Isoenzymatic characterization of Phlebotomus ariasi and P. perniciosus of canine leishmaniasis foci from Eastern Pyrenean regions and comparison with other populations from Europe

An entomological survey was carried out in 2007 in two Pyrenean counties of Lleida province (north-eastern Spain), where cases of autochthonous canine leishmaniasis have been recently reported. Phlebotomus ariasi and P. perniciosus, vectors of Leishmania infantum in the Mediterranean area, were captured. The aim of the present study was to compare these phlebotomine populations with others captured in known leishmaniasis foci in Europe. Populations of these species were studied by analysing the polymorphism of seven enzymatic systems (HK, PGI, PGM, MDH, 6PGD, FUM and ACO) and compared with other specimens from endemic regions of France, Italy, Malta, Portugal and Spain captured in other campaigns, and also with previously published results. Phlebotomus ariasi was more polymorphic than P. perniciosus. Only the ACO locus had diagnostic alleles, but some other alleles show high characteristic frequencies for each species. The neighbour-joining trees separated two population groups in both species. On the basis of the isoenzyme study results, sand fly populations of the Pyrenean region in Lleida province are closely related to those of other nearby leishmaniasis endemic regions in France and Spain.


Introduction
In recent years, several studies on phlebotomine fauna have been carried out in the Mediterranean area, mainly on the vectors of Leishmania infantum in the context of the expansion or emergence of leishmaniasis [4,5,24]. In Spain, two sand fly species are proven vectors of L. infantum, Phlebotomus (Larroussius) ariasi and P. (L.) perniciosus [26,32,35]. P. (L.) ariasi is associated with cooler and more humid zones, while P. perniciosus is ubiquitous and has been found in a wide variety of climatic zones ranging from semi-arid to sub-humid [2,5,6,17,36].
Nevertheless, few studies have been published on population diversity in the new focus areas, including the Pyrenean region of Lleida province in Spain. In this region, two potential vectors with different environmental requirements have been identified, P. ariasi and P. pernicious [6]. The first is more abundant in the Catalan Pyrenees, where leishmaniasis was not considered endemic until the recent discovery of cases of autochthonous canine leishmaniasis [4,5]. During an entomological survey in 2007 in two Pyrenean counties of Lleida province (north-eastern Spain), sand flies were captured and kept in liquid nitrogen for isoenzymatic characterization of their populations. The use of isoenzymes as genetic markers is well suited to the analysis of a large number of samples. The technique can be used to identify closely related species as well as those nonidentifiable via morphological criteria alone [13]. The aim of the present study was to compare these populations with others in the neighbouring leishmaniasis foci in France and other European populations. Some of P. perniciosus specimens had been studied for three enzymatic systems beforehand (PGI, PGM, HK). In this work, isoelectrofocusing was used to analyse the polymorphism of seven enzymatic systems previously shown to be polymorphic within species of the subgenus Larroussius [7,10,11,29], and a comparison was made with other nearby endemic regions and previously published results [8,10,28,30].

Material and Methods
Sand fly collection Population samples were collected from different Mediterranean countries in distinct periods of time (from 1989 to 2009), specifically in France, Italy, Malta, Portugal and Spain, including those in the two Pyrenean counties of Lleida province. The geographical locations with details of the associated code, year of capture, latitude, longitude and altitude are shown in Table 1. A total of 13 populations of P. perniciosus (6 from France, 1 from Italy, 1 from Malta, 4 from Spain and 1 from Portugal), and 21 of P. ariasi (7 from France, 13 from Spain and 1 from Portugal) were studied. Sand flies were captured using miniature CDC light traps (Centers for Disease Control) [1] set in different locations near farms, houses and kennels. The traps were set for one night at each site at sunset, and left in operation all night (from 6 p.m. to 6 a.m.). The CDC light traps were recovered and grouped according to sampling site and date of capture. Sand flies were immediately cryopreserved in liquid nitrogen (À196°C) until the analysis [10,29].

