G en e t ic p o l y m o r p h is m in s y m p a t r ic s p e c ie s
OF THE GENUS PHLEBOTOMUS, WITH SPECIAL REFERENCE
t o P h lebo to m us perniciosus a n d P hlebo to m us lo ng icuspis (D ip t e r a , P h l e b o t o m id a e )
MARTÍN-SÁNCHEZ J.*, GRAMICCIA M.**, PESSON B.*** & MORILLAS-MARQUEZ F.*
S u m m a ry :
The Random Amplified Polymorphic DNA assay was used to study genetic variation within and between five Phlebotomus species belonging to three subgenera: P. (Larroussius) ariasi, P. (L.) longicuspis, P. (L.) perniciosus, P.(Paraphlebotomus) sergenti and P. (Phlebotomus) papatasi sympatric in southern Spain and proven vector of leishmaniasis. Two cluster analysis were proposed: one according to sandfly species and populations, the second according individual specimens of Phlebotomus perniciosus, Phlebotomus longicuspis s.I. and intermediate morphological specimens between these species. The results obtained are closely correlated with the taxonomy classically accepted for the subgenera and with the automatic classifications made by other authors which use morphological and isoenzymatic data. The validity of the species Phlebotomus longicuspis is also discussed.
KEY WORDS : Phlebotomidae, sandfly, Phlebotomus, sympatric species, genetic polymorphism, Random Amplified Polymorphic DNA (RAPD).
R ésu m é : Po l y m o r p h is m e g é n é t iq u e d’e s p è c e s s y m p a t r iq u e sd e Ph l e b o t o m u s, a v e c u n e é t u d ep a r t ic u l iè r e p o r t a n tsu r P . PERNICIOSUS ET P . LONGICUSPIS ( D IPTERA, PHLEBOTOMIDAE)
Le polymorphisme d'amplification de l'ADN par amorces aléatoires (RAPD) a été utilisé pour étudier la variabilité génétique inter et intraspécifique de cinq espèces de Phlebotomus
appartenant à trois sous-genres : P. (Larrousssius) ariasi, P. (L.) longicuspis, P. (L.) perniciosus, P. (Paraphlebolomus) sergenti et P.
(Phlebotomus) papatasi. Ces espèces sont sympatriques dans le sud de l'Espagne et sont des vecteurs reconnus de leishmanioses.
Deux classifications automatiques sont proposées : la première compare des populations des cinq espèces et la seconde, des individus de P. pernidosus, P. longicuspis s.l. ainsi que des spécimens présentant des caractères morphologiques
intermédiaires entre ces deux espèces. Les résultats montrent une corrélation étroite avec la taxonomie classiquement admise pour les sous-genres et avec les classifications automatiques réalisées par d’autres auteurs sur les caractères morphologiques et isoenzymatiques. La validité du statut spécifique de P. longicuspis est également discutée.
MOTS CLÉS : Phlebotomidae, phlébotome, Phlebotomus, espèce sympatrique, polymorphisme génétique, RAPD.
INTRO DUCTIO N
S
andflies ( D i p t e r a , Nematocera, Phlebotomidae) play an important role in human diseases mainly as vectors of leishmaniasis and several viroses.Their identification and classification is usually based on morphological criteria (Artemiev & Neronov 1984;
Lewis 1982). From the turn of the century, the species P hlebotom us (Larroussius) perniciosu s, P. (L.) ariasi, P. (P araphlebotom u s) sergenti, P. (Phlebotom us) p a p a tasi, and Sergentom yia m in u ta have been found in Spain, and it is easy to differenciate among them.
However, in recent years the species P. (P a.) a lex a n -
* D ep artam ento de Parasitología, Facultad de Farm acia, U niversidad d e G ranad a, C am pus U niversitario d e Cartuja, 1 8 071, G ranad a, Spain.
** Laboratorio di P arassitologia, Istituto Su periore di Sanità, Viale Regina Elena 299, 00161, Rom a, Italy.
*** Laboratoire d e Parasitologie, Facu lté d e P harm acie, Université Louis Pasteur, Strasbourg I, B.P. 24, 67401 Illkirch, France.
