Parasitol Res (2004) 94: 416–420 DOI 10.1007/s00436-004-1231-4
O R I GI N A L P A P E R
Ana M. Aransay Æ Johann M. Testa Francisco Morillas-Marquez Æ Javier Lucientes Paul D. Ready
Distribution of sandfly species in relation to canine leishmaniasis from the Ebro Valley to Valencia, northeastern Spain Received: 29 August 2004 / Accepted: 7 September 2004 / Published online: 14 October 2004 Springer-Verlag 2004
Abstract In Spain, only two of the 12 recorded species of sandflies, Phlebotomus (Larroussius) ariasi Tonnoir and P. (L.) perniciosus Newstead, are proven vectors of Leishmania infantum Nicolle, the causative agent of endemic leishmaniasis. Studies of the distributions of phlebotomine sandflies are important for evaluating the possible effects of climate warming on any northward or altitudinal range shifts of leishmaniasis or the other diseases they transmit. We describe a recent sandfly survey in Spain, starting in the northern Ebro Valley and continuing southeast into the Levante region of the Mediterranean coast. Sandflies (P. ariasi only) were found for the first time in the northern province of Alava, in the upper Ebro Valley, where cases of canine leishmaniasis have been described during the last decade. Throughout the provinces sampled, P. ariasi predominated over P. perniciosus in cooler bioclimatic zones, and this statistically significant pattern was more marked than that with higher altitudes.
Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s00436-004-1231-4 A. M. Aransay Æ J. M. Testa Æ P. D. Ready (&) Molecular Systematics Laboratory, Entomology Department, Natural History Museum, Cromwell Road, London, SW7 5BD, UK E-mail:
[email protected] Fax: +44-20-79425229 A. M. Aransay Servicio de Parasitologı´ a, Instituto de Salud Carlos III, Centro Nacional de Microbiologı´ a, Ctra. Majadahonda-Pozuelo Km.2, 28220 Majadahonda, Madrid, Spain F. Morillas-Marquez Departamento de Parasitologı´ a, Facultad de Farmacia, Universidad de Granada, Granada, Spain J. Lucientes Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain
Introduction Arias Encobet (1911) first reported the occurrence of phlebotomine sandflies (Diptera: Psychodidae, Phlebotominae) in Spain. Later, Pittaluga and De Buen (1917) related these flies to leishmaniasis in Spain. Since then, many studies have described the distributions of sandfly species in regions of Spain where human and canine leishmaniasis have been found (Najera-Angulo 1943; Houin 1965; Morillas-Marquez et al. 1983, 1991; Gil Collado et al. 1989; Lucientes-Curdi et al. 1991; Gallego Berenguer et al. 1992; Arnedo-Pena et al. 1994). Only two sandfly species out of the 12 identified in Spain, Phlebotomus (Larroussius) perniciosus Newstead, 1911 and P. (L.) ariasi Tonnoir, 1921, are proven vectors of Leishmania infantum Nicolle, 1908, which is the causative agent of most, if not all, autochthonous cases of leishmaniasis (Rioux et al. 1986; Lucientes-Curdi et al. 1988; Martin-Sanchez et al. 1996). More systematic surveys of sandfly distributions are required for Spain, partly for the following reasons. Earlier distribution records probably contain errors, because of misidentifications made before detailed morphological characters were described (Martı´ nezOrtega and Conesa-Gallego 1987), especially for species of the subgenus Larroussius that are morphologically similar. Furthermore, knowledge of sandfly distributions could be affected by the variety of capture methods used and the seasons when collections were made (LucientesCurdi et al. 1991). The present report is an update on the sandfly species collected and identified in and between the Ebro Valley and the Levante region of northern and eastern Spain, respectively, based on new collections during the summers of 2000 and 2001 and a literature review. We describe for the first time the presence of sandflies in the province of Alava (upper Ebro Valley), where cases of canine leishmaniasis were reported during the last decade (Sa´ez de Santamarı´ a et al. 1997), and relate the distributions of P. ariasi and P. perniciosus to bioclimate and altitude.
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Materials and methods Study area The collections were carried out in the provinces of Alava and La Rioja, in July and August 2000, and Zaragoza, Tarragona, Castello´n and Valencia in June and July 2001. The 38 collecting locations are listed and described in Table X (Electronic Supplementary Material). Sample collection Sandflies were mostly captured overnight in CDC (Centers for Disease Control, Atlanta, USA) miniature light-traps (Sudia and Chamberland 1962) placed peridomestically, inside or near stables and houses. Traps were placed on two consecutive nights in some of the locations, because adverse weather conditions or any failure of the CDC traps could bias results. On a few occasions [Table X (Electronic Supplementary Material)], captures were made with manual aspirators, castor oil impregnated A4 papers (sticky traps) placed for at least 3 days in holes of walls surrounding stables, or funnel traps—also known as exit–entrance traps (Yuval and Schlein 1986)—placed upside-down in rabbit burrows. Specimens were stored in 96% ethanol at 20C, after killing by freezing.
