Sand Fly Sampling and Identification

Sand flies were collected starting on SD 0 in both locations. Follow-up collections in Heraklion (Crete) were performed between SDs 78 and 80, SDs 134 and 136, SDs 318 and 320, SDs 409 and 411, SDs 533 and SD 534 and SD 661 and 663. Follow-up collections in Leros were performed between SDs 77 and 80, SDs 144 and 146, SDs 338 and 340, SDs 424 and 426, SDs 539 and 542 and SDs 675 and 678. The US Center for Disease Control (CDC) miniature light traps and mechanical aspirators were used for all collections. The light traps were placed close to animal-inhabited biotopes. The traps operated overnight (for 2–4 consecutive nights each time), and the sand flies collected were kept either dry or in 70% ethanol until examination.
All captured sand flies were counted. For species identification, the head and the terminal part of the abdomen of all collected female sand flies were mounted on permanent microscopy slides and subsequently identified using the appropriate keys based on the morphology of the pharynx and the genitalia armature and documented [11 , 12 ]. Upon species identification, female sand flies of the same species and collected at the same time point from the same trap were homogenised in groups of five and kept frozen at − 20 °C until further molecular analysis.