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.

Keywords: DNA barcode; Divergent regions; Mites; Molecular identification; Universal primers; rDNA 28S.

Fig. 1

Adult morphology of the nine mite species. aDermatophagoides farinae (10 × 10); bPsoroptes cuniculi (10 × 4); cSarcoptes scabiei canis (10 × 10); dOrnithonyssus bacoti (10 × 10); eCheyletus malaccensis (10 × 10); fDemodex folliculorum (10 × 40); gDemodex brevis (10 × 40); hDemodex canis (10 × 40); iDemodex caprae (10 × 40). Scale-bars: 100 µm

Fig. 2

Molecular phylogeny of the nine mite species and the closely related species based on data for the three divergent regions of 28S rDNA: D5 (a); D6 (b); D8 (c). Support values are shown above branches. The numbers in parentheses represent ML conflicting support values (which means the conflicting topology). Phytoseius sp. (GenBank: KP276390) was used as the outgroup. Triangles indicate sequences retrieved from GenBank

Fig. 3

Frequency histogram plots of divergence for the three candidate DNA barcodes. a D5; b D6; c D8