Functional Implication of Heat Shock Protein 70/90 and Tubulin in Cold Stress of Dermacentor Silvarum

Background: The tick Dermacentor silvarum Olenev (Acari: Ixodidae) is a vital vector tick species mainly distributed in the north of China and overwinters in the unfed adult stage. The knowledge of the mechanism that underlies its molecular adaptation against cold is limited. In the present study, genes of hsp70 and hsp90 cDNA, named Dshsp70 and Dshsp90, and tubulin were cloned and characterized from D. silvarum, and their functions in cold stress were further evaluated.

Methods: The genome of the heat shock proteins and tubulin of D. silvarum were sequenced and analyzed using bioinformatics methods. Each group of 20 ticks were injected in triplicate with Dshsp90-, Dshsp70-, and tubulin-derived dsRNA, whereas the control group was injected with GFP dsRNA. Then, the total RNA was extracted and cDNA was synthesized and subjected to RT-qPCR. After the confirmation of knockdown, the ticks were incubated for 24 h and were exposed to - 20 °C lethal temperature (LT50), and then the mortality was calculated.

Results: Results indicated that Dshsp70 and Dshsp90 contained an open reading frame of 345 and 2190 nucleotides that encoded 114 and 729 amino acid residues, respectively. The transcript Dshsp70 showed 90% similarity with that identified from Dermacentor variabilis, whereas Dshsp90 showed 85% similarity with that identified from Ixodes scapularis. Multiple sequence alignment indicates that the deduced amino acid sequences of D. silvarum Hsp90, Hsp70, and tubulin show very high sequence identity to their corresponding sequences in other species. Hsp90 and Hsp70 display highly conserved and signature amino acid sequences with well-conserved MEEVD motif at the C-terminal in Hsp90 and a variable C-terminal region with a V/IEEVD-motif in Hsp70 that bind to numerous co-chaperones. RNA interference revealed that the mortality of D. silvarum was significantly increased after injection of dsRNA of Dshsp70 (P = 0.0298) and tubulin (P = 0.0448), whereas no significant increases were observed after the interference of Dshsp90 (P = 0.0709).

Conclusions: The above results suggested that Dshsp70 and tubulin play an essential role in the low-temperature adaptation of ticks. The results of this study can contribute to the understanding of the survival and acclimatization of overwintering ticks.