C orresp on d en ce: J . M artín-Sánchez. E-mail: joaq u in a@ p laton.u gr.es
dri, P. (P a.) ch a b au d i, P. (P a.) riouxi, P. (L.) longi
cuspis, P. (L.) lan geron i and P. (Transphlebotom us) m ascittii have also been recorded although in limited numbers. The differentiation between some of them is sometimes very difficult; for example, after the des
cription of P. riouxi (Depaquit et al., 1998) there is doubt if the P. c h a b a u d i cited in Spain are certainly P .ch a b a u d i or P. riouxi. Similar doubt can be raised about the species P. longicuspis and P. perniciosus, and to contribute to solve this doubt is one of the objec
tives of the present paper.
P. longicuspis was described in 1930 by Nitzulescu as a variety of P. lan geron i in northern Africa and has always been considered to be very similar to P. p e r niciosus. The males of P. perniciosu s and P. longicuspis have always been distinguished by the different mor
phology of their aedeagus which culminate in a single point in P. longicuspis and are bifurcate in P. p ern i
ciosus. For some time, the female specimens were only distinguished by biometric data (Parrot, 1936) until Leger et al. 1983 demonstrated that they could be mor
phologically distinguished by the shape of the recep-
Parasite, 2000, 7, 247-2 5 4 7 4 7
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2000074247
MARTIN-SANCHEZ J., GRAMICCIA M., PESSON B. & MORILLAS-MARQUEZ F.
1 : T h ey are 11 LCA and 7 LCB sp ecim en s.
2 : T h ey are LCB.
3 : It is LCA.
4 : T h ey are n o t includ ed eigh t sp ecim en s m ale P.p./P.l. from T orvizcó n (2 ) and Turre (6 ), w h ich m ake 174 individual sp ecim en s.
T ab le I. - Individual sp ecim en s from natural p op u lations studied by applying th e RAPD-PCR tech n iq u e. T h e sp e cies to w h ich th ey belon g, th e se x and th e locality in w h ich th ey w ere captured are recorded . P.p. = P. p e rniciosus, P.l. = P. longicuspis, P.a. = P. ariasi, P.s. = P.
sergenti, P.pa. = P. papatasi, M = m ale, H = fem ale.
tacle at the base of the sperm ducts. In Spain, although P. p em ic io su s has been found since around the begin
ning of the century, P. lon gicu spis was only first recorded in 1982 (Morillas et al., 1982) and the exis
tence of female specimens has not yet been irrefutably demonstrated. Later, male specimens with interme
diate forms between the two species were found (Morillas et al., 1991) and the impossibility to diffe
rentiate the two Spanish species using biometric data was demonstrated (Collantes & Martinez Ortega, 1997).
Recently, Benabdennbi et al. (1999) found in Morocco two populations of P. longicuspis that differed in the shape of the penean valves, the number of hairs on the internal surface of the coxites and the allelic fre
quencies o f some isoenzymes. According to these authors, the morph male denominated LCB corres
ponds to P. longicuspis s. str. whereas one denomi
nated LCA corresponds to a variant of:P. pem iciosu s.
In a previous study (Martin-Sanchez et al., 1995), we showed that RAPD-PCR could be an interesting mole
cular technique for taxonomical, ecological and genetic population studies on Old World sandflies, and that the patterns obtained with the chosen nine primers were reproducible. By using several primers a high number of molecular characters can be generated that can be used to identify different taxa. The amplified products by RAPD-PCR can be classified into two groups: variable (polymorphic) or conserved (non- polymorphic) (Hadrys et al., 1992). Both products can be used to establish associations between different spe
cies and populations.
The objectives of this study were as follows: 1) to determine the genetic divergence between P h lebo- tom us species vectors of leishmaniasis sympatric in southern Spain; 2) to evaluate the relationships among geographic populations within species and
3) to provide new data on the controversial taxo
nomic status of the species P. p e m ic io s u s and P. lo n gicuspis.
MATERIAL A N D METHODS
Sa n d f l y s a m p l e s a n d r a n d o m a m p l i f i c a t i o n o f DNA f r a g m e n t s
S
pecimens from natural populations of the species P. pem iciosu s, P. longicuspis, P. ariasi, P. sergenti, P. p a p a ta si and some specimens with intermediate morphological characters between the species P . p em ic io su s and P. longicuspis (P.p./P.l.) were cap
tured using CDC traps, manual techniques and adhe
sive traps. The captures were made in several locations in southern Spain (Almería, Granada and Huelva pro
vinces) during 1994-1995. In addition to these, other specimens were captured in northern Morocco (Chef- chaouene and Ouezzane) in 1995 with CDC traps.
