Molecular Typing of Sand Fly Species (Diptera, Psychodidae, Phlebotominae) from Areas Endemic for Leishmaniasis in Ecuador by PCR-RFLP of 18S Ribosomal RNA Gene

Abstract

ABSTRACT. Surveillance of the distribution of sand fly species is important for prediction of the risk and expansion of Leishmania infection in endemic and surrounding areas. In the present study, a simple and reliable method of typing New World Lutzomyia species circulating in endemic areas in Ecuador was established by using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique. PCR-RFLP of 18S ribosomal RNA (rRNA) genes with the restriction enzyme AfaI and subsequently HinfI successfully identified seven sand fly species in nine endemic areas in Ecuador. Although intraspecific genetic-diversity affecting the RFLPpatterns was detected in a species, the patterns were species specific. The method promises to be a powerful tool for the classification of New World Lutzomyia species.

Distribution of Lutzomyia ayacuchensis, the vector of Andean-type cutaneous leishmaniasis, at different altitudes on the Andean slope of Ecuador

“…trapidoi were dominant, corresponding to our previous findings (Terayama et al, 2008). Therefore, the habitat of Lu.…”

Genotyping of sand fly species in Peruvian Andes where leishmaniasis is endemic

“…A similar observation indicating that Lu. nuneztovari from Bolivia was located in the Helcocyrtomyia clade was made on the basis of the 18S rRNA gene sequence (Terayama et al, 2008). In addition, a discrepancy in the classification of Lutzomyia species including the Verrucarum group between the morphological examination and phylogenetic analyses of 12S and 28S rRNA gene sequences was reported (Beati et al, 2004), suggesting the necessity for careful reconsideration of the classification of some Therefore, only a few steps will be required for actual field surveillance.…”

“…The amplification of 18S rRNA gene fragments was performed with sand fly 18S rRNA gene-specific primers (Lu.18S 1S: 5'-TGCCAGTAGTTATATGCTTG-3' and Lu.18S AR: 5'-CACCTACGGAAACCTTGTTAC-3') (Terayama et al, 2008;Kato et al, 2007. The PCR was carried out in a volume of 15 l with the primers (0.4 M each), Ampdirect Plus (Shimadzu Biotech, Tsukuba, Japan), and Taq polymerase (Ex Taq; Takara Bio, Shiga, Japan).…”

“…To overcome these problems, molecular markers have been explored to develop easier and accurate ways of classifying sand flies, and most results of DNA sequence analyses support the morphology-based grouping (Aransay et al, 1999;Torgerson et al, 2003;Beati et al, 2004;Kato et al, 2007Kato et al, , 2008Terayama et al, 2008;Kuwahara et al, 2009;Cohnstaedt et al, 2011). Since a DNA sequence analysis requires no special storage conditions for samples and less expertise with minimum risk of different interpretations among researchers, the usability of this method for sand fly taxonomy is now widely accepted (Kato et al, 2010a).…”

“…Recently, we developed a genotyping method to classify 7 sand fly species 4 distributed in Ecuador by using a PCR-restriction fragment length polymorphism (RFLP) analysis of 18S rRNA genes (Terayama et al, 2008).…”