Sand Fly Morphological Identification Protocol

The head, thorax and genitalia of the sand flies were cut off and placed in a drop of ethanol. Soft tissues were lysed in a bath of 10% potassium hydroxide (KOH), then bleached in a Marc-André solution. After dehydration in successive alcoholic baths, these parts of the insects were then mounted between microscope slide and cover slide in Euparal for species identification. The first abdominal tergites of each specimen were dried before any processing and stored in a vial at -20°C for later molecular analysis. The specimens were observed with an Olympus BX50 microscope coupled with a DP 26 Olympus camera allowing photographs to be taken. Measurements and counting of several characters used in systematics [8 (link)] have been realized using Stream Essentials software (Olympus, Japan). The sketches were made using the camera lucida installed on the microscope. The drawings were then finalized using Indian ink.