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Demodex canis is an important parasitic mite causing skin lesions in dogs. However, molecular studies on this species are limited because of the lack of transcriptomic data. To obtain functional genes of D. canis, mites were collected and RNA was extracted for transcriptome sequencing and functional annotation. Coding sequences (CDSs) of important functional genes were screened and identified using bioinformatics strategies. Six complete CDSs were amplified by specific primers, cloned and sequenced. Results demonstrated that the quantity of the RNA extracted from 100 mites was 12.8 ng and the RNA integrity number was 5.4, indicating that it could be used to construct a cDNA library. Transcriptome sequencing yielded 263,619 unigenes, of which 151,048 (57.3%) were functionally annotated. Using bioinformatics strategies, 58 total CDSs were identified as homologous to those of closely related mite species, including 16 types of allergen genes, 13 types of protease genes, and nine types of motion-related genes. The verified CDSs were almost identical to the CDSs of unigenes. Phylogenetic analyses of tropomyosin further revealed that the verified CDSs clustered successively with those of Demodex species and Tetranychus species in Raphignathae, which agreed with the morphological classification, demonstrating the reliability of the transcriptomic data. In conclusion, this study is the first to successfully sequence transcriptome of D. canis, perform functional annotation, and verify CDSs, which will provide ample data for further studies on the functional genes and molecular pathogenic mechanism of D. canis. [ABSTRACT FROM AUTHOR]

Demodex are among the tiniest organisms in Acari and are important mammalian parasites. However, differences in pathogenicity between two human parasites, Demodex folliculorum and Demodex brevis, remain unknown. Related genetic studies are limited by RNA extraction difficulties and molecular data deficiencies. In this study, RNA extraction, de novo sequencing, functional annotation, and differential gene expression analyses were performed to compare D. folliculorum and D. brevis. This yielded 67.09 and 65.10 million clean reads, respectively, with similar annotations. Bioinformatics analyses and manual alignments identified 237 coding sequences comprising 48 genes from 29 families, including five important functional classes. Of these, 30 genes from 20 families related to metabolism, motion, detoxification and stress response, and allergic reaction were differentially expressed between the two species. Cathepsin type 1, serine protease inhibitor, arginine kinase, triosephosphate isomerase, muscle-specific protein 20-2, myosin alkaline light chain, troponin C, tropomyosin, and heat shock protein 90 were highly expressed in D. folliculorum, whereas cathepsin type 2, aspartic protease, serine protease, myosin heavy chain type 2, and alpha tubulin type 1C were highly expressed in D. brevis. Verified coding sequences were nearly consistent with unigene clusters. Further, absolute quantification results demonstrated that differentially expressed genes followed the predicted expression trend. Therefore, the first RNA sequencing and functional annotation analysis of two Demodex species was successful. Differential expression of important functional genes is likely implicated in pathogenicity disparities between these two species. Our study provides molecular data and technical support for further studies on human Demodex pathogenicity and functional genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Demodex is a type of parasitic mite which could cause serious dermatoses in 11 orders of mammals. However, due to the tiny body with thick chitin hard to be ruptured as well as the difficulty in obtaining a large number of mites, the quantity and quality of extracted RNA could hardly satisfied for transcriptome sequencing. This has hampered the research on functional genes and molecular pathogenesis of Demodex for a long time. To solve the problems above, the present study established a new RNA extraction method in combination Azanno method with liquid nitrogen grinding using 16 human and canine Demodex mite samples. The RNA quality detection results of Agilent 2100 Bioanalyzer showed that 8 of 16 RNA samples met the requirements for trace RNA-Seq, with RIN of 5.0–6.5 and RNA quantity of 1.1–16.0 ng. RNA quality was affected by grinding process and parasitic position of Demodex. Enough grinding number (≥2000) in moderate time (≤20 min) was significant for mites' complete rupture and RNA degradation prevention. D. brevis (100%, 3/3) parasitizing in human sebaceous glands had significantly higher RNA qualification rate than D. folliculorum (57.14%, 4/7) parasitizing in human hair follicles. Yet D. canis parasitizing in dog had lower RNA qualification rate (16.67%, 1/6) as mites were embedded in skin tissues and blood clots. It should be pointed out that microplate reader had defects with a lower RNA qualification rate of 6.25% (1/16) unmatched with 2100 Bioanalyzer, reminding that it could be only used as reference in RNA quality evaluation. Image 1 • Azanno method & liquid nitrogen grinding successfully extracted RNA from pure Demodex. • grinding process and mites' parasitic position mainly affected the quality of extracted RNA. • microplate reader could be used only as a reference in RNA quality evaluation. • 2100 Bioanalyzer should be the more reliable method to detect RNA quality for RNA-Seq. [ABSTRACT FROM AUTHOR]