Enzyme analysis
Sand flies were removed from liquid nitrogen, the last abdominal segments were separated for morphological identification by the keys of Gállego et al. (1992) [16] after clearing in Marc-André solution. The rest of the thorax and abdomen were homogenized in 50 mL of distilled water for the isoenzymatic study of the protein extract [11,29].
Isoelectrofocusing was carried out in ultrathin agarose gels with the ampholytes at pH 4.6-5 and 3-10, according to the protocols previously described [7,10]. We studied eight loci of seven polymorphic enzymes in sand flies of the  [10,11,30].
Allele frequencies, tests for deviation from Hardy-Weinberg equilibrium at each locus in each population, and Nei's genetic distance were calculated using Biosys-2 [38]. PHYLIP version 3.6a2 [14] was used for neighbourjoining phenetic analysis and to calculate bootstrap by majority-rule consensus tree. GENEPOP [34] was used to test genotypic differentiation and to estimate Fst values between each pair of populations. FSTAT v. 2.9.3 [18] was used to test Fst pairwise significance after Bonferroni correction.

Results
Allele frequencies at eight polymorphic loci are reported separately for P. perniciosus and P. ariasi (Supplementary Documents 1 and 2). The two species were found to share common alleles. The most polymorphic loci were HK, PGI, PGM and 6PGD, and P. ariasi was found to be more polymorphic than P. perniciosus. Most of the populations analysed were in Hardy-Weinberg equilibrium, except two out of 13 populations at one locus for P. perniciosus (MDH-1), and five out of 21 populations of P. ariasi at two loci (PGM and 6-PGD).
Nei's genetic distances and Fst values between pairs of populations of P. perniciosus and P. ariasi are given in Tables 2 and 3 , respectively. Nei's genetic distances calculated for the 13 populations of P. perniciosus were very low, between 0.000-0.085 and the neighbour-joining tree separated the next groups: one group formed by all the populations of France, Portugal and Spain and other group constituted by Malta and Italy populations, showing a bootstrap value of 60% ( Figure 1). The genotypic differentiation for each population pair was highly significant for these two populations. Fst values seemed to confirm the results of the phenetic analysis and point to an intermediary status of P. perniciosus populations in France.
Nei's genetic distances for P. ariasi were between 0.001-0.159 and the neighbour-joining tree ( Figure 2) separated the western populations (Cheires in Portugal and Río Tinto in Spain), with a bootstrap value of 100%, from the rest of Spain and France, where we found bootstrap values >60%. The genotypic differentiation for each population pair was highly significant for Cheires and Río Tinto populations. As for P. perniciosus, Fst values seemed to confirm the results of the phenetic analysis.

Population
Geographical coordinates

Code
Year of Capture (July-August)

