Ticks are important ectoparasites and vectors of a variety of pathogens in both animals and humans, and their increasing global distribution poses a growing health risk. Unlike other blood-feeding vectors, ticks feed for an extended period at each life stage and rely exclusively on blood for development and reproduction. Blood digestion in ticks is mediated by a complex multienzyme network within the endolysosomal system of the midgut (MG) epithelial cells. Previous studies have focused largely on protein digestion during the slow feeding phase. However, the processing of the blood meal after the mating-induced rapid engorgement ("big sip") remains unclear, although the rapid turnover of proteins from host blood proteins into yolk proteins in fully fed females is a crucial step for tick reproduction. In this study, we performed a label-free quantitative proteomic analysis of MG tissue extracts and MG contents of the hard tick Ixodes ricinus to characterize proteases and protease inhibitors expressed during selected timepoints of female feeding and off-host digestion. In addition, we analyzed the distribution of digestive enzymes by activity profiling in MG extracts and contents with specific diagnostic substrates. Our results show that the multienzyme network, mainly based on aspartic acid and cysteine cathepsins and complemented by specific types of serine proteases and metalloproteases, is involved in the intracellular and probably also in the luminal digestion of blood meal proteins in fully engorged female ticks. We also detected different types of protease inhibitors and proposed their regulatory role in controlling both endogenous (tick-derived) and host protease activities in the MG tissue and luminal contents storing ingested blood. These results provide comprehensive insights into the physiology of the tick MG and offer new opportunities for the development of future control strategies against ticks and tick-borne diseases.

Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Praha Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice Czech Republic; Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic

Vector Biology Section Laboratory of Malaria and Vector Research National Institute of Allergy and Infectious Diseases Bethesda Maryland USA

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