LSU rDNA D5 region: the DNA barcode for molecular classification and identification of Demodex

Whether ribosomal genes can be used as DNA barcodes for molecular identification of Demodex (Acariformes: Demodicidae) is unclear. To examine this, Demodex folliculorum, D. brevis, D. canis, and D. caprae were collected for DNA extraction, rDNA fragments amplification, sequencing, and analysis. The V2 and V4 regions of SSU rDNA; D5, D6, and D8 regions of LSU rDNA; and ITS region were obtained from the four morphospecies. BLAST analysis showed that the obtained sequences matched those of Demodex or Aplonobia (Acariformes: Tetranychidae) in Raphignathae. Phylogenetic trees derived from V2, V4, D5, D6, and D8 regions, but not from ITS region, showed that the four species of Demodex clustered independently. Sequence divergence analysis further demonstrated that D5, D6, and D8 regions had obvious barcoding gap between intraspecific and interspecific divergences, with the gap of D5 (16.91%) larger than that of D6 (11.82%) and D8 (4.66%). The V2 and V4 regions did not have a barcoding gap, as the intraspecific and interspecific divergences partially overlapped. For the ITS region, intraspecific and interspecific divergences completely overlapped. These results suggest that the D5, D6, and D8 regions of LSU rDNA, especially D5, are suitable DNA barcodes for Demodex.