Fig. 1 Proportion of Phlebotomus ariasi (light grey) and P. perniciosus (dark grey) collected at each location [see Table X (Electronic Supplementary Material) for codes], in relation to altitude (meters above sea level; graph with black diamond data-points) and bioclimatic zone (a grey bar at the bottom: T temperate, SM supra-Mediterranean, MM meso-Mediterranean, TM thermo-Mediterranean)
Mounting and identification of sandflies Sandflies were washed for a few minutes, once in 5% detergent solution and twice in distilled water, before being dissected: each thorax and attached anterior abdomen was stored at 70C in individual cryotubes for subsequent molecular studies; the head and genitalia of the same individual were slide-mounted in Berlese fluid (ASCO Laboratories, Manchester, UK) for morphological identification following the characters described by Martı´ nez-Ortega and Conesa-Gallego (1987). Voucher specimens were placed in the phlebotomine collection of the Department of Entomology, Natural History Museum, London.
Results Geographical distributions of all sandfly species A total of 1,643 phlebotomine sandflies was collected and morphologically identified as follows: 1,003 (61.05%) P. (L.) perniciosus, 556 (33.84%) P. (L.) ariasi, 42 (2.56%) P. (Phlebotomus) papatasi (Scopoli, 1786), 12 (0.73%) P. (Paraphlebotomus) sergenti Parrot, 1917, one (0.06%) P. (L.) langeroni Nitzulescu, 1930 and 29 (1.77%) Sergentomyia minuta (Rondani, 1843). The distribution of sandflies per location is given in Table X (Electronic Supplementary Material), and the relative frequencies of P. perniciosus and P. ariasi per location are also summarized in Fig. 1. The results are described from the northwest to the southeast, descending the Ebro Valley to the Mediterranean coastal provinces. For the first time, sandflies were collected in Alava province, up to Sojo (435¢N, 37¢W) near the boundary of Vizcaya province in the Basque country. In Alava, at the northwestern limit of the Ebro Valley, all specimens were identified as P. ariasi. A further 29 km south, in Cuzcurrita de Rio Tiron (La Rioja province),
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P. perniciosus was also collected. Continuing southeast in the Ebro Valley, P. papatasi (one male) and P. sergenti (four males, one female) were collected in the most southeastern village sampled in La Rioja province, Igea (RIO-Ige/01, 02/03 August 2000). These species had not been reported in this province previously, although they had been captured in Zaragoza province (Lucientes 1986) close to the mentioned locality. In La Rioja province, S. minuta was only found in RIO-Rob/01 (four males, 12 females). In Zaragoza province, further down the Ebro Valley, all of the species previously reported (Gallego Berenguer et al. 1992) except P. ariasi were found in different localities: P. perniciosus as stated in Table X (Electronic Supplementary Material); P. papatasi in ZAR-ElB/01 (one male, one female), ZAR-ElB/02 (two males, nine females), ZAR-Ate/01 (three males, 12 females); P. sergenti in ZAR-ElB/02 (1 male); and S. minuta in ZAR-Ate/01 (four males, two females). A single male, identified as P. langeroni, was collected in a rabbit burrow in ZAR-ElB/02. This species had previously been reported in this region by Lucientes et al. (1995). In Castellon province, to the south of the Mediterranean delta of the River Ebro, we collected P. perniciosus and P. ariasi as reported in Table X (Electronic Supplementary Material), P. papatasi in CAS-ElM (four males, nine females), P. sergenti in CAS-Ara (three males, three females) and S. minuta in CAS-Ara (three males, two females). In Valencia province, further south on the Mediterranean coast, P. perniciosus was the only species captured (Table X, Electronic Supplementary Material), except for two females of S. minuta found in VAL-LaC. No sandflies were collected in the localities close to sea level [San Carlos de la Rapita in Tarragona province (between Zaragoza and Castellon provinces), Oropesa in Castellon province and Meliana in Valencia province]. This result was independent of the environments of the locations (outside a house at the foot of a mountain, a horse-riding school close to the sea shore, and a garden in a residential area, respectively).
Table 1 Contingency table for three categories of the relative abundance of Phlebotomus ariasi (as % of the total number of P. ariasi and P. perniciosus) in three bioclimatic zones. Values are the observed number of localities characterized by each pair of categories and, in brackets, the expected values based on the total ratios. M Mediterranean bioclimatic zone. v2=33.0313,df=4, P500 m.a.s.l. [Table X (Electronic and Supplementary Material), Fig. 1]; and, its predominance was more marked in the supra-Mediterranean bioclimatic zone (Table 1) than it was at higher altitudes (Table 2). The same number of light traps were set in all locations, so collection effort does not explain the observations that only P. ariasi occurred at the northwestern end of the Ebro valley and that this species was not found at altitudes of
