Mem Inst Oswaldo Cruz, Rio de Janeiro, 109(4) June 2014
Original Article

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

Azzedine Bounamous1,2, Véronique Lehrter1, Leila Hadj-Henni1, Jean-Claude Delecolle3, Jérôme Depaquit1,+

1Transmission Vectorielle et Épidémiosurveillance de Maladies Parasitaires EA 4688-USC, Agence Nationale de la Sécurité Sanitaire de lu2019Alimentation, de lu2019Enviromment 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
3Université de Strasbourg, Strasbourg, France

Page: 466-472 DOI: 10.1590/0074-0276130584
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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.

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).



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.



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 ( 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.



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.





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.

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.



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.



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).



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.



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



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+ Corresponding author:
Received 16 December 2013
Accepted 17 March 2014

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