Table I shows the size and geographical origin of natural populations of phlebotomine species studied by RAPD-PCR. Samples were first conserved in liquid nitrogenum (- 196° C) until their morphological classi
fication, after which they were kept in alcohol (70° C).
Specimens captured with adhesive traps were only conserved in alcohol. The distal portion o f the abdomen of each specimen was conserved for the morphological identification of the species. Classifica
tion of the two populations of P. longicuspis s.l. was made according to Benabdennbi et al., 1999. Pooled and single Italian specimens of P. p e m ic io s u s and P. p a p a ta si from cultured colonies and a pool of Spa
nish P. p e m ic io s u s (Turre, Almería province) were used as reference samples for RAPD-PCR.
7 4 8 Parasite, 2000, 7, 247-254
G eograp h ical reg io n
Species
Spain
M o ro cco
Total 1664
G ranada Alm ería Huelva
Loja
M F
Restabal
M F
T orvizcón
M F
Turre
M F
Higuera
M F
C h efch aou en e
M F
O u ezzan e
M F
P. p e m ic io s u s _ _ _ _ 13 9 8 4 - - - - 4 45
P. longicuspis - - - - 23 - l 3 - - - 1 8 1 6 3 2 - 30
P. a ria si 2 - - - 1 - 1 11 13 1 - - - - 29
P. sergenti 5 1 18 4 2 - - - - - 1 - - - 31
P. p a p a ta s i 1 - - - 4 1 6 19 - - - - - — 31
Genetic polymorphism in species o f the genus Phlebotomus
The DNA purification technique and the amplification conditions were the same previously reported (Martín- Sánchez et al., 1995) and carefully standardized in order to overcome the criticized lack of reproducibi
lity of the RAPD-PCR (Black IV & DuTeau, 1997).
Da t a a n a l y s is
Each amplified fragment was coded indicatively by its molecular weight in base pairs followed by the name of the primer used. The presence or the absence of the fragments in the individual specimens of each species was used to decide w hether this was a conserved or a polymorphic fragment. The amplified fragments were coded as “0” (absent) or “1” (present) for each individual (dendrogram 2) or group of indi
viduals (dendrogram 1), generating a matrix of binary data (matrix 1 for dendrogram 1 and matrix 2 for den
drogram 2, data not shown). The results obtained were used to perform a cluster analysis. Dendrograms were constructed using Jaccard’s similarity index and clustering by average linkage. A correspondence ana
lysis comparing P. p e rniciosus and P. longicuspis ampli
fied fragments was performed using matrix 2 and STAF-ITCF, 1991.
RESULTS
Ge n e r a l d a t a
R
andom amplification of DNA extracted from 174 individual specimens (114 males and 60 females) of natural sandfly populations of the genus Phle- botom us with each of the nine primers produced a variable number of DNA fragments of different longitude. In Table II the conserved fragments in each of the species studied are recorded. There are a total of 143 fragments of conserved DNA in the species of which 11 are shared by at least two species. No dif
ferences were observed between the overall patterns obtained from males and females in each species exa
mined: no band was constant for one sex and not for the other sex.
In t r a s p e c i f i c d a t a
P hlebotom us (P araphlebotom u s) sergenti
A total of 31 specimens, 26 males and five females were studied. Among them, 30 were Spanish and one was of Moroccan origin (Chefchaouene). The band pat
tern obtained with the primers P94B and M13 showed a high degree of homogeneity. The greatest hetero
geneity was shown by primer 006. No differences were observed between the populations with the exception of the fragment 463-004 which was conser
ved in all the populations except for the one from Loja.
