ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera, Psychodidae): possible consequences in their ability to transmit Leishmania tropica.

Abstract

An intraspecific study on Phlebotomus sergenti, the main and only proven vector of Leishmania tropica among the members of the subgenus Paraphlebotomus was performed. The internal transcribed spacer 2 (ITS2) sequences of 12 populations from 10 countries (Cyprus, Egypt, Italy, Lebanon, Morocco, Pakistan, Portugal, Spain, Syria, and Turkey) were compared. Samples also included three species closely related to P. sergenti: Phlebotomus similis (three populations from Greece and Malta), Phlebotomus jacusieli and Phlebotomus kazeruni. Our results confirm the validity of the taxa morphologically characterised, and imply the revision of their distribution areas, which are explained through biogeographical events. At the Miocene time, a migration route, north of the Paratethys sea would have been followed by P. similis to colonise the north of the Caucasus, Crimea, Balkans including Greece and its islands, and western Turkey. Phlebotomus sergenti would have followed an Asiatic dispersion as well as a western migration route south of the Tethys sea to colonise North Africa and western Europe. This hypothesis seems to be well supported by high degree of variation observed in the present study, which is not related to colonisation or to intra-populational variation. Two groups can be individualised, one oriental and one western in connection with ecology, host preferences and distribution of L. tropica. We hypothesise that they could be correlated with differences in vectorial capacities.

Full text

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ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus
similis (Diptera, Psychodidae): possible consequences in their ability to
transmit Leishmania tropica
Je ´ro ˆme Depaquita,*, Hubert Ferte ´a, Nicole Le ´gera, Fabienne Lefranca, Carlos Alves-Piresb,
Hanafi Hanafic, Michele Marolid, Francisco Morillas-Marqueze, Jean-Antoine Riouxf,
Milena Svobodovag, Petr Volfg
aLaboratoire de Parasitologie, Faculte ´ de Pharmacie, 51 rue Cognacq-Jay, 51096, Reims Cedex, France
bInstituto de Higiene e Medicina Tropical, Lisbon, Portugal
cUS Naval Medical Research Unit no. 3, Cairo, Egypt
dLaboratorio di Parassitologia, Istituto Superiore di Sanita `, Rome, Italy
eDepartamento de Parasitologia, Facultad de Farmacia, Granada, Spain
fInstitut de Botanique, Montpellier, France
gLaboratory of Parasitology, Charles University, Praha, Czech Republic
Received 18 March 2002; received in revised form 30 April 2002; accepted 30 April 2002
Abstract
An intraspecific study on Phlebotomus sergenti, the main and only proven vector of Leishmania tropica among the members of the
subgenus Paraphlebotomus was performed. The internal transcribed spacer 2 (ITS2) sequences of 12 populations from 10 countries (Cyprus,
Egypt, Italy, Lebanon, Morocco, Pakistan, Portugal, Spain, Syria, and Turkey) were compared. Samples also included three species closely
related to P. sergenti: Phlebotomus similis (three populations from Greece and Malta), Phlebotomus jacusieli and Phlebotomus kazeruni. Our
results confirm the validity of the taxa morphologically characterised, and imply the revision of their distribution areas, which are explained
throughbiogeographical events.At the Miocene time, amigration route,north of the Paratethys sea would havebeen followed by P.similis to
colonise the north of the Caucasus, Crimea, Balkans including Greece and its islands, and western Turkey. Phlebotomus sergenti would have
followed an Asiatic dispersion as well as a western migration route south of the Tethys sea to colonise North Africa and western Europe. This
hypothesis seems to be well supported by high degree of variation observed in the present study, which is not related to colonisation or to
intra-populational variation. Two groups can be individualised, one oriental and one western in connection with ecology, host preferences
and distribution of L. tropica. We hypothesise that they could be correlated with differences in vectorial capacities. q 2002 Australian
Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.
