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. 2010 Dec 14:3:119.
doi: 10.1186/1756-3305-3-119.

Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

Zdeněk Franta  1 , Helena Frantová, Jitka Konvičková, Martin Horn, Daniel Sojka, Michael Mareš, Petr Kopáček
Affiliations

Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

Zdeněk Franta et al. Parasit Vectors. .

Abstract

Background: Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes.

Results: This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding.

Conclusions: Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens.

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Figures

Figure 1
Figure 1
Overview of the feeding phases, midgut morphology and overall hemoglobinolysis in the gut of a Ixodes ricinus female during feeding on the host. An adult Ixodes ricinus female feeds for about 7 to 8 days. The slow feeding period starts one day post-attachment, during which the female ingests about one third of the total blood meal. The major portion of the host blood (about two thirds) is ingested by the mated female during the rapid engorgement phase, taking place during the last 24-48 hours before the engorged tick drops off the host. Light microscope panel: The semi-thin sections were stained with toulidine blue; scale bar = 20 μm. UF - unfed ticks; 2d, 4d, 6d - 2, 4, 6 days of feeding, respectively; FF - fully fed (engorged) ticks. Midgut epithelium scheme panel: RC - reserve cells (stem cells); DCN - digestive cells persisting from the nymphal stage; DCI - initial digestive cells (prodigest cells); DC - digestive cells; DDC - detached digestive cells; RB - residual bodies (hemosomes); E - endosomes; blue circles - cell nuclei; yellow circles - lipid vacuoles. Relative hemoglobinolysis panel: Quantification of relative hemoglobinolysis in gut tissue extracts was measured using the fluorescamine derivatization assay at pH 4.2 and normalized to one tick gut according to the method described in reference [12].
Figure 2
Figure 2
Active-site titration of digestive peptidases in the Ixodes ricinus gut tissue extracts during feeding on the host. The groups of 15-20 females were forcibly removed and collected from four guinea pigs at the indicated feeding time points. The guts from individual ticks were subsequently dissected and the gut tissue extracts were prepared from the pool of longitudinally cut midgut halves. The absolute molarities of the individual peptidases in gut homogenates were determined by stoichiometric titration with the appropriate active-site inhibitors (Table 1). Bars represent the average value from a triplicate measurement. Standard deviations were ≤10% of the average value and are not shown. The molarity of cathepsin L (IrCL) was not determined because an appropriate inhibitor was lacking. For details, see Material and Methods section.
Figure 3
Figure 3
Dynamic profiles of mRNA expression and enzyme activities of individual peptidases during the feeding of Ixodes ricinus females. For each time point, the guts were dissected from 15-20 females removed from four guinea pigs. Half of the guts were processed either for total RNA isolation or gut tissue extraction as described above. Gene expression profiles of the indicated enzymes were determined by qRT-PCR, using elongation factor 1 as a reference gene. The expressions were related to the maximum mRNA level set as 100%. Columns correspond to the average value of triplicate technical determinations, while error bars indicate the corresponding standard deviations. The enzyme activity curves of indicated peptidases were determined using specific fluorogenic substrates and shielding inhibitors to mask possible interference from other peptidases (see Table 1). Enzymatic activities in the homogenates were normalized per one gut tissue. The circles display the average activities obtained from triplicate measurements. The standard deviations were ≤5% of the average value and are not shown.
Figure 4
Figure 4
Immunolocalization of cathepsin B (IrCB) in the gut of an Ixodes ricinus female during feeding. Panel A: Western blot analysis of authentic IrCB in gut homogenates electro-transferred onto a PVDF membrane. LMW - protein markers; UF - unfed ticks; 2 d - 2 days fed ticks (0.2 gut/lane); 4 d, 5 d, 6 d - 4, 5, 6 days fed ticks, respectively (0.1 gut/lane); FF - fully fed ticks (0.08 gut/lane). CBB - Coomassie stained membrane; Ab@IrCB - rabbit antibody against recombinant IrCB (Ra×IrCBIg, dilution 1:100). Secondary antibody - Swine anti-rabbit-peroxidase conjugate (1:1000). Panel B: Immunolocalization of IrCB in the gut of I. ricinus on semi-thin sections using affinity purified rabbit antibody Ra×IrCBACP (30 μg/ml). Secondary antibody - Goat anti-rabbit with conjugated Alexa Fluor® 488 (1:200). Merged images with DAPI nuclei staining; scale bars = 20 μm. Panel C: Control images without primary antibodies.
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