The colony of human body lice (Pediculus humanus humanus) were provided by Kosta Y. Mumcuoglu from the Kuvin Center for the Study of Infectious and Tropical Diseases, Hebrew University-Hadassah Medical School, Jerusalem, Israel. This laboratory-reared colony adapted to rabbit blood were fed four times per week for 20–30 min on rabbits and maintained at 30 ± 1°C and 60–70% relative humidity without exposure to any drugs [90]. Survival and immobilization were monitored by microscopical observations at 1, 2, 3 and 24 h after contact of 44 lice at different stages of the life cycle with insecticides at concentrations ranging from 1 to 100 μM or 1% DMSO on a Whatman grade 1 filter paper in a 55 mm Petri dish following the knockdown classification from KD+ to KD++++ (S1 Table), as previously described [12]. Nits (50 to 190 per condition) were incubated with lotilaner or ivermectin and hatching was monitored between six and nine days. Water and solution of 1% DMSO were used as negative controls. Pictures were recorded with a Motic Digital Microscope 143 Series and the Motic Image Plus 3.0 software.

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