New location for Bulinus truncatus (Audouin, 1827) (Gastropoda: Planorbidae) intermediate host of Schistosoma haematobium and its distribution in the Iberian Peninsula

Elías D. Dana¹*, Juan García-de-Lomas², José L. Juan Bañón³, Encarnación Esteban³, María A. A. Grácio⁴, Emilio González-Miras⁵, Francisco Rodríguez-Luque⁶ & Guillermo Ceballos⁷

¹Grupo de Investigación, Transferencia I+D en Recursos Naturales, Universidad de Almería (Almería, España)

²Grupo de Investigación Estructura y Dinámica de Ecosistemas Acuáticos, Universidad de Cádiz (Cádiz, España)

³Instituto Valenciano de Microbiología. Ctra. de Bétera a San Antonio Km. 0,3-46117 Bétera (Valencia, España)

⁴Unidade de Parasitologia Médica e Unidade de Microbiologia e Parasitologia Médicas (UPMM). Instituto de Higiene e Medicina Tropical-Universidade Nova de Lisboa, Rua da Junqueira N100-1349-008 (Lisboa, Portugal)

⁵Sociedad para el Estudio y Recuperación de la Biodiversidad Almeriense (SERBAL), C/Baqueira Beret nº 2, 04720 Aguadulce (Almería, España)

⁶Asociación Naturalista Almeriense. C/ Murcia 84, 04004 (Almería, España)

⁷Servicio de Geo y Biodiversidad. Consejería de Medio Ambiente y Ordenación del Territorio, Av. Manuel Siurot 50, 41071 (Sevilla, España)

*Autor para correspondencia/Corresponding author: edana@ual.es

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The genus Bulinus O.F. Müller, 1781 (Mollusca, Gastropoda, Planorbidae) comprises a group of species mainly distributed throughout warm regions. At least 35 species of Bulinus have been described subdivided between Physopsis and Bulinus s.s. -the latter including the old Phyrgophysa genus (Mandahl-Barth, 1965). Within the genus Bulinus Brown (1994) recognizes four species groups; a group of taxa related to B. africanus, another group related to B. forskalii, a third group with taxa closely related to B. reticulatus and finally a complex of forms which includes B. truncatus and B. tropicus. As in other hololimnic species, this group shows wide genetic variation between populations in great contrast with the greatly reduced variation at the morphological level (Jones et al., 2001; Stothard et al., 1997). This fact causes phylogenetic relationship problems, and thus the apparition of lineages of reduced geographical distribution and frequent synonyms (Hubendick, 1954, 1962; Brown, 1994; Nalugwa et al., 2010).

Bulinus truncatus (Audouin, 1827) [Syn.: Physa truncata (Auduoin, 1827), Bulinus contortus (Michaud, 1829)] can be found dispersedly and irregularly throughout Africa, islands and coastal areas of the Mediterranean basin and the Irano-Turanian region (Grácio, 1981; Rolán et al., 1987; Brown, 1994). It is the principal host of Schistosoma haematobium, which causes urinary schistosomiasis or bilharzia, thus its worldwide distribution has aroused great interest generating most of the international scientific literature about the species ecology and distribution (Mandahl-Barth, 1965). Bilharzia is the most important human disease caused by parasitic worms, with around 200 million infected (accumulating all variations of the illness) and up to 250.000 deaths a year. It is the second cause of morbidity and mortality worldwide, after malaria, and according to the World Health Organization, around 12% of the human population is infected (Santos et al., 2004).

In this paper, the geographical distribution of the species in the Iberian Peninsula is shown and a new location is reported.

In march 2014 several hundred individuals of the genus Bulinus were found in an artificial pond –formed by excavation– in El Ejido, Almería (UTM ED50 30 S, X=518335 E, Y=4068085, 90×50 m², Fig. 1). Original material was deposited at the Museo Nacional de Ciencias Naturales (MNCN 15.05/72320). Ten specimens (n=10) were frozen to be analysed by molecular techniques (PCR). The use of these techniques is of maximum interest in the genus Bulinus, as identification based on morphological characters can induce confusion with other species both native and introduced (in the study site, a few specimens of Physa sp. were visually detected). This is especially relevant in this group as different species are specifically related to the life cycle of human or cattle parasites (López-Soriano, 2004; Andree & López, 2013).

