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

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).

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.

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

CDC: miniature light traps; cyt B: cytochrome b; SP: sticky papers; UV: ultraviolet CDC.