Mild-Vectolysis: A Nondestructive DNA Extraction Method for Vouchering Sand Flies and Mosquitoes.

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, tested in a verified barcode (cytochrome oxidase I gene), while preserving their morphological integrity. Application of the method to sand flies allowed successful insect identification post DNA extraction, as all basic taxonomical structures necessary for identification (pharynx, cybarium, and genitalia) remained intact. The development of the methodology was more challenging in mosquitoes, due to the fragility of key morphological characters (scales and color). A small modification of the lysis buffer concentration, in combination with the adjustment of the incubation time, a postlysis 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, preserving of the original insect specimen post DNA extraction is highly advantageous, as it allows for 1) utilization of the specimen for further analysis and 2) storage for vouchering.