Leishmania Infection Dynamics in Sand Fly

The colony of P. papatasi was maintained at 26°C on 50% sucrose and 14 h light/10 h dark photoperiod as described previously (Benkova and Volf, 2007 (link)). Sand fly females were infected by feeding through a chick-skin membrane on heat-inactivated rabbit blood containing 10⁶ promastigotes ml⁻¹. Engorged sand flies were maintained in the same conditions as the colony and dissected 2, 5, 9 and 12 days PBM. The location of Leishmania infections in the sand fly digestive tract (foregut, SV, thoracic and abdominal midgut, and endoperitrophic and ectoperitrophic space) was determined by dissection and examination by light microscopy. Parasite loads were estimated by two methods: infections seen in the gut in situ were graded according to Myskova et al. (2008 (link)) as light (< 100 parasites per gut), moderate (100–1000 parasites per gut) and heavy (> 1000 parasites per gut). Alternatively, 30–40 guts from females with late infections (10–12 days PBM) were individually dissected into NET 50 and stored in −20°C for qPCR. Sand fly infection experiments were repeated four times for combinations of wild-type (FVI), KO and Kin lines and twice for combinations of FVI, +HASPB and +SHERP lines.