Introduction

The soft tick, Ornithodoros savignyi Audouin, more commonly known as the sand tampan, is distributed throughout the North Western parts of Southern Africa as well as Egypt, Ethiopia, Kenya and Zimbabwe (Paton and Evans 1929). The bites of these ticks cause irritation, blisters and bruising (Estrada-Pena and Jongejan 1999). They cause large losses of livestock especially young calves and lambs in the areas where they occur and are of special relevance in Southern Africa (Mans and Neitz 2003). Pathogenesis associated with these ticks is caused by secretion of a toxin during feeding and can lead to cardiac failure (Mans et al. 2004). The toxin also induces a serious allergic reaction in humans (Howell et al. 1975). No infectious diseases are transmitted by these ticks, although it has been reported that O. savignyi caused relapsing fever in humans (Paton and Evans 1929).

Invertebrates only have an innate immune system which is the first line of defense against infections. It is activated by host proteins that recognize conserved surface determinants of pathogens, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN) from bacteria and β-1,3-glucans from fungi (Medzhitov and Janeway 2002). Both cellular and humoral immune responses are activated to eliminate pathogens (Imler and Hoffmann 2000). In contrast to the well investigated immune response in insects, research into tick innate immunity is rather limited (Sonenshine and Hynes 2008). In ticks, cellular responses include phagocytosis and encapsulation, while humoral responses lead to the secretion of antimicrobial peptides and proteins. Most of the research in this field has focused on the identification and characterization of antimicrobial peptides (AMPs) in various hard tick species and in one soft tick, O. moubata.

Antimicrobial peptides and other immune-related proteins are synthesized within the fat bodies or hemocytes and secreted into the plasma. Both mRNA-based and proteomic approaches should be combined to decipher the complexity of innate immunity (Levy et al. 2004), however, proteomic analysis allows the investigation of innate immunity at the protein level, the real mediators of physiological function. Moreover, genomic approaches cannot be used for fluids such as hemolymph. The proteins in the hemolymph of Drosophila melanogaster have been extensively researched. The second dimension (2D) -map of hemolymph proteins of Drosophila was constructed to serve as a reference database for researchers investigating the changes that occur at protein level in different developmental stages, physiological conditions or after infection (Vierstraete et al. 2003). The 2D-database was updated by de Morais Guedes et al. (2003). In one of the first studies of this kind, Vierstraete et al. (2004) employing 2D difference gel electrophoresis (DIGE) identified 10 differential proteins that appear in the fruit fly hemolymph very early after immune-challenge with LPS. Using 2D-gel electrophoresis (2DE), Levy et al. (2004) found that more than 70 out of a total of 160 hemolymph proteins from Drosophila were modulated by at least fivefold after microbial infection. MS and data bank searches revealed the identity of proteins directly involved in innate immunity such as proteases, protease inhibitors and recognition molecules. Proteins with a potential function in the immune response as well as molecules resulting from the cleavage of proteins were also identified in this study.

Since the first paper was published by Madden et al. (2002) highlighting the complications associated with using proteomics approaches to characterize protein profiles of two related tick species, Amblyomma americanum and A. maculatum, several papers using the above approach have appeared in the tick field (Untalan et al. 2005; Vennestrøm and Jensen 2007; Oleaga et al. 2007; Francischetti et al. 2008a, b; Rachinsky et al. 2007, 2008; Wickramasekara et al. 2008). Most of these papers described the identification of either salivary gland or larval proteins from a few hard and soft tick species. Only two papers have reported using differential protein expression to identify proteins modulated in Babasia bovis- infected Rhipicephalus (Boophilus) microplus ovaries and midgut (Rachinsky et al. 2007, 2008).

Hemolymph molecules associated with tick immunity that have been described thus far include lysozyme, defensins, histidine-rich peptides, non-cationic peptides as well as an alpha-macroglobulin like glycoprotein (reviewed in Sonenshine and Hynes 2008; Taylor 2006). To date no information on innate immunity has been published for O. savignyi. To our knowledge no proteomics investigations have been performed to identify hemolymph proteins involved in tick innate immunity. This paper is the first report of a differential analysis performed in an attempt to identify hemolymph proteins involved in the immediate defense response of O. savignyi to Candida albicans challenge. In addition preliminary characterization of hemolymph proteins involved in the recognition and binding of the yeast cells is also described.

Materials and methods

Ticks

Ornithodoros savignyi ticks were collected from the Upington region (Northern Cape, South Africa), by sifting of sand, placed in plastic containers containing sterile sand and kept at room temperature. Adult female ticks were used in all experiments.

Culturing of microorganisms

Candida albicans in pellet form was obtained from the American type culture collection (ATCC catalog no. 90028TM, USA) and cultured according to the supplier’s specifications. For long term storage, 20 ml of the yeast culture was aliquoted into Eppendorf tubes (500 μl of yeast each) and 500 μl of 25% glycerol. The tubes were stored at −70°C. For the challenge of ticks with C. albicans, an aliquot of the yeast culture was thawed at room temperature and 500 μl of the culture added to 9.5 ml of potato dextrose broth (PDB) and grown overnight at 30°C in a shaking incubator. The culture (1 ml) was then centrifuged at 8,200g for 15 min, the supernatant removed and the pellet reconstituted in 1 ml of sterile saline (0.9% NaCl) to obtain an OD600 nm = 0.25. The suspension was inactivated by boiling at 95°C for 5 min (heat killed).