Keywords: Phlebotomus sergenti; Phlebotomus similis; Internal transcribed spacer 2; rDNA; Vector; Leishmania; Biogeography
1. Introduction
Phlebotomus sergenti Parrot, 1917 is the only proven
vector of Leishmania tropica (Wright, 1903) within the
subgenus Paraphlebotomus (Al-Zahrani et al., 1988; Guil-
vard et al., 1991). It has a more widespread distribution than
the parasite (Depaquit et al., 1998). The presence of this
sandfly in L. tropica free areas and the varied prevalence
of anthroponotic cutaneous leishmaniasis in endemic areas
suggest the possible existence of closely related sandfly
species (cryptic species) with different vectorial capacities.
The history of the species since its first description is
confused. The male was described from the Constantine
area (Algeria), based on having a pyriform style with a
terminal and a sub-terminal spine (Parrot, 1917). A descrip-
tion of a female caught in copula in Portugal (Franc ¸a, 1918)
was based only on external characters, an approach consid-
ered useless for the diagnosis of species. The same authors
redescribed the male at the same time and provided an
original drawing in which the aspect of the style is in agree-
ment neither with the text, nor with the original description.
Does it belong to the same species?
Adler and Theodor (1929) redescribed the male and
female from specimens belonging to Parrot’s collection
and from their own collections from Palestine, Syria and
International Journal for Parasitology 32 (2002) 1123–1131
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.
PII: S0020-7519(02)00088-7
www.parasitology-online.com
* Corresponding author. Tel.: 133-3-26-91-37-23; fax: 133-3-26-91-35-
97.
E-mail address: jerome.depaquit@univ-reims.fr (J. Depaquit).

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Mesopotamia, without taking into account that the latest
specimens did not come from ‘the type locality’. Similar to
Parrot(1917),theynoticedinthemaleaglobulousstylewith
two slightly shifted terminal spines. In the description of the
female they stressed the presence of internal morphological
characters such as the pharyngeal armature of long teeth and
the reservoir of the spermatheca formed by five to six rings,
which were not observed in the Algerian specimens.
Perfiliev(1963,inLewis,1982),inthedescriptionofPhle-
botomus sergenti similis from the Caucasus, Crimea and
southern Ukraine, and later (Perfiliev, 1968), stressed that
the style of this subspecies has a sub-apical spine on a tuber-
cle inserted slightly under the apical one. He reported that in
Phlebotomus sergenti sergenti, the two spines are generally
at the same level, and concluded that the presence of a sub-
apical spine as it appeared in Parrot (1917) should not be
considered as characteristic. For him, the differentiation
between the two sub-species was based on the form of the
basal process and length of the lateral lobe. After Artemiev
and Neronov (1984) and Depaquit et al. (1998), we consider
P.sergentiandP.similisasrepresentingtwodistinctspecies.
Regarding their vectorial capacities, P. sergenti is a
proven vector of L. tropica in Saudi Arabia (Al-Zahrani et
al., 1988) and in Morocco (Guilvard et al., 1991). Except in
eastern Africa and in Namibia, P. sergenti is the most prob-
able vector in all foci of anthroponotic cutaneous leishma-
niasis, where it is always found as a dominant species
(Killick-Kendrick, 1990). Concerning P. similis, its role
(considered by Garifallou et al. (1984) as P. sergenti) in
the transmission of L. tropica was evoked in the Greek
Ionian Islands but has never been proven.
The present molecular study concerns four species of the
sub-genus Paraphlebotomus Theodor. Focussing on the
type-species P. sergenti Parrot, and including material
from various origins, it constitutes the first contribution to
the knowledge of molecular variation within the main
vector of L. tropica. The use of rDNA, has many advantages
including high mutation rhythm (e.g. internal transcribed
spacer 2, (ITS2)), speed and ease of use, multiple target
sites, predefined marker systems (e.g. ITS2), known poly-
merase chain reaction (PCR) primers, as well as a small
knowledge base for sandflies (Depaquit et al., 2000 and
Di Muccio et al., 2000). This multicopy gene involves
homogenisation processes usually called molecular drive
(Dover, 1982).
