried this method on the Algerian sandflies, including P. papatasi. The method is not able to distinguish all the species and the combined double digestion of two different markers is needed for the specific identification. Due to the conservation of some parts of DNA sequences, the simple digestion of PCR products cannot separate some species belonging to different genera (like P. ariasi and S. schwetzi) or subgenera (like P. sergenti and P. ariasi). Consequently, this method is not enough discriminant to be used in routine all over the Mediterranean Basin. In fact, it is easier to firstly identify the species easy to recognise by a microscopical examination and secondly, to apply efficient PCR-RFLP methods to identify the species for which morphological identification is difficult, like the females of the Perniciosus complex, those of P. chabaudi and P. riouxi and for some Sergentomyia males.

ACKNOWLEDGEMENTS

To Kaouther Jaouadi, Mohammad Akhoundi and Mireille Cousinat, for their help, and to Sylvette Gobert, for proofreading this paper.

Supplementary data

Sampling species 

                  GenBank accession
Genus Subgenus Species Area City Catching method Catching date Gender Specimen cytB COI
Phlebotomus Phlebotomus P. bergeroti Tamanrasset Tamanrasset UV 15 Sep 2006 BER1TAM KJ480973 KJ481079
  Paraphlebotomus P. sergenti Aurès Menaa CDC 15 Sep 2007 SE670 KJ480971 KJ481077
          CDC 15 Sep 2007 SE669 KJ480966 KJ481072
          CDC 26 Sep 2007 SE621 KJ480960 KJ481066
        Tighargar SP 30 Aug 2006 SE335 KJ480968 KJ481074
      Bordj Bouarreridj Khola SP 8 Jul 2007 SE675 KJ480972 KJ481078
          SP 29 Jun 2007 SE627 KJ480965 KJ481071
      Ghardaia Ghardaia SP 20 Sep 2006 SE304 KJ480967 KJ481073
          SP 20 Sep 2006 SE307 KJ480969 KJ481075
          SP 20 Sep 2006 SE305 KJ480962 KJ481068
          SP 20 Sep 2006 SE312 KJ480970 KJ481076
        Metlili SP 6 Sep 2007 SE539 KJ480963 KJ481069
          SP 06 Sep 2006 SE540 KJ480964 KJ481070
          SP 19 Sep 2006 SE394 KJ480961 KJ481067
    P. alexandri Aurès El-kantra CDC 20 Sep 2007 AL646 KJ480988 KJ481093
        Ghoufi SP 30 Aug 2006 AL300 KJ480985 KJ481090
        Menaa SP 13 Sep 2007 AL607 KJ480987 KJ481092
      Batna Barika SP 1 Sep 2005 AL142 KJ480981 KJ481086
          SP 1 Sep 2005 AL215 KJ480984 KJ481089
          SP 1 Sep 2005 AL117 KJ480980 KJ481085
          SP 1 Sep 2005 AL202 KJ480983 KJ481088
          SP 1 Sep 2005 AL116 KJ480979 KJ481084
          SP 1 Sep 2005 AL114 KJ480978 KJ481083
          SP 1 Sep 2005 AL200 KJ480982 KJ481087
          SP 1 Sep 2005 AL110 KJ480977 KJ481082
      Ghardaia Metlili SP 19 Sep 2006 AL372 KJ480986 KJ481091
      Tamanrasset Tamanrasset UV 15 Sep 2006 AL7TAM KJ480990 KJ481095
          UV 15 Sep 2006 AL28TAM KJ480989 KJ481094
    P. chabaudi Aurès Menaa SP 31 Aug 2006 CB2 EU935791 FJ196432
          SP 31 Aug 2006 CB1 KJ480974 FJ196431
          CDC 26 Sep 2007 CB3 EU935792 FJ196433