P hlebotom us (P hlebotom us) p a p a ta si
A total o f 31 samples of Spanish origin were studied, and 20 Italian laboratory reared specimens were used as reference samples. The greatest individual variabi
lity was observed with primers 005 and 006 with which it was not possible to define conserved frag
ments on a species level. We did not detect any dif-
Species Fragments o f conserved DNA P. pemiciosus * 8 4 3 -0 3 6 , 1150-036
* 250-P 94B . 345-P 94B , 465-P 94B , 617-P94B . 7 13 -P 9 4 B . 858-P 94B , 1337-P 94B
* 7 8 3 -0 0 4 , 9 9 4 -0 0 4
* 6 6 0 -P 9 4 , 7 8 3 -P 9 4 , 9 9 4 -P 9 4 , 1 4 2 7 -P 9 4 1956-P 94
* 4 3 2 -0 0 5 , 505-005, 579-005, 7 4 1 -0 0 5 , 8 31- 0 05, 9 6 5 -0 0 5 , 1129-005
* 6 8 5 -0 0 6 , 1018-006
* 300-78, 3 4 4 -7 8 , 530-78, 6 5 0 -7 8 P. longicuspis s.l. * 994-P 94, 1956-P 94
* 530-78 P. ariasi * 7 5 6 -0 3 7
* 2 9 8-036, 388-036, 6 0 9 -0 3 6
* 306-M 13, 468-M 13, 505-M 13, 780-M 13
* 250-P 94B , 352-P 94B , 477-P 94B , 515-P94B , 7 13 -P 9 4 B , 1244-P 94B , 1500-P 94B
* 5 8 0 -0 0 4 , 2399-004
* 660-P 94. 9 7 0 -P 9 4 . 1375 -P 9 4 . 1 5 83-P 94
* 6 2 4-005, 699-005, 8 3 1 -0 0 5 , 1 3 4 5 -0 0 5
* 8 0 4-006, 9 9 4 -0 0 6
* 300-78, 530-78, 650-78 P. sergenti * 3 5 8 -0 3 7 , 3 8 1 -0 3 7 . 6 26-0 3 7
* 1 6 8 -0 3 6 , 2 2 0 -0 3 6 , 362-036, 4 5 1 -0 3 6 , 579- 036, 9 9 7 -0 3 6 , 1122-036
* 236-M 13, 387-M 13, 408-M 13, 456-M 13, 535-M 13, 761-M 13, 821-M 13, 863-M 13, 891- M 13, 960-M 13, 1055-M 13, 1168-M 13
* 265-P94B , 358-P94B , 404-P 94B , 530-P94B , 755-P 94B , 834-P 94B , 925-P 94B , 1306-P 94B
* 311-004. 3 7 5 -0 0 4 . 4 1 5-004, 6 5 9 -0 0 4 , 775- 0 0 4 , 2 1 8 1 -0 0 4
* 2 5 5 -P 9 4 , 32 1 -P 9 4 , 502-P 94, 556-P 94, 644- P94, 767-P 94, 1168-P 94, 1 2 8 5 -P 9 4
* 5 5 3 -0 0 5 , 6 0 9 -0 0 5 , 7 2 6 -0 0 5 7 5 6 -0 0 5 , 947- 005, 9 83-005, 1018-005, 2 1 8 1 -0 0 5
*3 53-006, 1 0 6 9 -0 0 6 , 1 1 4 5 -0 0 6 , 1 2 4 7 -0 0 6
* 206-78, 217-78, 300-78, 456 -7 8 , 530-78, 564-78, 801-78, 850-78, 947 -7 8 , 1156-78, 1264-78
P. papatasi * 3 5 1 -0 3 7 , 3 7 4 -0 3 7 , 7 1 3 -0 3 7
* 2 8 4-036, 3 23-036, 337-036, 3 6 2-036, 4 1 8 - 0 3 6 , 5 7 9 -0 3 6 , 7 4 1-036, 8 16-036, 1122-036
* 550-M 13, 840-M 13
* 370-P 94B , 617-P 94B , 677-P 94B
* 5 4 8 -0 0 4 , 7 2 7 -0 0 4 , 1 2 9 5 -0 0 4
* 332-P 94, 6 2 0 -P 9 4 , 6 5 2 -P 9 4 . 1255-P94
* 330-78, 444 -7 8 , 468-78, 530-78, 714-78 T ab le II. - Fragm ents o f conserved DNA in th e sp e cies studied. Each fragm ent is d enom in ated b y its M olecular w eight in b a se pairs fol
low ed by th e n am e o f th e prim er used. T h e m ost easily identifiable fragm ents, in general o f th e greatest intensity and, preferentially, not shared b y oth er sp ecies, are m arked in bold.