Phlebotomine sandflies constitute a good model for
biogeography because larvae live in the soil and imagos fly
on very short distances. Variation observed in sequences
results from biogeographical or vicariant events, which
explain the choice of distance and phylogeny as analysis
methods.
2. Materials and methods
Our sample included 12 populations of P. sergenti from
10 countries. In order to study the inter-populational differ-
ences, we also included three closely related species (Depa-
quit et al., 2000): P. similis Perfiliev (from three locations),
Phlebotomus jacusieli Theodor and Phlebotomus kazeruni
Theodor and Mesghali (Table 1).
To appreciate the existence of intra-populational varia-
tion, a pilot study was carried out on Turkish specimens
caught in two distinct localities: Adana, located at the foot
of the Eastern slope of the Taurus, and Sanliurfa, 300 km
eastwards, a well known focus of cutaneous leishmaniasis
caused by L. tropica (Volf et al., 2002). From the latter
location, wild flies as well as colonised flies were compared
(Table 1).
The majority of the studied specimens were caught with
CDC miniature light traps, but some flies were caught on
oily papers or came from laboratory colonies (Table 1).
Phlebotomine sandflies were dried before DNA extrac-
tion. The head and genitalia of a single sandfly were cut off
in a drop of ethanol then cleared in boiling Marc-Andre ´
solution and mounted between slide and cover slide for
identification (these slides are available on request from
the senior author). Genomic DNA was extracted from the
thorax, wings, legs and abdomen. Body parts were crushed
and incubated at 608C in a solution containing cetyltri-
methylammonium bromide, following the method used by
Depaquit et al. (2000). RNase (0.5 units) was added to the
aqueous phase, which was then incubated at 378C for
30 min. to remove RNA. Total genomic DNA was precipi-
tated by the addition of isopropanol. After centrifugation,
the pellet was washed, dried, and resuspended in 50 ml of
water. PCR was performed in a 50 ml volume using 5 ml of
extracted DNA solution and 50 pmol of each of the two
primers used in a previous study (Depaquit et al., 2000).
Primer sequences are C1a : 50-CCT GGT TAG TTT CTT
TTC CTC CGC T-30and JTS3 : 50-CGC AGC TAA CTG
TGT GAA ATC-30. The PCR mix contained (final concen-
trations) 10 mM Tris HCl, pH 8.3, 1.5 mM MgCl2, KCl
50 mM, Triton X-100 0.01%, 200 mM dNTP each, and
0.25 ml (1.25 units) of Taq polymerase (Eurobio). Initial
denaturation at 948C for 5 min was followed by 35 cycles
of denaturation at 948C for 30 s, annealing at 628C for 1 min
and extension at 728C for 1 min with a final elongation time
of 10 min at 728C. Amplicons were analysed by electro-
phoresis in 1.5% agarose gel containing ethidium bromide.
PCR products were both direction sequenced directly by
Qiagen using the universal C1a and JTS3 primers used for
DNA amplification, or cloned into pUC 18 vectors using the
‘sure clone ligation kit’ (Amersham Pharmacia) following
the manufacturer’s instructions before sequencing three
times. Identical sequences (one direction only) were always
obtained within individuals, except one Moroccan specimen
which needed a fourth sequencing because of a doubtful
nucleotide.