Discussion
Recently, several studies on phlebotomine fauna have been carried out in Europe regarding the trends for dispersion of leishmaniasis disease [4,5,24]. In the new focus areas, few studies have been published on population diversity. However, studying population diversity more closely in these areas is crucial to detect possible emergence and expansion of the disease. One of these recently discovered foci was the Pyrenean region of Lleida province in Spain, where two potential vectors have been identified, P. ariasi and P. pernicious [6]. In field work in Lleida, both species were captured at two different times at the same sampling sites, Bastida and Guardia, on cattle and pig farms, respectively, showing its syntopy. In these cases, both species occur sympatrically living in the same geographical area and sharing the possible role of vector species of leishmaniasis [6,35].
In this study, eight gene loci were examined in sand flies from Eastern Pyrenean areas, as in a recent publication [10]. Although three of the enzymes were known to be sufficiently polymorphic for studying populations of P. perniciosus (HK, PGI and PGM) [28], there was no previous data available on P. ariasi. In our study, all the loci were polymorphic for P. ariasi of Lleida, particularly PGI, PGM, ACO and 6-PGD. In the case of the two populations of P. perniciosus from Lleida, the most polymorphic were MDH-1 and 6-PGD, whilst HK, FUM, ACO and MDH-2 were monomorphic. The isoenzymatic analyses showed possible diagnostic alleles between the two species only on the ACO locus. ACO 1 was revealed only in P. perniciosus. This allele cannot be considered a fixed allele in P. perniciosus since one heterozygotic specimen was found in Morocco [10]. Some alleles showed elevated frequencies characteristic for each species: PGI 3, PGM 7, FUM 4 and 6-PGD 2 for P. ariasi and PGM 2, FUM 1 and 6-PGD 1 for P. perniciosus.
Most of the populations analysed were in Hardy-Weinberg equilibrium, except two out of 13 populations at one locus for P. perniciosus (MDH-1), and four out of 21 populations of P. ariasi at two loci (PGM and 6-PGD). This disequilibrium could be due to the presence of rare alleles in several individuals with a lack of heterozygotes with the most common allele (MDH-1 for P. perniciosus and 6-PGD for P. ariasi). In the enzymatic analysis of P. perniciosus, only 13 populations were studied because this species is poorly represented in the Pyrenean region (two populations in Lleida and two in France), where high altitudes correlate negatively with its presence [6]. P. perniciosus showed low genetic variability. All of the alleles identified in Lleida were found in the other populations, as well as being reported in previous studies [28,29]. The Western populations (Portugal and Huelva) were differentiated by the frequency of allele 2 of the PGI locus, as already observed in other localities of Andalusia and Portugal [7,28,30]. PGM is a polymorphic enzyme that does not structure populations, even on a European scale [28]. In the HK locus, allele 2 was a rare allele of three populations (Italy, Malta and Portugal) and was absent from the other ten populations of Spain and France. However, this allele allowed two groups of P. perniciosus to be differentiated, one from North Africa, Malta and Italy, and the other from France and the Iberian Peninsula [28,30]. These results conflict with molecular studies that group populations of France with others of Italy and Malta, suggesting the hypothesis of introgression in France of two lineages from Mediterranean refuges (southern Italy and Spain) during their postglacial dispersal [28]. Our Fst values, like the recent findings Table 2. Below diagonal: Fst values between each pair of populations were estimated using GENEPOP [34]. Above diagonal: Nei (1972) [27] genetic distance. Values calculated from the isoenzyme data at the seven polymorphic loci for the 13 populations of P. perniciosus. Population codes are given in Table 1.  Table 3.
Below diagonal: Fst values between each pair of populations were estimated using GENEPOP [34]. Above diagonal: Nei (1972) [27] genetic distance. Values calculated from the isoenzyme data at the seven polymorphic loci for the 21 populations of P. ariasi.
Population codes are given in Table 1. with approaches using matrix-assisted laser desorption/ ionisation (MALDI-TOF), support an intermediary status of populations in France [23]. The enzymatic analysis of P. ariasi involved 21 populations, as this species is well represented in the Pyrenean region (10 populations in Lleida, 1 population in Girona and 4 in France), and correlates positively with increasing altitude [6]. The genetic variability of P. ariasi is greater than P. perniciosus in the same distribution area [30]. The enzymatic analysis revealed a geographical structuration in two groups: Huelva in south-western Spain and Portugal, supported by allele frequencies of PGI 1, No data are available on the vectorial role of P. perniciosus in Lleida, but this species has been found naturally infected by L. infantum in Tarragona and other  regions in Spain, as well as in France, Portugal, Italy and Malta [9,12,19,20,21,25,31,33]. Our results, which include the analysis of a greater number of enzymatic systems, are consistent with those obtained previously with three of these enzymes grouping populations of the Iberian Peninsula and France. In contrast with P. perniciosus, more molecular than enzymatic data are available for P. ariasi [15,22], and at least four haplogroups have been identified in the European area of distribution. The role of southern Spain as a refuge for P. ariasi during the last glaciation has been confirmed [14]. The hypothesis of a complementary refugial area in the northern Pyrenees [22] is supported by our enzymatic analysis, which revealed three rare alleles (PGM 5, HK 8 and 6-PGD 3) only in the Ariège population (Aston) and one (PGM 5) in the Pyrénées-Orientales populations (Vira) and not present in the southern Pyrenean populations in Lleida. As seen before in previous studies, enzymatic and molecular analyses of P. perniciosus have yielded differing results. Thus, a combination of different techniques would be recommendable for future studies on the characterization of P. ariasi in the Lleida populations. The grouping of the Lleida Pyrenean populations with those analysed from the French Pyrenees (Pyrénées-Orientales and Ariège) and from Girona and Tarragona suggests P. ariasi could act as a vector on either side of the mountain range in Spain and France. In fact, the vectorial role of P. ariasi in France has been proven [37], while in Lleida only one study has been carried out in a Pyrenean location, where some specimens yielded positive PCR results [3].

Conclusions
The study of eight loci of seven polymorphic enzymes enabled us to determine that sand fly populations of the Pyrenean region in Lleida province (north-eastern Spain) and of neighbouring Eastern Pyrenees areas are closely related to those of nearby leishmaniasis endemic regions in the other European foci studied.

Supplementary Material
Tab. S1. Allelic frequencies at the eight polymorphic loci characterized in 13 populations of P. perniciosus. Population codes are given in Table 1. Tab. S2. Allelic frequencies at the eight polymorphic loci characterized in 21 populations of P. ariasi. Population codes are given in Table 1.