Parasite, 2000, 7, 247-2 5 4
Mémoire 249
MARTIN-SANCHEZ J., GRAMICCIA M„ PESSON B. & MORILLAS-MARQUEZ F,
ferences between the different Spanish populations stu
died although differences were observed between Spa
nish and Italian populations with primers P94B and 78.
For example, fragment 650-78 was conserved in the specimens of Spanish origin but not in reared Italian specimens.
P hlebotom us (Larroussius) a riasi
A total of 29 specimens were studied from three Spa
nish provinces (Almería, Granada and Huelva). Diffe
rences were observed between the specimens from eastern (Almería and Granada) and western Andalu
sian populations (Huelva) (E.A.P. and W.A.P., respec
tively). In some cases a fragment conserved in the W.A.P. is polymorphic (e.g. 305-004) or absent (as with 580-P94) in E.A.P., and sometimes the opposite occurs (e.g. 456-78 and 548-P94, respectively).
P hlebotom us (Larroussius) p e rniciosus
We used 45 individual specimens, 11 of Moroccan ori
gin (Ouezzane) and the rest from two Spanish pro
vinces (Almería and Granada). A pool of ten females of Spanish origin (Turre), and another pool of 11 males and 15 females of Italian reared specimens were used as reference samples.
The band pattern obtained with primers P94 and 005 shows a high degree of homogeneity between the dif
ferent populations and specimens.
With primers 037, 036 and M13 a marked variability was expressed by the presence of bands with slight differences in their molecular weights. This variability was also observed between individuals captured from the same geographical area. In general, there was greater similarity between the Spanish and the Italian specimens than between the former and the Moroccan ones.
P hlebotom us (Larroussius) longicuspis s.l.
A total of 30 specimens, 24 males and six females were studied. These were identified by morphological cha
racteristics as P. longicuspis s.l. according to classical criteria: males with pointed non-bifurcated aedeagus and females with a characteristic dilation of the distal portion of the sperm ducts (Parrot, 1936; Leger et al., 1983).
P. longicuspis s.l. (Table I) can be divided into two morphologically distinct types, in accordance with Benabdennbi et al., 1999: specimens with the penean valves with a curved distal portion denominated LCA, and specimens with a straight distal end denominated LCB. Of the 21 Moroccan males, 11 have the LCA mor
phology and ten specimens are LCB; the only three specimens from the Ouezzane locality have the LCB morphology. The three Spanish specimens are of LCA type.
P94 is the primer with the highest degree of homo
geneity within P. longicuspis s.l. and together with 78 we obtained three specific fragments, 994-P94, 1956- P94 and 530-078. The first and second fragments are also conserved in the species P. p em icio su s which also presents, with the same primer, another additional three conserved fragments (660-P94, 783-P94 and 1427- P94). It is not possible to distinguish between these two species since some specimens of P. longicuspis s.l.
present the same conserved fragments as P. p ern i- ciosus. We obtained similar results with the other pri
mers but with a greater variability and, for the same reason, it was not possible either to differentiate bet
ween this species and P. pem iciosu s.
In relation to the two morphological types mentioned, group LCA, including the three Spanish specimens, has nine conserved fragments: 660-P94*, 505-005*, 778- 006**, 300-078*, 801-078, 617-P94B*, 713-P94B*, 1427- 004** and 564-P94**. The fragments marked* are conserved in the species P. p e m ic io s u s and those marked** are conserved in all the populations of P. p er- niciosus studied with the exception of the Moroccan ones from Ouezzane. Finally, fragment 801-078 is variable in P. p em iciosu s from Turre and is conserved in the remai
ning populations of this species. The Spanish popula
tion of LCA has another five fragments in common with the species P. p em iciosu s although one should take into account that only three specimens are studied.
In the morphological type LCB which includes the six female studied, we did not detect any conserved frag
ment.
Specimens intermediate between P hlebotom us p em ic io su s and P hlebotom us longicuspis (P.p./P.l.) We studied eight male specimens from two Spanish provinces which presented intermediate morpholo
gical characteristics between both species, i.e. a penean valve characteristic of P. p em iciosu s and another typical of P. longicuspis s.l., or more specifically, type LCA.