Sequence alignment was performed using the MUST
package (Philippe, 1993). Maximum parsimony, bootstrap
and distance analysis using Neighbour-Joining (Saitou and
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1125
Table 1
Specimens sequenced
SpeciesOrigin (country and area or
locality)
Method and year of catchingNumber of studied specimensa
Sequencing strategya
GenBank accession number
Phlebotomus sergenti
Cyprus
Egypt
Israel
Italy
Lebanon
Morocco
Pakistan
Portugal
Spain
Syria
Turkey
Turkey
Turkey
Paphos
South Sinaı ¨
West bank
Sicily
Qaa
Rif
Pringabad
Mertola
Granada
Kassab
Cukurova
Sanliurfa
Sanliurfa
CDC miniature light trap, (1993)
Colony, (1997)b
CDC miniature light trap, (1999)
Oily paper, (1999)
CDC miniature light trap, (1996)
CDC miniature light trap, (1998)
Colony, (1996)c
CDC miniature light trap, (1996)
CDC miniature light trap, (1994)
CDC miniature light trap, (1995)
Oily paper, (1997)
Colony, (1999)d
CDC miniature light trap, (1999)
Two males
Two males
One male; one female
One male
Two males
Three males
Two males; one female
Two males
Three males
Two males
Two males
Three males
Two males; one female
C 1 DS
C
DS
C
C
C 1 DS
C
C
C
C 1 DS
DS
C 1 DS
DS
AF462323
AF462329
AF462325
AF462330
AF462326
AF462331
AF218323
AF462327
AF462324
AF462328
AF462332
AF462332
AF462332
Phlebotomus similis
Greece
Greece
Malta
Crete
Macedonia
Gozo
CDC miniature light trap, (1991)
CDC miniature light trap, (1994)
CDC miniature light trap, (1989)
Three males
Two males
Two males
DS
C 1 DS
C
AF462333
AF218324
AF462334
Phlebotomus jacusieli
LebanonNachgura CDC miniature light trap, (1996)One maleC AF218317
Phlebotomus kazeruni
Egypt Sinaı ¨ Colony, (1997)e
Two malesCAF218318
aDS, direct sequencing of the PCR product; C, cloning before sequencing of the insert.
bFifth generation.
cKillick-Kendrick et al. (1995).
dEighth generation.
eHanafi et al. (1999).

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Nei, 1987) were performed using Phylogenetic Analysis
Using Parsimony (PAUP*) 4.0 beta 8 version software
(D.L. Swofford, PAUP*. (* and other methods). Version
4. Sinauer Associates, Sunderland, Massachussets. 1998)
on the complete dataset, as well as on this dataset, from
which were excluded sites 281–314 to account for a variable
poly (AT) insertion (or deletion) within P. sergenti. Gaps
were treated as a fifth base, even in the complete dataset.
Phlebotomus kazeruni was used as outgroup, following the
results obtained by Depaquit et al. (2000).
3. Results
The size of amplified fragments was species dependent,
and varied between 480 and 516 nucleotides from which
274–310 nucleotides were attributed to ITS2. The alignment
was 547 sites long including 112 variable sites, of which 45
were parsimony uninformative and 67 were parsimony
informative (Fig. 1). The region was AT rich, with the
proportion of AT greater than 70% (70.83–73.84% for the
complete amplified fragment, and 80.73–84.28% for ITS2
only).
Sequences were identical for specimens sampled from the
same location. Topologies obtained from a maximum parsi-
mony analysis carried out on the complete dataset (547
nucleotides) are given in Fig. 2. Phlebotomus sergenti and
P. similis are both monophyletic and strongly supported by
bootstrap values of 92 and 87%, respectively.
Phlebotomus similis is the sister species of P. jacusieli,
confirming the results of Depaquit et al. (2000). Bootstrap
support is moderate (71%), but their grouping is supported
by an AA indel at sites 192–193 and two nucleotide substi-
tutions at sites 240–241 (Fig. 1).
The grouping of P. similis from Greek Macedonia, Crete
and Gozo (Malta) is supported by two A indel changes at
sites 461–462. Additional indels (189–191 and 225) group
populations from Gozo with those from Macedonia. The
observed clusterings within P. sergenti are consistent for
populations from Israel, Egypt, Sicily and Morocco, inde-
pendently of the analytical methods used (maximum parsi-
monyor neighbour-joining).
bootstrap support using parsimony (Fig. 2). Populations
from Sicily and Morocco appear as sister populations
(bootstrap value 100%). Both taxa have a long poly(AT)
insertion for sites 289–296. They also share a common
poly(AT) stretch (sites 297–306), one indel site (386) and
two substitutions (391 and 442). Populations from Egypt
and Israel appear as sister populations with strong boot-
strap support (98%).
