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An increasing number of researchers have applied secondary-structure based multiple alignments of rDNA genes in phylogeny. These studies mostly depended on a few valuable divergent domains in LSU and SSU rDNA. Yet other divergent domains, e.g. D1, were poorly investigated and rarely used. However, these domains might contain additional evolutionary data and play a vital role in DNA-based phylogenetic study. Here, we investigated all available D1 sequences of Arachnida taxa and predicted corresponding secondary structures to help identify homologous positions in the D1 region. Long insertions were found exclusive to Eriophyoidea and folded into three newly proposed helices. Non-Acari taxa were all GC rich. In Acari, most Trombidiformes and all Mesostigmata (Parasitiformes) taxa were AT rich and Ixodida (Parasitiformes) GC rich; however there was no consistent base bias in Sarcoptiformes sequences. For Eriophyoid mites, genera Cecidophyopsis and Aceria were both well supported in MP, NJ, ME and ML tress based on D1 sequences, and clusters of Cecidophyopsis species were identical with former study. This demonstrated that the D1 region could act as a valuable molecular marker in phylogenetic reconstruction of Eriophyoidea. Additionally, D1 has been proven suitable in phylogenetic analysis at the family and genus level in Acari, but not in Opiliones.

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.

The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.