In general, with all the primers these specimens show an indistinguishable band pattern from that of the species P. pem iciosu s, with a few small exceptions. For example, fragment 579-005, which is conserved in P.
pem iciosu s, is absent from the two specimens P.p./P.l.
from Turre (Almeria province). This fragment is also absent from all the Moroccan specimens of P. longi
cuspis s.l. and from one specimen of Spanish origin.
C
l u s t e r a n a l y s isAND CORRESPONDENCE ANALYSIS
From the band patterns obtained by RAPD, we obtained the Jaccard’s similarity index and constructed two dendrograms to summarize the relationships among different groups. In dendrogram No 1 (Fig. 1) we considered the following 14 OTUs, in relation to
2 5 0
Mémoire
Parasite, 2000, 7, 247-254Genetic, polymorphism in species o f the genus Phlebotomus
Fig. 1. - Cluster analysis according to sp ecies and geographical popu
lations. D endrogram constru cted using Ja c c a rd ’s sim ilarity ind ex and clustering b y average linkage. N um ber o f observation s = 153 (DNA fragm ents, data not sh ow n ), nu m b er o f variables = 14 (p o p u lations). 1 is P. pap atasi, 2 is P. sergenti, 3 is P. ariasi from so u theastern Spain, 4 is P. ariasi from south w estern Spain, 5 is P. lon
gicuspis fem ale from C h efch ao u en e (M o ro cco ), 6 is P. longicuspis m ale LCB from C h efch ao u en e, 7 is P. longicuspis m ale LCA from C h efch ao u en e, 8 is P. longicuspis LCB from O u ezzan e (M o ro cco ), 9 is P. p e rnicosus from O u ezzan e, 10 is P. longicuspis LCA from Spain, 11 is interm ediate P. pem icosus/P . longicuspis LCA from Spain, 12 is P. p em iciosu s from T urre (Sp ain ), 13 is P. pem iciosu s from N aples (Italy), and 14 is P. p em iciosu s from T orvizcon (Spain).
species, morphological type and geographical origin:
P. p ap a ta si, P. sergenti, P. a ria si from southeast Spain, P. a ria si from southwest Spain, Italian P. p e rniciosus, Spanish P. p em icio su s from Turre, Spanish P. p e m i ciosus from Torvizcon, Moroccan P. p em ic io su s from Ouezzane, Spanish LCA P. longicuspis, Moroccan LCB P. longicuspis from Ouezzane, Moroccan female P. lon gicuspis from Chefchaoune, Moroccan male LCB P. lon gicuspis, Moroccan male LCA P. longicuspis from Chef
chaoune and the intermediate Spanish P. pem iciosus/P . longicuspis LCA. We considered it more appropriate to study females of P. longicuspis s.l. independently from the LCB males of this species in order to avoid the interference of previous information which could have produced inaccurate results. The number of individuals in each group is recorded in Table I. We analysed a total o f 153 fragm ents w hich are conserved for someone of the 14 OTUs (data not shown) and the dendrogram obtained (Fig. 1) revealed the following:
- the species P. sergenti and P. p a p a ta si belonging to the subgenera P araphlebotom u s and Pblebotom us, res
Parasite, 2000, 7, 247-254
pectively, are clearly separate from the remaining group belonging to the subgenus Larroussius (J = 0,980);
- within Lairoussius, 1) the species P. a ria si is clearly independent of the other members of this subgenus (J = 0,886). The two Spanish populations of P. aria si are separated by a distance of 0.211. 2) At a distance of J = 0,793 two groups begin to separate, one includes female P. lo n g icu sp is and male LCB from Chef
chaouene and the other is comprised of LCB from Ouezzane and LCA in addition to all the P. p em icio su s populations. It should be noted that the LCB group from Ouezzane is comprised of only three individuals.
The similarity between the Italian P. p em icio su s and the Spanish specimens from Torvizcon is noteworthy, and the intermediate position of the Spanish LCA P.
longicuspis and of P.p./P.l. with respect to P. p e m i ciosus from northern Morocco and the group of Spa
nish and Italian P. p em icio su s is also noteworthy.