According to maximum parsimony analysis (Fig. 2), the
phylogenetic position (paraphyletic or monophyletic) of
populations from Spain and Portugal is not resolved, as
evidenced by the lack of bootstrap support. In the neigh-
bour-joining analysis, they appear as monophyletic, and as
the sister group of populations from Israel, Egypt, Sicily and
This clade has 100%
Morocco. Their sequences are identical except for Portu-
guese flies which have a shorter poly(AT) pattern (two
nucleotides) than the Spanish specimens (positions 307
and 308), and a G nucleotide at site 360, that is probably
autapomorphic (position 360).
The Lebanese population appears as sister group of the
previous ones, independently of the analytical method used.
This position is well supported by bootstrap values (98%).
The sequences obtained from Cypriot, Pakistani, Syrian,
and Turkish flies were the shortest: 480 bp (Fig. 1) because
they include very few poly(AT) patterns. They constitute
the most basal populations within P. sergenti. The Turkish
populations shared a plesiomorphic site (387). Two addi-
tional changes (substitutions) at sites 85 and 327 distinguish
the Syrian population.
The observed clusterings are supported in part by the
insertion of 13–47 nucleotides in poly (AT) patterns,
including positions 269–316 (Fig. 1). To measure the
impact of these poly(AT) patterns, a separate analysis
using the same methodology was undertaken with sites
281–314 excluded. The new dataset (513 sites) includes
78 variable sites, of which 41 were parsimony informative
and 37 parsimony uninformative. The topology of trees
obtained following both maximum parsimony (Fig. 3)
and neighbour-joining analyses is similar. These new
analyses confirm the monophyly of P. sergenti and P.
similis, which are supported by bootstrap values of 100
and 91%, respectively.
Phlebotomus similis is again placed as the sister species
of P. jacusieli. Within P. similis, the populations from
Greek Macedonia and Malta are the most closely related,
with the population from Crete as their sister group.
In comparison with the results from complete database
(Fig. 2), the clustering within P. sergenti are consistent for
populations from Israel, Egypt, Sicily and Morocco (Fig. 3).
However, the phylogenetic position of populations from
Spain and Portugal is very different. They appear as the
sister group of populations from Pakistan, Cyprus, Lebanon,
Syria, and Turkey (Fig. 3), not as the sister group of popula-
tions from Israel, Egypt, Sicily and Morocco as previously
emphasised (Fig. 2). This clustering is supported by a boot-
strap value of only 50%. Accordingly, the position of popu-
lations from Spain and Portugal, as sister-group of those
from Israel, Egypt, Sicily and Morocco, is only supported
by the poly(AT) indel events.
The two P. sergenti groups are not correlated to micro-
satellite region only. A study not taking into account this
part of the gene, gave a similar bifurcating tree. However,
the phylogenetic status of the AT microsatellite is of the
highest interest. It resolves relationships into the western
populations of P. sergenti. The more western the popula-
tions, the longer is the AT microsatellite, which could be
correlated to evolutionary processes. Further studies seem
necessary to explain why Iberic specimens have a shorter
AT microsatellite than African and Sicilian species, which
do not permit to resolve their phylogenetic position.
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J. Depaquit et al. / International Journal for Parasitology 32 (2002) 1123–1131
1127
Fig. 1. ITS2 sequence alignment (from positions 131 to 472, written in bold print) with flanking 5.8 and 28S rDNA (written in thin print). Identities are denoted
by dashes and gaps by asterisks. Sequences from Turkish flies are identical and include two populations from Sanliurfa (wild and colony) and another from
Cukurova.

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4. Discussion
The ITS2 is known to be informative at the intraspecific
level for Rhipicephaline ticks (Barker, 1998) or South
American mosquitoes (Marrelli et al., 1999; Malafronte et
al., 1999). In Europe, considerable variation in ITS2 was
described for six sibling species from the Anopheles macu-
lipennis complex (Proft et al., 1999). However, in the taxon-
omy of Phlebotomine sandflies, ITS2 was used in the study
of two subgenera of Phlebotomus. In the subgenus Larrous-
sius (Di Muccio et al., 2000), ITS2 contained no intraspe-
cific variability among seven populations of P. perniciosus
(from Italy and Morocco) as well as in populations of P.
perfiliewi from Italy and Greece. In the subgenus Paraphle-
botomus, a similar observation was made for two popula-
tions of P. sergenti from Cyprus and Pakistan (Depaquit et
al., 2000). In the study reported here, two or three (excep-
tionally one) specimens sequenced from each population
had no intra-populational variability (identical sequences).
