title: DNA sequence analysis suggests that cytb-nd1 PCR-RFLP may not be applicable to sandfly species identification throughout the Mediterranean region.
abstract: Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification.
authors.affiliation: Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, World Health Organization Collaborating Center for Leishmaniasis, Ctra. Majadahonda-Pozuelo Km2, Majadahonda, 28220, Madrid, Spain. pamellanes@isciii.es.
authors.forename: Ivonne Pamela
authors.initials: IP
authors.lastname: Llanes-Acevedo
authors.affiliation: Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, World Health Organization Collaborating Center for Leishmaniasis, Ctra. Majadahonda-Pozuelo Km2, Majadahonda, 28220, Madrid, Spain. c.arcones@isciii.es.
authors.forename: Carolina
authors.initials: C
authors.lastname: Arcones
authors.affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain. r.galvez@ucm.es.
authors.forename: Rosa
authors.initials: R
authors.lastname: Gálvez
authors.affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain. oihane.martin.s@gmail.com.
authors.forename: Oihane
authors.initials: O
authors.lastname: Martin
authors.affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain. rocichec@ucm.es.
authors.forename: Rocío
authors.initials: R
authors.lastname: Checa
authors.affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain. amontoya@ucm.es.
authors.forename: Ana
authors.initials: A
authors.lastname: Montoya
authors.affiliation: Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, World Health Organization Collaborating Center for Leishmaniasis, Ctra. Majadahonda-Pozuelo Km2, Majadahonda, 28220, Madrid, Spain. cchichar@isciii.es.
authors.forename: Carmen
authors.initials: C
authors.lastname: Chicharro
authors.affiliation: Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, World Health Organization Collaborating Center for Leishmaniasis, Ctra. Majadahonda-Pozuelo Km2, Majadahonda, 28220, Madrid, Spain. susi_11@hotmail.com.
authors.forename: Susana
authors.initials: S
authors.lastname: Cruz
authors.affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain. gmiro@ucm.es.
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Miró
authors.affiliation: Neglected Tropical Diseases Programme, Foundation for Innovative New Diagnostics-FIND, Chemin des Mines 9, Campus Biotech, 1202, Geneva, Switzerland. isra.cruz@finddx.org.
authors.forename: Israel
authors.initials: I
authors.lastname: Cruz
mesh_terms: D000818:Animals; D001483:Base Sequence; D002980:Climate; D045303:Cytochromes b / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D030561:Databases, Nucleic Acid; D042967:Electron Transport Complex I / Q000235:genetics*; D005260:Female; D008297:Male; D019083:Mediterranean Region; D008877:Middle East; D010691:Phlebotomus / Q000145:classification / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011576:Psychodidae / Q000145:classification* / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013030:Spain
chemical_list: D004247:DNA; D045303:Cytochromes b; D042967:Electron Transport Complex I
keywords: Mediterranean phlebotomine sandflies; Mitochondrial DNA; PCR-RFLP; cytb