Molecular identification of ticks, lice, and fleas
Molecular identification was performed on 15 single specimens for which morphologic identification to species level was equivocal. We amplified three target genes: the internal transcribed spacer-2 (ITS2), cytochrome oxidase 1 (CO1), and 16S ribosomal (r)RNA (Supplementary Table 1). The PCRs were performed in a SimpliAmp PCR Thermal Cycler (Applied Biosystems, Singapore) in 10-µl reactions that consisted of 2 µl of 5x HOT FIREPol® Blend Master Mix (Solis BioDyne, Estonia), 2 µl of template, and 0.5 µl of 10 µM primer. Molecular grade water was included as a negative control on each run. The cycling conditions have been described before in detail (Mwamuye et al., 2017), with the exception that the final extension step for the three fragments was seven minutes. Amplicons of the correct size were visualised alongside Quick-Load® 100-bp DNA Ladder (Biolabs, UK) by electrophoresis on 1.6 % ethidium stained agarose gels under UV light. Bi-directional sequencing of amplicons purified by Exo 1-rSAP combination (Biolabs, UK) was performed by Macrogen (Netherlands). Sequence chromatograms were inspected, edited, and aligned using Geneious Prime version 2019.0.4 software (Biomatters, New Zealand). The resulting sequence contigs were used in nucleotide BLAST searches (Altschup et al., 1990) against the GenBank nr database (www.ncbi. nlm.nih.gov/blast) to identify sequence matches.