Tick saliva, an essential chemical secretion of the tick salivary gland, is indispensable for tick survival owing to the physiological influence it exerts on the host defence mechanisms via the instrumentality of its cocktail of pharmacologically active molecules (proteins and peptides). Much research about tick salivary proteome has been performed, but how most of the individual salivary proteins are utilized by ticks to facilitate blood acquisition and pathogen transmission is not yet fully understood. In addition, the phosphorylation of some proteins plays a decisive role in their function. However, due to the low phosphorylation level of protein, especially for a small amount of protein, it is more difficult to study phosphorylation. Maybe, for this reason, the scarcity of works on the phosphorylated tick salivary proteomes still abound. Here, we performed a phosphoproteomic analysis of Haemaphysalis longicornis tick saliva via TiO2 enrichment and the most advanced Thermo Fisher Orbitrap Exploris 480 mass spectrometer for identification. A total of 262 phosphorylated tick saliva proteins were identified and were subjected to functional annotation/enrichment analysis. Cellular and metabolic process terms accounted for the largest proportion of the saliva proteins, with the participation of these proteins in vital intracellular and extracellular transport-oriented processes such as vesicle-mediated transport, exocytic process, cell adhesion, and movement of cell/subcellular component. “Endocytosis”, “Protein processing in endoplasmic reticulum”, and “Purine metabolism” were the most significantly enriched pathways. The knockdown (RNAi) of Tudor domain-containing protein (TCP), actin-depolymerizing factors (ADF), programmed cell death protein (PD), and serine/threonine-protein kinase (SPK) resulted in the dissociation of collagen fibers and the pilosebaceous unit, increased inflammatory infiltrates/granulocytes (possibly heterophiles), and the depletion of the epithelium. Ticks injected with SPK dsRNA engorged normally but with a change in skin colour (possibly an autoimmune reaction) and the failure to produce eggs pointing to a possible role of SPK in reproduction and host immune modulation. Ticks injected with ADF dsRNA failed to acquire blood, underscoring the role of ADF in facilitating tick feeding. The results of this study showed the presence of phosphorylation in tick saliva and highlight the roles of salivary phosphoproteins in facilitating tick feeding.

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.

Haemaphysalis longicornis (Neumann), a tick of public health and veterinary importance, spend the major part of their life cycle off-host, especially the adult host-seeking period. Thus, they have to contend with prolonged starvation. Here, we investigated the underlying molecular mechanism of tick starvation endurance in the salivary glands, midguts, ovaries, and Malpighian tubules of starved H. longicornis ticks using the data-independent acquisition quantitative proteomic approach to study the proteome changes. Essential synthases such as glutamate synthase, citrate synthase, and ATP synthase were up-regulated probably due to increased proteolysis and amino acid catabolism during starvation. The up-regulation of succinate dehydrogenase, ATP synthase, cytochrome c oxidase, and ADP/ATP translocase closely fits with an increased oxidative phosphorylation function during starvation. The differential expression of superoxide dismutase, glutathione reductase, glutathione S-transferase, thioredoxin, and peroxiredoxin indicated fasting-induced oxidative stress. The up-regulation of heat shock proteins could imply the activation of a protective mechanism that checks excessive protein breakdown during starvation stress. The results of this study could provide useful information about the vulnerabilities of ticks that could aid in tick control efforts.

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