Abstract Ticks are external parasitic arthropods that can transmit a variety of pathogens by sucking blood. Low‐temperature tolerance is essential for ticks to survive during the cold winter. Exploring the protein regulation mechanism of low‐temperature tolerance of Haemaphysalis longicornis could help to explain how ticks survive in winter. In this study, the quantitative proteomics of several tissues of H. longicornis exposed to low temperature were studied by data independent acquisition technology. Totals of 3 699, 3 422, and 1 958 proteins were identified in the salivary gland, midgut, and ovary, respectively. The proteins involved in energy metabolism, cell signal transduction, protein synthesis and repair, and cytoskeleton synthesis changed under low‐temperature stress. The comprehensive analysis of the protein regulation of multiple tissues of female ticks exposed to low temperature showed that maintaining cell homeostasis, maintaining cell viability, and enhancing cell tolerance were the most important means for ticks to maintain vital signs under low temperature. The expression of proteins involved in and regulating the above cell activities was the key to the survival of ticks under low temperatures. Through the analysis of a large amount of data, we found that the expression levels of arylamine N‐acetyltransferase, inositol polyphosphate multikinase, and dual‐specificity phosphatase were up‐regulated under low temperature. We speculated that they might have important significance in low‐temperature tolerance. Then, we performed RNA interference on the mRNA of these 3 proteins, and the results showed that the ability of female ticks to tolerate low temperatures decreased significantly.
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