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Title: Mild-Vectolysis: A nondestructive DNA extraction method for vouchering sand flies and mosquitoes

item GIANTSIS, IOANNIS - American Farm School
item CHASKOPOULOU, ALEXANDRA - European Biological Control Laboratory (EBCL)
item BON, MARIE-CLAUDE - European Biological Control Laboratory (EBCL)

Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2015
Publication Date: 5/1/2016
Citation: Giantsis, I., Chaskopoulou, A., Bon, M. 2016. Mild-Vectolysis: A nondestructive DNA extraction method for vouchering sand flies and mosquitoes. Journal of Medical Entomology. 53:3, 692-695.

Interpretive Summary: Mosquitoes undoubtedly constitute one of the world’s most serious nuisance and disease related pests that are responsible for the suffering of more than 500 million people per year, with ~ 1 million deaths annually. Mosquitoes are responsible for the transmission of many medically important pathogens and parasites such as bacteria, protozoans, nematodes and viruses that can cause serious diseases such as malaria, dengue, yellow and Chikungunya fever, encephalitis or filariasis. Sand fly species of the genera Phlebotomus (Old World) and Lutzomyia (New World) are responsible for the transmission of Leishmania parasitic protozoans. Even though leishmaniasis is not common in the United States (occasional cases have been acquired in Oklahoma and Texas), the disease constitutes a major threat to the U.S. military during their overseas operations. Not all mosquito and sand fly species are competent disease vectors and species differ in their behavior and ecology. Therefore, proper identification of species through classical and molecular techniques is very important. Classical taxonomic methods for vector identification are time consuming, require specialized expertise, and sometimes do not provide conclusive results. It is therefore important to design novel, efficient and cost-effective molecular techniques that will provide reliable information for vector taxonomy. However, most molecular genetic techniques destroy the specimen. Here we developed a nondestructive DNA extraction methodology for mosquitoes and sand flies, called Mild-Vectolysis, which provides sufficient DNA for genetic bar coding and preserves the insect's body for morphological study and for retaining as a museum voucher specimen.

Technical Abstract: Nondestructive techniques allow the isolation of genomic DNA, without damaging the morphological features of the specimens. Though such techniques are available for numerous insect groups, they have not been applied to any member of the medically important families of mosquitoes (Diptera: Culicidae) and Phlebotomine sand flies (Diptera: Psychodidae). This study presents Mild-Vectolysis, the first nondestructive DNA extraction methodology for vouchering taxa of mosquitoes and sand flies, which provided sufficient amounts of DNA to produce a verifiable barcode (cytochrome oxidase I gene), while preserving their morphological integrity. Application of the method to sand flies, allowed successful identification of the species based on morphological characters after DNA extraction, since all basic taxonomical structures necessary for identification (pharynx, cybarium, genitalia) remained intact. The development of the methodology was more challenging for mosquitoes due to the fragility of key morphological characters (scales, color). A small modification of the lysis buffer concentration, in combination with the adjustment of the incubation time, a post-lysis freezing stage and the avoidance of ethanol, achieved the extraction of sufficient DNA quantity, while preserving the integument of the mosquitoes, although a small proportion of the scales and the color still appeared to have been lost. In addition to the practicality and efficiency of our methodology, preservation of the original insect specimen post DNA extraction is highly advantageous as it allows for a) utilization of the specimen for further analysis and b) storage for vouchering.