Improvement on the extraction method of RNA in mites and its quality test.

To solve the long-existing difficult problems in extracting RNA and constructing a complementary DNA (cDNA) library for trace mites, we conducted a further comparative experiment among three RNA extraction methods (TRIzol method, Omega method, and Azanno method) based on our previous attempts at the construction of cDNA library of mites, with Psoroptes cuniculi still used as the experimental subject. By subsequently decreasing the number of mites, the least number of mites needed for RNA extraction of each method were found by criteria of completeness, concentration, and purity of the extracted RNA. Specific primers were designed according to the allergen Pso c1, Pso c2, and Actin gene sequences of Psoroptes to test the reliability of cDNA library. The results showed that Azanno method needed only 10 mites with sensitivity 204 times higher than previously used TRIzol method and 20 times higher than Omega method; clear RNA band was detected by agarose gel electrophoresis; and ultraviolet spectrophotometer determination showed that RNA concentration, 260/280, and 260/230 were in the range of 102 to 166 ng/µl, 1.83 to 1.99, and 1.49 to 1.72, respectively. Finally, specific primers detection showed that the amplified sequences had 98.33, 98.19, and 99.52% identities with those of P. cuniculi or Psoroptes ovis in GenBank, respectively, indicating that the cDNA library constructed using 10 mites was successful and it could meet the requirements for molecular biology research. Therefore, we concluded that Azanno method was more effective than TRIzol method and Omega method in RNA extraction and cDNA library construction of trace mites.

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

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.

Squamarina (lichenised fungi) species described from China belong to at least three unrelated genera.

New collections of six Squamarina species from type localities in China were studied. The comparison of morphological characteristics and secondary metabolites with those of the type specimens and phylogenetic analyses suggest that S. callichroa and S. pachyphylla belong to Rhizoplaca, S. semisterilis belongs to Lobothallia and S. chondroderma should be retained in Lecanora temporarily. Only two species, S. kansuensis and S. oleosa, remain in Squamarina. The new combinations Lobothallia semisterilis (H. Magn.) Y. Y. Zhang, Rhizoplaca callichroa (Zahlbr.) Y. Y. Zhang and R. pachyphylla (H. Magn.) Y. Y. Zhang are proposed. Detailed descriptions to aid the identification of these species, distributions and phylogenetic trees, based on multiple collections, are presented. The generic concept of Squamarina is recircumscribed in this study.

Diethyl 2,6-dimethyl-4-phenyl-1,4-dihydro-pyridine-3,5-dicarboxyl-ate.

The title mol-ecule, C(19)H(23)NO(4), was synthesized by the reaction of benzaldehyde, ethyl acetoacetate and NH(4)HCO(3). The dihydro-pyridine ring adopts a flattened boat conformation and the plane of the base of the boat forms a dihedral angle of 88.78 (9)° with the phenyl ring. The packing is stabilized by strong inter-molecular N-H⋯O and weak inter-molecular C-H⋯O hydrogen bonds.

The Construction of Full-Length cDNA Library for Otodectes cynotis.

BACKGROUND: Otodectes cynotis (Hering, 1838) is the pathogen of otodectic mange distributed worldwide. The mite mainly infests carnivores and, sometimes, humans. However, due to the lack of cDNA library, research on its pathogenesis has been challenging. METHODS: To solve this problem, the present study first sampled O. cynotis mites from an infested cat from Xi'an, China, for RNA extraction. Then, the full-length cDNA library was constructed using the SMART technique. Finally, positive clones > 500 bp and Hsc70-5 gene fragment specifically amplified from the cDNA library were sequenced and analyzed to verify the library's reliability. RESULTS: Results showed that RNA extracted from 300 mites had good quality with a concentration of 149 ng/µl and OD260/OD280 of 1.99. The library satisfied the quality standard of a good library with a titer of 5.02 × 105 PFU/ml and a combination rate of 97.61%. In addition, clone 4 and Hsc70-5 showed 98.38% and 99.72% identity with Ef1-α and Hsc70-5 gene sequences of O. cynotis in GenBank, respectively. CONCLUSION: The cDNA library of O. cynotis constructed here was successful and reliable, creating the basis for research on RNA sequencing and functional genes of O. cynotis.