Moreover, in Turkey, where we had access to three popula-
tions (two wild and one colony; Table 1), the sequences
obtained from all the specimens were identical. Based on
these results within populations, we can assume recency of
common descent and hence origin for populations geogra-
phically distant more than 300 km.
Results presented here further demonstrate the informa-
tional potential of ITS2 at the intraspecific level for a Phle-
botomine species that is widespread in the Old World. At
the present time, only one molecular study concerning Phle-
botomine sandflies, carried out on Lutzomyia longipalpis
emphasised intraspecific variation (Uribe Soto et al.,
2001). The other ones focussed on interspecific and/or inter-
generic variation (Esseghir et al., 1997, 2000; Aransay et al.,
1999, 2000; Martin-Sanchez et al., 2000).
Phlebotomus sergenti and P. similis are demonstrably
monophyletic on the basis of several molecular synapomor-
phies. This result confirms a study previously carried out
using morphological and morphometric approaches for
examining the species status of these two taxa (Depaquit
et al., 1998). These two species are allopatric at the present
time. The revised distribution of P. similis (Fig. 4) includes
Azerbaijan, Russia, Ukraine, Romania, ex-Yugoslavia,
Albania, Greece, Turkey and Malta. Turkey is the only
country in which both species are present, but P. similis
should occur only west of Taurus and Antitaurus and P.
sergenti only to the east (Fig. 4). In Greece all of the
previous continental or insular records of P. sergenti are
actually P. similis. The same is true for Malta (Gozo),
where P. similis is present, not P. sergenti, as reported by
Le ´ger et al. (1991). Indeed, the presence of P. similis is
difficult to explain on this last island because P. sergenti
(not P. similis) occurs in Sicily, which implies two different
establishments. Should a human importation of P. similis be
considered in Malta? The significant variability in ITS2
J. Depaquit et al. / International Journal for Parasitology 32 (2002) 1123–1131
1128
Fig. 3. Most parsimonious phylogram (length 90) obtained after a Branch
and Bound search of PAUP* performed on the database with sites 281–314
excluded.Phlebotomuskazeruniis usedas outgroup. CI ¼ 0:92;RI ¼ 0:93.
Bootstrap values (1000 replications) are indicated on the branches.
Fig. 2. Two most parsimonious phylograms (length 134) obtained after a
Branch and Bound search of PAUP* performed on the complete database
(547 nucleotides). Phlebotomus kazeruni is used as outgroup. CI ¼ 0:88;
RI ¼ 0:91. Bootstrap values (1000 replications) are indicated on the
branches.

Page 7

sequence among populations from Crete, continental
Greece and Malta would suggest that this is not a recent
importation by human transport.
Based on the groupings observed in the present study, and
on their present distributions, P. similis and P. sergenti
would have followed generalised tracks at the Miocene
time (Fig. 5).
From the middle-eastern centre of dispersion of Para-
phlebotomus (Depaquit et al., 2000), a migration route
circumventing the Paratethys Sea by the north could have
been followed by P. similis (or its ancestor), and explains
today its distribution in the north of the Caucasus, Crimea,
Balkans, Greece and its islands (by routes then open in the
Mesogea), and Turkey where its eastern extension have
been blocked by the orogenesis of the Taurus and Antitaurus
mountains (15–20 millions years ago).
If we look for intraspecific P. sergenti variations, two
branches are individualised. One is related to the north–east-
ern Mediterranean area (Cyprus, Pakistan, Syria and
Turkey), while the other is South and West of the first one
(Egypt, Morocco, Sicily, Spain and Portugal). The Lebanese
flyinsertstwoadditionalAT,whichplacesitonthetree(Fig.
