Function of heat shock protein 70 in the thermal stress response of Dermatophagoides farinae and establishment of an RNA interference method.

Dermatophagoides farinae are an important mite species that cause stored product deterioration and allergic diseases. They widely breed in human habitats because of their strong tolerance to extreme external temperatures. However, mechanisms underlying the stress response and tolerance of D. farinae are unclear. We hypothesized that heat shock protein 70 plays an important role in the heat stress response of D. farinae. In this study, we determined the survival rates of D. farinae at high temperatures (37 °C-45 °C) by performing temperature-gradient experiments in vitro and assessed the expression level of HSP70 by performing RT-qPCR. First, we confirmed that HSP70 regulated the heat stress response of D. farinae, with maximum heat stress regulation observed at 41 °C. Next, we confirmed the presence of a Dicer enzyme-mediated RNA interference (RNAi) pathway in D. farinae by searching the NCBI database and a Dicer site prediction website. Finally, we performed RNAi in D. farinae by using an immersion method with screened dsHSP70 fragments. Moreover, we performed concentration-gradient experiments to determine that 600 ng/μl was the minimal effective concentration of dsHSP70 for silencing HSP70. These results confirm that HSP70 regulates the heat stress response of D. farinae. The present study is the first to report the use of the non-invasive and highly sensitive immersion method for performing RNAi in D. farinae. The results of the present study provide a technical foundation for performing functional gene research and for developing molecular prevention and control strategies against medically important mites.