There has been no widely accepted DNA barcode for species identification of Demodex. In this study, we attempted to solve this issue. First, mitochondrial cox1-5′ and 12S gene fragments of Demodex folloculorum, D. brevis, D. canis, and D. caprae were amplified, cloned, and sequenced for the first time; intra/interspecific divergences were computed and phylogenetic trees were reconstructed. Then, divergence frequency distribution plots of those two gene fragments were drawn together with mtDNA cox1-middle region and 16S obtained in previous studies. Finally, their identification efficiency was evaluated by comparing barcoding gap. Results indicated that 12S had the higher identification efficiency. Specifically, for cox1-5′ region of the four Demodex species, intraspecific divergences were less than 2.0%, and interspecific divergences were 21.1-31.0%; for 12S, intraspecific divergences were less than 1.4%, and interspecific divergences were 20.8-26.9%. The phylogenetic trees demonstrated that the four Demodex species clustered separately, and divergence frequency distribution plot showed that the largest intraspecific divergence of 12S (1.4%) was less than cox1-5′ region (2.0%), cox1-middle region (3.1%), and 16S (2.8%). The barcoding gap of 12S was 19.4%, larger than cox1-5′ region (19.1%), cox1-middle region (11.3%), and 16S (13.0%); the interspecific divergence span of 12S was 6.2%, smaller than cox1-5′ region (10.0%), cox1-middle region (14.1%), and 16S (11.4%). Moreover, 12S has a moderate length (517 bp) for sequencing at once. Therefore, we proposed mtDNA 12S was more suitable than cox1 and 16S to be a DNA barcode for classification and identification of Demodex at lower category level. [ABSTRACT FROM AUTHOR]

Aspartic proteases (ASPs) are important hydrolases for parasitic invasion of host tissues or cells. This was the first study on Demodex ASP. First, the complete coding sequence (CDS) was amplified, cloned and sequenced. Then, the protein physical and chemical properties was analysed. Finally, the recombinant plasmid, expression and purification system was established. Results showed that the lengths of CDS of Demodex folliculorum and D. brevis were 1161 and 1173 bp, respectively. The molecular weight of the protein was approximately 40 KDa. It contained an aspartic acid residue, a substrate-binding site and signal peptide, yet lacked a transmembrane domain and was located in the membrane or extracellular matrix. The phylogenetic and conserved motif analyses showed that D. folliculorum and D. brevis clustered separately and then formed a single branch, which finally clustered with other Acariformes species. The prokaryotic expression systems for recombinant ASP with His-tag (rASP-His) and GST-tag (rASP-GST) were constructed. The inclusion bodies of rASP-His were renaturated by gradient urea and purified using NI beads, while those of rASP-GST were renaturated by sarkosyl and Triton X-100 and purified using GST beads. Conclusively, the prokaryotic expression and purification system of Demodex rASP was successfully established for further pathogenic mechanism research. • The first study on Demodex ASP gene amplification, expression and purification. • ASP CDSs were successfully amplified by OE-PCR from the two human Demodex species. • ASP of two species predicted to be a secretory protein with similar conserved motifs. • Modified urea gradient and S-T methods were suitable to renaturate inclusion bodies. • It provides a basis for study on molecular pathogenic mechanism of ASP in Demodex. [ABSTRACT FROM AUTHOR]