          CDC 26 Jan 1900 CB572 EU935793 FJ196434
        Ain-Zaatout CDC 20 Sep 2007 CB573 EU935794 FJ196435
          CDC 20 Sep 2007 CB583ZAT EU935795 KJ481172
    P. riouxi Ghardaïa Ghardaïa SP 20 Sep 2006 RX1 EU935815 FJ196436
          SP 20 Sep 2006 RX2 EU935816 KJ481173
          SP 20 Sep 2006 RX3 EU935817 FJ196437
        Metlili SP 19 Sep 2006 RX4 EU935818 FJ196438
          SP 19 Sep 2006 RX5 EU935819 KJ481174
          SP 19 Sep 2006 RX7 EU935821 FJ196439
          SP 19 Sep 2006 RX8 EU935822 FJ196440
          SP 19 Sep 2006 RX9 EU935823 FJ196441
  Larroussius P. perniciosus Aurès Ain Zaatout CDC 11 Sep 2007 PN538 KJ481033 KJ481136
          CDC 15 Sep 2007 PN668 KJ481034 KJ481137
        Menaa CDC 20 Sep 2007 PN597 KJ481035 KJ481138
          CDC 26 Sep 2007 PN582 KJ481036 KJ481139
        Tazoult CDC 16 Sep 2007 PN517 KJ481037 KJ481140
          CDC 16 Sep 2007 PN518 KJ481038 KJ481141
      Ghardaia Metlili CDC 5 Sep 2007 PN660 KJ481039 KJ481142
          CDC 5 Sep 2007 PN656 KJ481040 KJ481143
      Jijel Beni Ahmed SP 10 Aug 2004 PNBE2-1 KJ481041 KJ481144
        Fdoules CDC 28 Aug 2007 PN643 KJ481042 KJ481145
          CDC 15 Sep 2007 PN632 KJ481043 KJ481146
          CDC 28 Aug 2007 PN642 KJ481044 KJ481147
          CDC 26 Aug 2007 PN641 KJ481045 KJ481148
      Bordj Bouarreridj Salama SP 1 Aug 2007 PN4 KJ481046 KJ481149
          SP 1 Aug 2007 PN677 KJ481047 KJ481150
      Mila Ferdjioua SP 10 Sep 2004 PNFE1-2 KJ481048 KJ481151
          SP 10 Sep 2004 PNFE3 KJ481049 KJ481152
        Sidi- Khalifa SP 20 Aug 2004 PNSKH2 KJ481050 KJ481153
        Vieux Mila SP 23 Jul 2004 PNVIM 2 KJ481051 KJ481154
    P. longicuspis Aurès Menaa CDC 13 Sep 2007 LC601 KJ481052 KJ481155
          CDC 13 Sep 2007 LC509 KJ481053 KJ481156
        El-Kantra CDC 20 Sep 2007 LC644 KJ481054 KJ481157
        Tighargar CDC 14 Sep 2007 LC633 KJ481055 KJ481158
      Bordj Bouarreridj Salama SP 1 Aug 2007 LC637 KJ481056 KJ481159
          SP 1 Aug 2007 LC2 KJ481057 KJ481160
        Madjana SP 1 Aug 2007 LC3 KJ481058 KJ481161
      Jijel Beni Ahmed CDC 10 Aug 2004 LCBE1 KJ481059 KJ481162
      Mila Vieux Mila SP 12 Jul 2004 LCVIM6 KJ481060 KJ481163
      M’Sila Boussaada CDC 15 Aug 2007 LC549 KJ481061 KJ481164
          CDC 15 Aug 2007 LC553 KJ481062 KJ481165
          CDC 15 Aug 2007 LC545 KJ481063 KJ481166
          CDC 6 Aug 2006 LC331 KJ481064 KJ481167
          CDC 15 Aug 2007 LC548 KJ481065 KJ481168
    P. perfiliewi Jijel Beni Ahmed SP 10 Aug 2004 PFBE1 KF680819 KJ481175
          SP 10 Aug 2004 PFBE2 KF680820 KJ481176
          SP 10 Aug 2004 PFBE7 KF680821 KJ481177
      Mila Ferdjioua SP 10 Sep 2004 PFFE1 KJ480975 KJ481080
      M’Sila Adama CDC 1 Aug 2007 PF635 KJ480976 KJ481081
    P. ariasi Aurès Ain Zatout CDC 11 Sep 2007 AR529 HM131125 KJ481169
          CDC 11 Sep 2007 AR526 HM131125 KJ481170
        Menaa CDC 26 Sep 2007 AR576 HM131125 KJ481171
    P. chadlii Aurès Ain Zaatout CDC 11 Sep 2007 CD525 HM131080 KJ499906
