During previous studies we have described the major constituents of the digestive machinery of the hard tick I. ricinus and deciphered how they are organized into a hemoglobinolytic pathway [8, 12]. Herein, we have focused on the dynamic profiling of the components of the tick digestive apparatus during the course of tick feeding. This information is particularly relevant since the feeding phase is decisive for pathogen transmission. We have provided a comprehensive insight into the gene transcription, activity and molar concentration of five digestive enzymes during blood feeding, as well as the localization of the IrCB, as the most abundant peptidase of the pathway. Placing these molecular data into the context of previous ultrastructural and histochemical studies on tick gut development performed in 1970's and 80's [3, 13, 18] improves significantly our understanding of the entire process of intracellular blood digestion.
Morphological changes of the midgut epithelium observed during blood feeding of I. ricinus by light microscopy (Figure 1) basically correspond to the numerous previous reports published on gut morphology in other ixodid tick species [14, 15, 19, 20]. In these studies, a variety of cells within the gut epithelium have been described and named according to their structure and/or putative functions, resulting in a rather disperse nomenclature in different tick species. Based on our observations (Figure 1), we tend to support the hypothesis of some authors [13, 15] that various cell types represent digestive cells at different stages of development, reflecting their growth, differentiation and gradually changing function in endocytosis, lysosomal digestion, secretion and handling of waste products via hemosome formation . This study also supports the recognition of distinct digestive phases previously based on the structural changes of the tick gut . The initial continual digestive phase takes place during the slow feeding period starting one to two days p.a. to the host and is characterized by the gradual maturation and detachment of digestive cells from the gut epithelium . During the rapid engorgement phase, when the mated female ingests about two thirds of the total blood meal, no more reserve cells or initial digestive cells are observed and the mature digestive cells remain associated with the epithelium.
We have previously characterized the hemoglobinolytic pathway in semi-engorged I. ricinus females that were fed for six days, comprising of a cascade of enzymatic activities of aspartic peptidase IrCD, cysteine peptidase IrCL, asparaginyl endopeptidase IrAE, the major endo/exopeptidase IrCB and dipeptidyl peptidase IrCC . The determination of the molar concentrations as well as activity measurements of the component peptidases presented in this work support that the hemoglobinolytic pathway described for the sixth day p.a. is preserved throughout the entire blood feeding period.
The quantitative determination of the overall hemoglobinolytic activity indicates that digestion is almost negligible for the first two days after tick attachment on the host and dramatically increases between days four and six, progressing towards the end of the slow feeding period (Figure 1). The maximum level of hemoglobinolytic capacity (activity normalized to the entire tick gut) was recorded in the guts of fully fed ticks that just dropped off the host. The trend of an increase in total hemoglobinolysis matches with the concomitant activity profiles of the majority digestive enzymes, namely IrCB, IrCC, IrAE and IrCD. The significant up-regulation of their respective mRNA levels could be monitored already at the second day of feeding, peaking towards the end of the slow feeding period between days four and six and then steeply declining during the rapid engorgement phase. This result is consistent with an earlier histological observation demonstrating that RNA is mainly accumulated in the stem and prodigest cells (herein referred to as reserve and initial digestive cells, respectively), that disappear during the rapid engorgement stage . The contrast of the significant down-regulation of mRNA expression to the peaking hemoglobinolysis and enzyme activities (except for IrCL) during the rapid engorgement suggests that the following second continuous digestive phase, i.e. blood digestion in fully fed ticks detached from the host, relies mainly on the enzymes synthesized and accumulated during the slow feeding period. Our results contradict the prevailing opinion that the rapid engorgement stage is associated with reduced hemoglobinolysis, which has been accordingly designated the reduced-digestion phase in the literature [18, 20, 21]. To our knowledge, the general recognition of the term 'reduced-digestion phase' originates from a 1973 report on the total proteolytic activity in gut homogenates of the hard tick Hyalomma excavatum. The authors reported a low activity in both the initial stage of feeding as well as in fully engorged females. However, it is important to take into consideration that they related the proteolytic activity to the total protein concentration in the gut extracts without any compensation for the proteins of the host origin. To our experience, it is impossible to free mechanically the dissected gut tissues of significant host blood contamination, since substantial portion of the host proteins is already present inside the digestive vacuoles. Moreover, a gentle washing has to be applied to prevent an undesired detachment of digestive cells from the epithelium. Therefore, the host proteins inevitably remain the most abundant components in the gut homogenates from the later feeding stages. Despite its simplicity, normalization of hemoglobinolytic and enzyme activities to one tick gut is more reliable than relation to the total protein or host hemoglobin concentrations (based on absorbance at 415 nm) and reflects better the real total proteolytic potential of the gut. In conclusion, this approach leads to a novel interpretation of the blood digestion capacity during the 'big-sip' which disproves the earlier opinions.
The only enzyme activity that drops significantly during the fast engorgement phase is that of IrCL. This finding was also supported by the Western blot analysis showing a substantial reduction of IrCL amount in fully engorged ticks (unpublished results). The reason for the decrease of IrCL activity in fully fed ticks remains still obscure. One possible explanation to this effect is a simultaneous increase of cathepsin L-specific inhibitors, such as cystatins found to be expressed in the midgut of the soft tick Ornithodoros moubata or the hard tick Haemaphysalis longicornis. Our results strongly imply that de novo protein synthesis of the digestive enzymes rather than activation of existing pro-enzymes underlies the overall increase of hemoglobinolytic activity in the gut of I. ricinus. The main evidence for this interpretation was provided by Western blot analysis of IrCB from gut homogenates dissected during the course of blood feeding (Figure 4A). At all time points, IrCB was mainly detected as an activated mature enzyme and the proportion of the IrCB pro-enzyme and mature active enzyme did not seem to be altered significantly in time (Figure 4A). The affinity purified antibodies made it possible to perform a clear immunolocalization of IrCB in I. ricinus midgut sections prepared at different time points of feeding (Figure 4B). Similar results were obtained for IrCL and IrCD (data not shown), indicating that the entire digestive apparatus remains localized intracellularly in the digestive cells during the feeding. This is in accordance with our previous single time point localization of IrAE in the midgut of semi-engorged I. ricinus and digestive enzymes localized in the digestive cells of other tick species [24, 26, 27]. On the other hand, it remains to be rigorously investigated whether or not intracellular digestion is preserved also in the guts of fully engorged ticks during the second digestive phase, following tick detachment and preceding oviposition.
Based on the results given herein we can conclude that all the major constituents of the tick digestive pathway are regulated at the transcriptional level. Certainly, the mechanisms triggering and regulating the expression of digestive enzymes remain to be explored. The negligible gene expression and enzyme activity at the early stage of tick feeding make the digestive system extremely vulnerable target for efficient impairment, for example via antibodies presents in vaccinated animals. Therefore, digestive enzymes as well as yet unknown regulating factors are promising candidates as concealed antigens for the development of efficient 'anti-tick' vaccines capable to control ticks and associated transmission of tick-borne pathogens [28, 29].