2) in an intermediate position, in agreement with the settle-
ment corridor role of this area in Miocene. From the middle-
eastern centre of dispersion, and according to its vast distri-
bution area, P. sergenti would have followed a western
migration route along the southern edge of the Tethys Sea
via Lebanon, Egypt and North Africa. From Morocco, it
J. Depaquit et al. / International Journal for Parasitology 32 (2002) 1123–1131
1129
Fig. 5. Hypothesis of settlement in the Old World by P. sergenti and P. similis from a centre of dispersion located in Asia. Phlebotomus similis would have
followed a northern way of migration, circumventing the Paratethys sea, to inhabit in due course the north-east of the Mediterranean basin. Phlebotomus
sergenti colonised Asia and would have followed a route along the southern edge of the Tethys sea to North Africa then to the west of Europe using the Betic
bridge.
Fig. 4. Distribution of P. sergenti (w) and P. similis (W W).

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would have colonised the Iberian peninsula probably before
theopeningoftheStraitofGibraltar,throughtheBeticbridge
(5 million years ago), then south of France and Corsica. Its
presenceinSicilycouldbeexplained(takingintoaccountthe
great homology of sequences from specimens from Sicily
and Morocco), by recent connections between these two
areas at the time of the Tethys drying, Messinian, 6 millions
years ago. In the East, there are no or little variation between
the sequences of flies from populations of Cyprus, Syria,
Pakistan and Turkey (where P. sergenti and P. similis seem
tobeseparatedbyTaurusandAntitaurus),whichconstitutea
homogeneous unit. Inside thisEastern group,we deduce that
P.sergentiwhichsettledinCypruscamefromtheeast,prob-
ablyfollowingtheCapeAndreasduringPleistocenetime,an
hypothesisthatwesuggestedrecently(Depaquitetal.,2001).
In light of our results, it seems judicious to consider the
variation of these molecular characters as intraspecific. In
another study carried out on populations of L. longipalpis,
Uribe Soto et al. (2001), emphasising molecular variations
in the mitochondrial ND4 gene, concluded the potential
existence of sibling species. Further studies like hybridisa-
tion will be necessary before sibling species can be firmly
characterised in P. sergenti. However, independently of this
taxonomic interpretation, the existence of these two popula-
tions can be correlated to the distribution and the dynamics
of the foci of cutaneous leishmaniasis caused by L. tropica
over the Old World. The most active of them are all located
in the eastern part of the distribution of the parasite:
Sanliurfa in Turkey (Volf et al., 2002), Aleppo in Syria
(Tayeh et al., 1997), Mazar-e-Sharif and Kabul in Afghani-
stan (Jalili et al., 1998; Hewitt et al., 1998), Timargara in
Pakistan (Rowland et al., 1999) and the traditional but not
well documented foci of Central Asia and India. In the
western part of the P. sergenti distribution area, the foci
are sporadic, less active with lower human prevalences
than in the eastern foci. In Morocco, the particular epide-
miological situation has been revised by Rioux (2001).
These differences could be correlated with the fact that
the vector does not have the same capacity to transmit the
parasite everywhere. It seems, however, that P. sergenti is
highly susceptible to L. tropica in the east (Killick-Kendrick
et al., 1995) as well as in the west where 10–20% sandfly
infection rates were found in a particular Moroccan focus
(Rioux, 2001). Recently, the vectorial competence of a
Jordanian strain of P. sergenti was experimentally shown
(Kamhawi et al., 2000). It would be interesting to reproduce
this work on P. sergenti populations from different geogra-
phical origins and on P. similis too. Other hypotheses could
relate to ecological data differing from populations, such as
biotope, relative abundance or host preferences.
Acknowledgements
The authors wish to thank M. Boutry and C. Grimplet for
their technical help, N. Haddad, R., H. Kasap and M. Kill-
ick-Kendrick and B. Pesson for providing us Phlebotomine
sandflies and M. Kaltenbach for proof-reading this manu-
script.
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