Direct multiplex PCR (dmPCR) for the identification of six Phlebotomine sand fly species (Diptera: Psychodidae), including major Leishmania vectors of the Mediterranean

Authors: GIANTSIS, IANNIS - AMERICAN FARM SCHOOL; CHASKOPOULOU, ALEXANDRA - EUROPEAN BIOLOGICAL CONTROL LABORATORY (EBCL); BON, MARIE-CLAUDE - EUROPEAN BIOLOGICAL CONTROL LABORATORY (EBCL)

Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/31/2016
Publication Date: 1/6/2017
Citation: Giantsis, I., Chaskopoulou, A., Bon, M. 2017. Direct multiplex PCR (dmPCR) for the identification of six Phlebotomine sand fly species (Diptera: Psychodidae), including major Leishmania vectors of the Mediterranean. Journal of Economic Entomology. tow269. DOI: https://doi.org/10.1093/jee/tow269.

Interpretive Summary: Phlebotomine sand flies are haematophagous insects responsible for transmitting several anthroponotic and zoonotic diseases. In order to develop sand fly control strategies and disease surveillance systems, fast, accurate and reliable identification at species level is crucial. Here we developed a novel molecular methodology for the discrimination of six common Eastern Mediterranean sand fly species that are present in an endemic focus of canine leishmaniasis in Northern Greece. This methodology is fast, cost-effective and reliable, and could be very useful in routine identification in entomological, epidemiological and systematic studies of sand flies.

Technical Abstract: Sand flies (Diptera: Psychodidae, subfamily Phlebotominae) are haematophagous insects that are known to transmit several anthroponotic and zoonotic diseases. Reliable identification of sand flies at species level is crucial for their surveillance, the detection and spread of their pathogens and the implementation of targeted pest control strategies. In the current study we designed a novel, time saving, cost effective and easy-to-apply multiplex PCR assay, that avoids sequencing, for the identification of 6 Eastern Mediterranean sand fly species: Phebotomus perfiliewi, P. simici, P. tobbi, P. papatasi, Sergentomyia dentata and S. minuta. This methodology is based on species-specific single nucleotide polymorphisms of the nuclear 18S rRNA gene, using one common and six diagnostic primers. Amplification products were easily and reliably separated in agarose gel yielding 1 single and clear band of diagnostic size for each species. Further, we verified its successful application on tissue samples immersed directly to the PCR mix, skipping DNA extraction. This direct multiplex PCR can be completed in less than 3 hours including all operating procedures, and costing no more than a simple PCR. The applicability of this methodology in the detection of hybrids is an additional considerable benefit.