Prokaryotic expression of DFP1 and DFP2 in Dermatophagoides farinae and their responses to temperature stress.

The functions of highly expressed genes DFP1 and DFP2 in Dermatophagoides farinae remain unknown. DFP1 and DFP2 have been abundantly annotated and were up-regulated under temperature stress at 43 °C and -10 °C in our previous RNA-seq study, indicating that DFP1 and DFP2 may have temperature stress response function. Here, we amplified, cloned, and sequenced to obtain the complete coding sequences of DFP1 and DFP2 and predicted their protein characteristics using bioinformatics analysis. Then, prokaryotic expression systems were constructed and found that DFP1 was expressed in Escherichia coli Rosetta-gami 2 (DE3) but not BL21 (DE3); DFP2 was expressed in both BL21 (DE3) and Rosetta-gami 2 (DE3), with higher expression in BL21 (DE3). Finally, the growth curves of bacteria were drawn and indicated that the DFP1- and DFP2-pET32a carrying recombinant bacteria grew better than the respectiveonly pET32a carrying control bacteria after heat and cold stress. This study confirms for the first time that DFP1 and DFP2 respond to temperature stress at the protein level. The constructed prokaryotic expression systems will provide an experimental foundation for future antibody preparation for western blotting detection to confirm the temperature-stress response functions of DFP1 and DFP2.

Identification and genetic characterisatin of cathepsin L in Demodex.

Owing to difficulties in obtaining functional gene sequences, molecular pathogenic mechanisms in Demodex have been understudied. In this study, overlap extension PCR was used to obtain the sequences of cathepsin L (CatL), a pathogenicity-related gene, to provide a foundation for subsequent functional research. Demodex folliculorum and Demodex brevis mites were obtained from the face skin of Chinese individuals, and Demodex canis mites were isolated from the skin lesions of a dog. RNA was extracted and used to synthesise double-stranded cDNA. PCR amplification, cloning, sequencing, and bioinformatics analysis of CatL were performed. CatL gene sequences of 1005, 1008, and 1008 bp were successfully amplified for D. brevis, D. folliculorum, and D. canis, respectively. These sequences showed 99.9 or 100% identity with templates previously obtained by RNA-seq. The Maximum Likelihood (ML) phylogenetic tree showed that D. folliculorum clustered with D. canis first, then with D. brevis, and finally with other Acariformes mite species. The three Demodex species had nine similar motifs to those of Sarcoptes scabies, Dermatophagoides pteronyssinus, and Dermatophagoides farinae, and motifs 10-13 were valuable for identification. CatL proteins of Demodex species were predicted to be approximately 38 kDa, be located in lysosomes, have a signal peptide but no transmembrane region, and have two functional domains, I29 and Pept_C1. However, interspecific differences were observed in secondary and tertiary protein structures. In conclusion, we successfully obtained CatL sequences of three Demodex species by overlap extension PCR, which creates conditions for further pathogenic mechanism studies.

Population identification and divergence threshold in Psoroptidae based on ribosomal ITS2 and mitochondrial COI genes.

Psoroptidae mites are a type of small mites with a wide range of hosts. The proliferation of Psoroptidae mites could cause symptoms such as severe itching, atopic dermatitis, and hair loss in infected animals. If severely infected, death can also occur. The morphological classification and identification of Psoroptidae mites is problematic due to the overlapping geographical distribution. In addition, there is no divergence threshold for molecular classification and identification. To solve this problem, gDNA was extracted from individual Psoroptes and Otodectes mites (China) for amplification of rDNA ITS2 and mtDNA COI. After that, the sequences obtained were aligned and analyzed with those retrieved from GenBank. Based on rDNA ITS2 sequences, Psoroptidae was divided into three genera, namely, Psoroptes, Chorioptes, and Otodectes, which was in accordance with morphological classification. The intraspecies, interspecies, and intergenera could be differentiated effectively, with thresholds ≤ 5.20, 6.18-14.86, and ≥15.72 %, respectively. However, based on mtDNA COI sequences, Psoroptidae was divided into four genera with Caparinia added, as Caparinia sp did not cluster with the other three genera. The intra- and interspecies could be differentiated effectively, but interspecies and intergenera could not. The intra- and interspecies identification thresholds were ≤ 2.12 and ≥10.93 %. Further analysis showed that host but not geographical isolation was found in Psoroptes and Chorioptes, whereas Otodectes mites parasitizing dogs and cats were the same species; neither host nor geographical isolation was observed. In conclusion, rDNA ITS2 is better than mtDNA COI for DNA barcoding in Psoroptidae.