In dendrogram No. 2 (Fig. 2) the OTUs are represented by 59 individuals belonging to P. p em ic io su s (11 from Ouezzane, 12 from Turre and two from Torvizcon), P. longicuspis s.l. (24 from Chefchaouene: six females, seven LCB males and 11 LCA males; three LCB from Ouezzane and one LCA from Turre) and six interme
diate specimens P. p./P. l. LCA. A total of 64 conserved and variable fragments was considered (all the ampli
fied fragments identified with accuracy in everyone of the 59 individuals, data not shown) to construct a binary matrix used to compute the Jaccard’s similarity index. Clusters were observed containing members of the species P. p em ic io su s (No. 5, 6 and 7), the inter
mediate specimens P.p./P.l. (No. 8) and the specimens of LCA P. longicuspis (No. 4 and 9), whereas specimens of LCB P. longicuspis (No. 2, 3) and the females P. lon gicuspis (No. 1) form five small groups. In this case the three LCB specimens from Ouezzane (No. 3) are grouped together with the other LCB specimens from Chef
chaouene (No. 1, 2) and become independent from the LCA. The specimens of LCA P. longicuspis (No. 4, 9) are integrated within both the Moroccan (No. 5) and the Spanish (No. 6, 7) P. p em iciosu s.
Correspondence analysis (Fig. 3) yielded similar results to ascending hierarchical classification. The plane formed by the first two axes (I1 = 20.7 %, I2 = 13.2 %) showed that P. p em icio su s specimens, LCA type and intermediate P.p./P.l. males, are grouped and markedly distinct from P. longicuspis females and LCB males, which were characterized by a high polymorphism.
DISCUSSION
A
pplication of the RAPD-PCR technique for the identification and genetic study of five species of the Ph lebotom us genus sympatric in southern M é m o ire251
MARTIN-SANCHEZ J„ GRAMICCIA M., PESSON B. & MORILLAS-MARQUEZ F.
Fig. 2. - Cluster analysis o f individual sp e cim ens o f th e sp e cies P. perniciosus, P. lon- gicuspis and sp e cim en s interm ed iate b e t
w e e n th e tw o (P .p ./ P .l.). D e n d ro g ra m co n stru cted using Ja c c a r d ’s sim ilarity and clustering b y average linkage. N um ber o f observation s = 64 (DNA fragm ents, data n ot show n), num ber o f variables = 59 (individual sp ecim en s). 1 are sp ecim en s fem ale P. lon- gicuspis from C h efch ao u en e; 2 are sp e ci
m en s m ale P. longicuspis LCB from C hef
c h a o u e n e ; 3 a r e s p e c i m e n s m a le P.
longicuspis LCB from O u ezzan e; 4 are sp e cim ens m ale P. longicuspis LCA from C hef
ch ao u en e; 5 are sp ecim en s P. perniciosus from O u ezzan e; 6 are sp e cim en s P. p ern i
ciosus from Turre; 7 are sp ecim en s P. p e r niciosus from T orvizcó n; 8 are sp ecim en s interm ediate P.p./P.l.; 9 is a sp ecim en P. lon
gicuspis from Spain.
Spain detected a high individual variability with, gene
rally, numerous fragments with only slight differences in their molecular weights. Black IV et al., 1993, sug
gested that the RAPD-PCR technique mainly amplifies highly variable regions of the genome which explains the large variability detected in a study on several aphid species. RAPD-PCR is biased in its amplification of repetitive regions (centromeric, telomeric, hetero- chromatic genomic regions and various classes of dis
persed repetitive and mobile elements), but amplifies many unique regions as well (Black IV & DuTeau, 1997; Williams et al., 1990). Polymorphisms at regions amplified by RAPD-PCR are typically manifest as the presence or absence of a band among individuals and most amplified RAPD bands are inherited in a Men- delian fashion as dominant alleles (Black IV & DuTeau, 1997).
The results of our numerical classification closely cor
related with the classically accepted subgenus classi
fication (Lewis, 1982) and with the automatic classifica
tions made by other authors which use morphological (Rispail & Leger, 1991) and isoenzymatic data (Pesson et al., 1991). Not all the species studied showed the same degree of genetic polymorphism. The most hete
rogenous was P. longicuspis s.l., and this was still the case after the morphological types LCA and LCB had been distinguished. In contrast, the most homoge
neous species was P. sergenti with 67 conserved frag
ments.