        Nara SP 30 Aug 2006 CD1 HM131079 KJ499904
          SP 30 Aug 2006 CD2 HM131079 KJ499905
Phlebotomus Phlebotomus S. fallax Aurès Ain Zaatout CDC 15 Sep 2007 FAL666 KJ480998 KJ481103
          CDC 20 Sep 2007 FAL542 KJ481000 KJ481105
        Menaa CDC 26 Sep 2007 FAL578 KJ480992 KJ481097
          CDC 26 Sep 2007 FAL581 KJ480995 KJ481100
          CDC 13 Sep 2007 FAL599 KJ480999 KJ481104
          CDC 13 Sep 2007 FAL610 KJ480996 KJ481101
          CDC 26 Sep 2007 FAL578 KJ480992 KJ481097
        Tazoult CDC 20 Sep 2007 FAL618 KJ480994 KJ481099
          CDC 20 Jan 1900 FAL612 KJ480997 KJ481102
          CDC 15 Sep 2007 FAL613 KJ480993 KJ481098
      Ghardaia Metlili CDC 6 Sep 2007 FAL519 KJ480991 KJ481096
    S. minuta Aurès Ain Zaatout CDC 20 Sep 2007 SM544 KJ481013 KJ481114
          CDC 11 Sep 2007 SM530 KJ481017 KJ481120
      Jijel Beni Ahmed SP 10 Aug 2004 SMBEN1 KJ481011 KJ481180
        Chemla CDC 13 Sep 2007 SM605 KJ481015 KJ481118
        Menaa CDC 20 Sep 2007 SM596 KJ481016 KJ481119
      Batna Barika SP 1 Sep 2005 SM213 KJ481009 KJ481181
      Ghardaia Metlili CDC 5 Sep 2007 SM515 KJ481012 KJ481115
          CDC 5 Sep 2007 SM561 KJ481014 KJ481117
      M’Sila Boussaada CDC 15 Aug 2007 SM554 KJ481013 KJ481114
      Mila Vieux Mila SP 10 Aug 2004 SMVIM3 KJ481019 KJ481122
          CDC 10 Sep 2004 SMVIM2 KJ481018 KJ481121
    S. antennata Aurès El-Kentra SP 20 Sep 2007 ANT522 KJ481007 KJ481112
          SP 12 Sep 2007 ANT630 KJ481008 KJ481113
      Batna Barika SP 1 Sep 2005 ANT209 KJ481006 KJ481111
      Ghardaïa Ghardaia SP 20 Sep 2006 ANTGARD KJ481005 KJ481110
    S. schwetzi Tamanrasset Tamanrasset UV 15 Sep 2006 SW5TAM KJ481020 KJ481123
          UV 15 Sep 2006 SW6TAM KJ481022 KJ481125
          UV 15 Sep 2006 SW14TAM KJ481021 KJ481124
          UV 15 Sep 2006 SW15TAM KJ481023 KJ481123
  Sintonius S. clydei Aurès Tazoult CDC 23 Sep 2007 CLY614 KJ481032 KJ481135
      Tamanrasset Tamanrasset UV 15 Sep 2006 CLY10TAM KC669793 KJ481179
          UV 15 Sep 2006 CLY17TAM KJ481031 KJ481134
          UV 15 Sep 2006 CLY24TAM KC669796 KJ481178
    S. christophersi Ghardaia Metlili CDC 5 Sep 2007 CHR654 KJ481025 KJ481128
          CDC 5 Sep 2007 CHR657 KJ481026 KJ481129
          CDC 6 Sep 2007 CHR651 KJ481024 KJ481127
          CDC 5 Sep 2007 CHR 653 KJ481027 KJ481130
          SP 19 Sep 2006 CHR351 KJ481030 KJ481133
          SP 19 Sep 2006 CHR349 KJ481029 KJ481132
          SP 5 Sep 2007 CHRMEK KJ481028 KJ481131
Grassomyia   G. dreyfussi Aurès Ain Zaatout CDC 11 Sep 2007 DRY527 KJ481001 KJ481106
          CDC 15 Sep 2007 DRY664 KJ481003 KJ481108
      Ghardaia Metlili CDC 5 Sep 2007 DRY560 KJ481004 KJ481109
          CDC 5 Sep 2007 DRY567 KJ481002 KJ481107

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Received: December 16, 2013; Accepted: March 17, 2014

+ Corresponding author: jerome.depaquit@univ-reims.fr

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