Classification and identification of mosquitoes in China based on rDNA 28S D5 region.

Accurate classification and identification of mosquitoes are essential for the prevention and control of mosquito-borne diseases. In this study, adult mosquitoes were collected from 15 cities across 14 provinces in China. They were identified morphologically with the dominant species determined. Furthermore, representative samples were identified at the molecular level based on rDNA 28S D5. In total, 880 adult mosquitoes were collected belonging to Culex (266), Aedes (473), Armigeres (13), and Anopheles (5). Aedes albopictus and "C. pipiens subgroup" were the dominant species. A total of 140 sequences of 28S D5 region (68 for "C. pipiens subgroup", 51 for Ae. albopictus, 18 for Ar. subalbatus, and three for An. sinensis) ranging from 148 to 161 bp were obtained, with 100 % success of amplification and sequencing. Molecular identification were consistent with morphological classification. Sequence analysis showed that "C. pipiens subgroup" was identified into three clades: the traditional C. pipiens subgroup (Clade I), the newly discovered C. cf. perexiguus (Clade II), and C. new sp. (Clade III). Clade I contained the most abundant haplotypes (16) widely distributed without geographical differences. Clade II included six haplotypes that were aggregately distributed south of the Yangtze River. Only three sequences in Clade III showed two haplotypes with no geographical differences. Further morphological comparisons demonstrated differences in body color, beaks, and abdomens among the three clades. In conclusion, the rDNA 28S D5 region could effectively distinguish Culex, Aedes, Armigeres, and Anopheles species at the lower category level, demonstrating its potential as a mini-DNA barcode for mosquito identification.

In vivo and in vitro characterization of a new Oya virus isolate from Culicoides spp. and its seroprevalence in domestic animals in Yunnan, China.

Biting midges are one of the most common hematophagous insects. They are capable of transmitting a wide range of arboviruses and have a significant impact on public health and veterinary medicine. Herein, from midge samples collected in 2013 in Yunnan, China, one sample induced a cell cytopathic effect (CPE) in BHK-21, MA104, and PK15 cell lines. Next-generation sequencing data, RACE and PCR determined the genome sequence of the sample and designated as an Oya virus (OYAV) isolate SZC50. Phylogenetic analysis of the sample revealed that it was cluster into viruses from species Orthobunyavirus catqueense. The open reading frames of S, M, and L segment of OYAV SZC50 were closest to those of OYAV SC0806. Moreover, 831 serum samples (736 pigs, 45 cattle, and 50 sheep) were gathered from 13 cities in Yunnan Province to detect neutralizing antibody of OYAV SZC50. A significant proportion of OYAV SZC50 antibody (more than 30%) was found in Yunnan pig populations, with the positive rate of OYAV SZC50 antibody in pigs from Malipo reaching 95%. To determine the pathogenicity of OYAV SZC50, we chose three animal models: specific pathogen-free Kunming mice, C57BL/6 mice lacking the interferon α/ß receptor, and chicken embryos. At 5, 6, and 7 days post-infection, all adult and suckling C57BL/6 mice, and specific pathogen-free suckling Kunming mice were dead. Our finding was expanding the knowledge about the infection and pathogenic risk of the neglected virus in the Orthobunyavirus.

Screening of reference genes and quantitative real-time PCR detection and verification in Dermatophagoides farinae under temperature stress.

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.