Within the species P. p a p atasi, in spite of the marked individual variability after amplification with some pri
mers, we did not detect genetic differences between the three Spanish populations studied. However, we did observe a degree of variability between these and
2 5 2
Mémoire
Parasite, 2000, 7, 247-254Genetic polymorphism in species o f the genus Phlebotomus
Fig. 3 . — C orrespondence analysis o f P. p e rniciosus/P.
longicuspis. Plane form ed by the first tw o axes. Visible points 1, 2, 3, 4, 5, 6, 7, 8, 9, 15 (h id d en point: 17), 16, 27, 30, 31 and 35 rep resen t m ales type LCB and fem ales o f P. longicuspis. V isible points 10 (hidden points: 12, 18, 19, 20, 25, 26, 29, 34, 41, 42, 43, 51, 54, and 56), 11, 13, 14 (h id d en points: 21 and 22), 23 (h id d en points: 24, 28, 32, 3 7 and 53), 3 6 (hidd en points: 39, 40 , 44, 45, 46, 47, 48, 50, 52, 55, 57 and 59), 38 (h id d en point: 4 9 ) and 58 represent P. p er
niciosus, interm ediate P.p/P.l and LCA type.
the Italian reared population which could be due to genetic selection by the culture process. Morrison et al. (1995) obtained very similar results to ours with isoenzyme electrophoresis.
L a rrou ssiu s is a clearly separate subgenus and the genetic divergence of the species P. aria si from the rest of the Larroussius is also clearly reflected. The diffe
rences we observed between specimens of this spe
cies from Huelva (W.A.P.) and those captured in the provinces of Almería and Granada (E.A.P.) suggest the existence of two populations resulting from genetic divergence that can be at least partially attributed to the climatic differences between both regions.
The study of P. p ern ic io sus and P. longicuspis is espe
cially interesting since both are vectors of L eisbm an ia infantum , previously considered to be the same spe
cies (Collantes & Martínez Ortega, 1997), and because P.longicuspis has been observed in two completely dif
ferent populations (LCA and LCB). The results obtained by RAPD-PCR show that both hypotheses can be valid:
1) P. longicuspis from Spain, the morphologically inter
mediate specimens P.p./P.I. and the LCA population of P. longicuspis specimens from Morocco are grouped together with the specimens of P. pern iciosu s and can, therefore, be considered as synonyms (P. p e r n i
ciosus species); 2) The females and the LCB males of P. longicuspis from Morocco are separated from these and are, therefore, considered as P. longicuspis s.str.;
the factorial analysis (Fig. 3) reinforce this hypothese.
Nevertheless, it is noteworthy that in dendrogram 1 the LCB P. lon gicu spis specimens from Ouezzane are grouped together with LCA P. longicuspis from Chef- chaouene instead of with the LCB specimens from the
same location. This is an apparent contradiction difficult to irrefutably explain but that could be due to the very few LCB specimens from Ouezzanne studied (number
= 3) and the high degree of polymorphism of this spe
cies. In any case, this apparent contradiction disappears when the individual specimens are considered (den- dogram 2). In this dendrogram 2 appear five marked groups in P. longicuspis females and LCB males, which indicate great heterogeinity and some groups seem to be more related to P. p ern iciosu s group; this hetero
geneity is confirmed by correspondence analysis.
Mitochondrial introgression between P. p ern iciosu s and P. longicuspis was inferred by Esseghir et al., 1999, based on mitochondrial and EF-a gene phylo- genies of Larroussius. Also Ready & Pesson, 1999 (personal communication) provide further evidence for introgression between wild populations of these two species and for the occurrence of cryptic species.
Hybridization in not uncommon among related insect species and it is known that genetic introgression in great enough to prevent the separation into two dis
tinct lineages of the two most important vectors of malaria, A nopheles g a m b ia e s.str. and An. arabien sis (Besansky et al., 1994). The heterogeneity of the P.
longicuspis s.str. group could reflect the occurrence of these features.
ACKNOWLEDGEMENTS
W e thank Dr. G. La Rosa (Laboratorio di Para
ssitologia, Istituto Superiore di Sanità, Roma, Italy) for his advice and helpful criticism of
Parasite, 2000, 7, 247-254
Mémoire 253
MARTIN-SANCHEZ J., GRAMICCIA M., PESSON B. & MORILLAS-MARQUEZ F.
this paper. We also thank Granada University for to sup
port the grant of Dr. Martín-Sánchez, and FIS (Madrid) by the project 99/0036.
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Reçu le 29 mars 2000 Accepté le 12 juillet 2000
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