DNA Extractions
We extracted louse DNA using the MasterPureTM Complete
DNA and RNA Extraction Kit (Lucigen, Middleton, WI, USA). First, each
louse specimen was prepared for DNA extraction by making a small
incision on their abdomen under a dissecting microscope. Each specimen
was then incubated overnight (<24hrs) at
520C in a solution of Tissue and Cell Lysis Solution
mixed with 1ul of 20ug/ul Proteinase K. Occasional vortexing of the
tubes facilitated cell lysis by allowing the solution to move into the
abdominal incision. After the incubation period, the exoskeletons of the
lice were removed from the tubes and stored in 70% ethanol for future
morphological analyses. We followed the manufacturer’s protocol to
purify the remaining digestion buffer. Using QUBIT dsDNA HS Assay Kit
(Thermo Fisher Scientific), we quantified DNA concentrations. As a
quality control step, to confirm the extraction of louse DNA, we ran a
polymerase chain reaction (PCR) using published louse cytochrome B
primers (Raoult et al., 2008) and PCR products were run on a 2% agarose
gel. Samples that failed to amplify (resulting in no visual bands on the
gel) were removed from any further analyses.