Divergent domains of 28S ribosomal RNA gene: DNA barcodes for molecular classification and identification of mites

Abstract

Background: The morphological and molecular identification of mites is challenging due to the large number of species, the microscopic size of the organisms, diverse phenotypes of the same species, similar morphology of different species and a shortage of molecular data. Methods: Nine medically important mite species belonging to six families, i.e. Demodex folliculorum, D. brevis, D. canis, D. caprae, Sarcoptes scabiei canis, Psoroptes cuniculi, Dermatophagoides farinae, Cheyletus malaccensis and Ornithonyssus bacoti, were collected and subjected to DNA barcoding. Sequences of cox1, 16S and 12S mtDNA, as well as ITS, 18S and 28S rDNA from mites were retrieved from GenBank and used as candidate genes. Sequence alignment and analysis identified 28S rDNA as the suitable target gene. Subsequently, universal primers of divergent domains were designed for molecular identification of 125 mite samples. Finally, the universality of the divergent domains with high identification efficiency was evaluated in Acari to screen DNA barcodes for mites. Results: Domains D5 (67.65%), D6 (62.71%) and D8 (77.59%) of the 28S rRNA gene had a significantly higher sequencing success rate, compared to domains D2 (19.20%), D3 (20.00%) and D7 (15.12%). The successful divergent domains all matched the closely-related species in GenBank with an identity of 74-100% and a coverage rate of 92-100%. Phylogenetic analysis also supported this result. Moreover, the three divergent domains had their own advantages. D5 had the lowest intraspecies divergence (0-1.26%), D6 had the maximum barcoding gap (10.54%) and the shortest sequence length (192-241 bp), and D8 had the longest indels (241 bp). Further universality analysis showed that the primers of the three divergent domains were suitable for identification across 225 species of 40 families in Acari. Conclusions: This study confirmed that domains D5, D6 and D8 of 28S rDNA are universal DNA barcodes for molecular classification and identification of mites. 28S rDNA, as a powerful supplement for cox1 mtDNA 5'-end 648-bp fragment, recommended by the International Barcode of Life (IBOL), will provide great potential in molecular identification of mites in future studies because of its universality.

First detection ofIxodiphagus hookeri(Hymenoptera: Encyrtidae) inIxodes ricinusticks (Acari: Ixodidae) from multiple locations of Hungary

“…25,26,59 In order to assess deeper knowledge of the variances of hookeri , or to identify specific traits of subgroups that can be better utilized by the biological control methods, the study of the I. hookeri genome, or at least specific genome regions, such as 28S rRNA or 16S rRNA genes may become inevitable, similarly to other weighty insect groups. 51,60,61…”

From qualitative to quantitative insect metabarcoding: an in tandem multilocus mosquito identification methodology

“…On the other hand, 28S rRNA revealed isles of conserved and hypervariable regions (D2, D8 and D10 in Diptera) (Figure 1) known before for their ability to distinguish closely related species. The D2 region has been used in the taxonomy of Acarina, Hymenoptera, Heteroptera and Diptera, including mosquitoes [21][22][23][24][25][26][27] . D8 and D10 regions have been used to a lesser extent in the classification and phylogeny of mites and mealybugs 22,27,28 .…”

“…The D2 region has been used in the taxonomy of Acarina, Hymenoptera, Heteroptera and Diptera , including mosquitoes 21-27 . D8 and D10 regions have been used to a lesser extent in the classification and phylogeny of mites and mealybugs 22, 27, 28 . Conserved regions flanking hypervariable regions were used for the design of three pairs of universal oligonucleotides (100% identity) ( Suppl.…”

Novel Demodex detection method involving non-invasive sebum collection and next-generation sequencing

“…For the time being, genetic diagnostics of Demodex parasites can be aided by PCR and second-generation sequencing that are far cheaper per patient yet less informative. Worth mentioning is the ongoing research for the most suitable barcoding regions [ 29 , 30 ].…”