Hebei Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
Parasit Vectors. 2021 Oct 19;14(1):542. doi: 10.1186/s13071-021-05056-y.
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.
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 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).
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.
壁虱 Dermacentor silvarum Olenev（蜱螨目：硬蜱科）是一种重要的媒介壁虱物种，主要分布在中国北方，以未进食的成虫越冬。对其分子适应寒冷机制的了解有限。本研究从 D. silvarum 中克隆并鉴定了 hsp70 和 hsp90 cDNA 基因，命名为 Dshsp70 和 Dshsp90，并进一步评估了它们在冷应激中的功能。
使用生物信息学方法对 D. silvarum 的热休克蛋白和微管蛋白基因组进行测序和分析。每组 20 只壁虱重复注射 Dshsp90、Dshsp70 和微管蛋白衍生的 dsRNA，而对照组注射 GFP dsRNA。然后提取总 RNA，合成 cDNA，并进行 RT-qPCR。在确认敲低后，将壁虱在 -20°C 的致死温度（LT50）下孵育 24 小时，然后计算死亡率。
结果表明，Dshsp70 和 Dshsp90 分别含有 345 和 2190 个核苷酸的开放阅读框，分别编码 114 和 729 个氨基酸残基。Dshsp70 的转录物与从 Dermacentor variabilis 鉴定的转录物具有 90%的相似性，而 Dshsp90 与从 Ixodes scapularis 鉴定的转录物具有 85%的相似性。多重序列比对表明，D. silvarum Hsp90、Hsp70 和微管蛋白的推导氨基酸序列与其他物种的相应序列具有非常高的序列同一性。Hsp90 和 Hsp70 显示高度保守和特征性氨基酸序列，在 Hsp90 的 C 末端具有高度保守的 MEEVD 基序，而在 Hsp70 的 C 末端具有高度可变的 C 末端区域，具有 V/IEEVD 基序，与众多共伴侣结合。RNA 干扰显示，注射 Dshsp70（P=0.0298）和微管蛋白（P=0.0448）dsRNA 后，D. silvarum 的死亡率显着增加，而干扰 Dshsp90 后（P=0.0709）则没有显着增加。
上述结果表明，Dshsp70 和微管蛋白在蜱的低温适应中发挥重要作用。本研究结果有助于了解越冬蜱的生存和适应。
