From the fat body to the hemolymph: Profiling tick immune and storage proteins through transcriptomics and proteomics.

Ticks are blood-feeding arachnids that are known to transmit various pathogenic microorganisms to their hosts. During blood feeding, ticks activate their metabolism and immune system to efficiently utilise nutrients from the host's blood and complete the feeding process. In contrast to insects, in which the fat body is known to be a central organ that controls essential metabolic processes and immune defense mechanisms, the function of the fat body in tick physiology is still relatively unexplored. To fill this gap, we sought to uncover the repertoire of genes expressed in the fat body associated with trachea (FB/Tr) by analyzing the transcriptome of individual, partially fed (previtellogenic) Ixodes ricinus females. The resulting catalog of individual mRNA sequences reveals a broad repertoire of transcripts encoding proteins involved in nutrient storage and distribution, as well as components of the tick immune system. To gain a detailed insight into the secretory products of FB/Tr specifically involved in inter-tissue transport and humoral immunity, the transcriptomic data were complemented with the proteome of soluble proteins in the hemolymph of partially fed female ticks. Among these proteins, the hemolipoglyco-carrier proteins were predominant. When comparing immune peptides and proteins from the fat body with those produced by hemocytes, we found that the fat body serves as a unique producer of certain immune components. Finally, time-resolved transcriptional regulation of selected immune transcripts from the FB/Tr was examined in response to experimental challenges with model microbes and analyzed by RT-qPCR. Overall, our data show that the fat body of ticks, similar to insects, is an important metabolic tissue that also plays a remarkable role in immune defense against invading microbes. These findings improve our understanding of tick biology and its impact on the transmission of tick-borne pathogens.

Insight Into the Dynamics of the Ixodes ricinus Nymphal Midgut Proteome.

Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

Functional characterization of the insulin signaling pathway in the hard tick Ixodes ricinus.

Ticks are blood-feeding arachnids transmitting a variety of pathogens to humans and animals. A unique trait in tick physiology is their ability to engorge and digest large amounts of host blood, ensuring their high reproductive potential. Activation of the blood digestive machinery in the tick gut, as well as processes controlling maturation of ovaries, are triggered upon blood meal uptake by still largely unknown mechanisms. Sensing of the nutritional status in metazoan organisms is facilitated by the evolutionarily conserved Insulin Signaling Pathway (ISP) and the interlinked Target of Rapamycin (TOR) pathway. Recently, we have identified three components of these pathways in the hard tick Ixodes ricinus midgut transcriptome, namely a putative insulin receptor (InR), and the downstream intracellular serine/threonine kinases AKT and TOR. In this study, we primarily focus on the molecular and functional characterization of the I. ricinus insulin receptor (IrInR), the first InR characterized in Chelicerates. A phylogenetic analysis across the major Arthropod lineages demonstrated that ticks possess only one gene encoding an InR-related molecule. Tissue expression profiling by quantitative PCR in semi-engorged I. ricinus females revealed that the IrInR, as well as AKT (IrAKT) and TOR (IrTOR) are expressed in various organs, with the highest expression being detected in ovaries. We have further evaluated the impact of RNAi-mediated knock-down (KD) of IrInR, IrAKT, and IrTOR on tick blood-feeding and reproductive capacity. Weights of engorged IrInR KD females and laid egg clutches were reduced compared to the control group, and these quantitative parameters clearly correlated with the efficiency of RNAi-KD achieved in individual ticks. The most striking phenotype was observed for IrAKT KD that impaired tick feeding and completely aborted egg production. A recombinant extracellular fragment of the IrInR α-subunit was used to produce antibodies in experimental rabbits to assess its potential as a protective antigen against tick feeding and reproduction. Our data clearly indicate the functionality of the ISP in ticks and demonstrate the need for further investigation of specific roles played by the endogenous insulin-like peptides in tick physiological processes.