Fifty 50 mg of tissue from each specimen were homogenized using a sterilized crystal homogenizer. DNA was extracted following the automatic method provided in the Maxwell 16 Viral Total Nucleic Acid Purification Kit (Promega Ibérica) and the solution was adjusted to 50 ng/μl. The amplification reaction was carried out over a total reaction volume of 50 μl containing 2 μg of DNA, 10 mM Tris HCL, 50 mM KCL, 0.1% Triton X-1’’, 2 mM MgCl₂, 0.5 mM for each dNTPs (Promega, Madison), 4 U Taq Polymerase (GenScript Taq, Bionova, Pisgataway, USA) and 0.5 μM for each primer (in both directions). The primer sequence was used to amplify the Mitochondrial cytochrome oxydase subunit I (Nalugwa et al., 2010). The amplification was performed as follows; initial phase 4 min at 95 ºC, followed by 40 cycles of 30 s at 94 ºC, 30 s at 50 ºC, 30 s at 72 ºC and final extension step with 7 min at 72 ºC, using a Sensoquest Labcycler termocycler. Amplicons were detected running an agarose 3% gel electrophoresis. PCR products were purified using the Purelink column (Invitrogen, Life Technologies, Löhne), following the instructions provided by the manufacturer. Amplified DNA sequences were obtained using the direct Big Dye sequencing method (Applied Biosystems, Life Technologies, Austin) on a genetic analyzer ABI PRISM 310 DNA sequencer (Applied Biosystems, Life Technologies) following the manufacturer’s recommendations and normal procedures. All samples were sequenced in both directions. Sequence similarity was compared with other gastropod sequences registered in the GenBank of the National Centre for Biotechnology Information and was determined using BLAST (Basic Local Aligned Search Tool) nucleotide. Material used for analyses and DNA extracted are kept frozen at −80 ºC and preserved at the Instituto Valenciano de Microbiología.

The analyses confirm the identification of the specimens as B. truncatus, with a 100% homology (sequence cover=100%, E. value=0.0, accession numbers of 100% ident: KJ135307.1, AM921807.1, AM286314.2). Hence, this is the southeastern most population detected in Spain, under the most arid climate conditions recorded in Europe: 260 mm mean annual precipitations and 17.8 ºC as mean temperature (Bayo, 2005, Fig. 2). These conditions are very similar to those populations studied by Yacoubi et al. (2007). Known distribution (Fig. 2) suggest a broader range of the species in the Iberian Peninsula. Nevertheless, it seems to be considered a rare species in the Iberian Peninsula as compared to the occurrence of other freshwater Planorbidae. A study of six species undertaken by Grácio (1983) in 62 different localities of the Portuguese Algarve only collected one site with the presence of Bulinus truncatus. There is a possibility that the species is in regression in some areas due to the alteration and destruction of its habitats and to the competitive displacement by species such as Physa acuta (Nobre, 1941; Grácio, 1983).

There is no information available about the current presence and life cycle of the associated parasite (Schistosoma haematobium) in the Iberian Peninsula, de Azevedo & Xavier (1966) considered likely that the life cycle of the parasite was being completed naturally in different sites of Portugal, where the authoctonous cases of schistosomiasis have occurred in the Iberian Peninsula. The latest case described (Grácio, 1981) occurred in the Algarve region and had Planorbarius metidjensis as intermediate host (possibility the Portuguese strain of S. haematobium had its origin in North Africa). These works conducted in Portugal have highlighted the role of Bulinus truncatus as potential intermediate host of S. haematobium. Recently, the possibility of a resurgence of the parasitic disease has emerged after the confirmation of several cases of transmission in Corsica (Berry et al., 2014; Holtfreter et al., 2014). This suggests the completion of the life cycle in the wild and the role of Bulinus truncatus in the transmission also for European countries. In Spain it has been considered an imported infection (mainly by immigrants, army personnel and travellers who have been in the endemic areas, Sánchez-Molina et al., 2010).

Due to the lack of specific studies, the presence of the parasitic worm in the natural environment cannot be ruled out, which confers a greater interest in knowing the distribution of this group of snails in areas not considered as schistosomiasis endemic regions. So far, available studies were focused on the description of patients -immigrants from African countries or travellers who had been in the areas where the parasite is endemic-, who showed the symptoms of infection (Sánchez-Molina et al., 2010). Some authors (Díaz & Florencio, 2001; Casimiro et al., 2006) suggest that in the actual scenario of global change, the rise in infected vectors increases the risk of transmission of the disease in areas where the adequate hosts exists is of greater importance than any rise in temperatures (Casimiro et al., 2006). This trend has been observed in the study area, where the prevalence of the disease is growing (more than 200 cases of vesicle schistosomiasis, Parrilla-Ruiz et al., 2004; Salas-Coronas et al., 2013). According to estimations of these authors only a small fraction of affected patients is being detected, thus the presence of the parasitic diseases and the risk of completion of the life cycle seem to be considerable. This fact recommends a better knowledge of the distribution of Bulinus truncatus in the Iberian peninsula.

This study is an example of the utility and social impact of increasing our knowledge on water bodies and their associated biota.

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This study was partially financed by the Consejería de Medio Ambiente y Ordenación del Territorio (Junta de Andalucía). We thank Dr. Rafael Araujo, curator of the malacological collection at the MNCN-CSIC for his help. We also thank the valuable comments made by anonymous referees, which allowed us to improve the original manuscript.

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