Challenge of ticks and hemolymph collection

Ticks were immobilized onto Petri-dishes with their ventral sides up using double sided tape. Before inoculation, tick integuments were wiped with ethanol. Ticks were injected into their idiosoma between their second and third coxa using a 10 μl Hamilton micro syringe (Hamilton Co., Reno, Nevada) and a 30 G1/2 sterile needle (Becton–Dickinson and Co.). Hemolymph was collected from ticks by piercing their legs with a needle after they were immobilized as described above. The hemolymph was extracted with a PCR micropipette (1–5 μl, Drummond Scientific Co., USA) and collected into Eppendorf tubes containing either lysis solution or tick bleeding buffer (TBB).

For 2D differential expression experiments, 25 ticks were injected with either 1 μl of sterile saline (aseptic injury control; S2) or heat-killed yeast cells (Y2) and the hemolymph collected after 2 h into a lysis solution containing 8 M urea, 5% (w/v) CHAPS and a mixture of protease inhibitors (Complete, Roche Diagnostics) at 4°C. Hemolymph obtained from 25 non-injected ticks served as the unchallenged control (UN). The samples were centrifuged for 15 min at 8,200g at 4°C and the protein content of the supernatants was determined using the Bradford method (1976).

For the isolation of yeast-binding proteins from hemolymph plasma, 50 ticks were injected with either 1 μl of sterile saline (S2), 5.4 μg β-1,3-glucan (B2) or heat killed yeast cells (Y2) and the hemolymph collected from the ticks 2 h post challenge. Hemolymph obtained from 50 non-injected ticks served as the unchallenged control (UN). For each of the four experimental conditions, the collected hemolymph (approximately 100 μl) was added to an equal volume of TBB (50 mM sodium hydrogen phosphate, 0.15 mM sodium chloride, 2 mM EDTA, 0.02% sodium azide, 2 mM phenylthiourea, protease inhibitor cocktail, pH 7.0) at 4°C. The hemolymph samples were centrifuged for 15 min at 8,200g (4°C) to obtain plasma samples.

Isolation of yeast-binding proteins from unchallenged and challenged hemolymph plasma samples

The procedure followed was adapted from the method described by Zhu et al. (2005). For saturated cultures, C. albicans was grown overnight at 30°C in a shaking incubator. Overnight cultures were washed once with 0.9% NaCl and twice with phosphate buffered saline (PBS). Cells were sedimented by centrifugation at 12,000g for 2 min after each wash. The washed cells were re-suspended in a small volume of PBS (1/4 of original volume) and incubated with the unchallenged or challenged hemolymph samples. As a control, yeast cells were incubated with TBB alone. After incubation of samples with rotation for 10 min, the yeast cells were pelleted by centrifugation for 2 min at 12,000g and washed twice with PBS. Bound proteins were eluted with 25 μl 0.15 M triethylamine, pH 11.5. The cells were pelleted and the eluted proteins were concentrated with acetone precipitation (one part protein: four parts acetone) and re-suspended in 10 μl first-dimensional electrophoresis (1DE) reducing buffer (0.06 M Tris, pH 6.8, 2% SDS, 0.1% glycerol, 0.05% β-mercaptoethanol and 0.025% bromophenol blue). Samples were analyzed by 1DE according to the method described by Laemmli (1970) using 12.5% polyacrylamide gels. Controls representing total hemolymph proteins (HL) and total yeast proteins (YC) were included. For the total hemolymph protein control, unchallenged hemolymph was used, while for the yeast protein control, heat killed yeast cells were sonified (Branson Sonic Power Company, USA), centrifuged at 8,200g for 15 min and the collected supernatant used for analysis. Electrophoresed proteins were detected by staining gels either with MS-compatible silver stain (Yan et al. 2000) or colloidal Coomassie G-250 (Candiano et al. 2004) for MS/MS analysis.

2DE: Isoelectric focusing

Hemolymph sample volumes were adjusted in order to analyze the same amount of protein (60 μg) for each set of experiments. Each sample was applied by including it in the rehydration solution [8 M urea, 0.5% (w/v) CHAPS, 0.125% (v/v) IPG buffer]. DTT was added to the rehydration solution prior to use to give a final concentration of 18 mM. Rehydration of 7 cm immobilized pH gradient strips (IPG-strips; non-linear pH gradient 3–10) and Isoelectric focusing (IEF) were performed at 20°C using the Ettan IPGphor II system (Amersham Bioscience). After rehydration for 12 h, IEF was performed at 100 V for 30 min, 500 V for 30 min, 1,000 V for 30 min and 5,000 V for 1 h 40 min. The current was limited to 50 μA/IPG strip.

In a separate experiment, 60 μg of the 2D protein standards: conalbumin (MW = 76 kDa, pI 6.6), albumin (MW = 66 kDa, pI 5.5), actin (MW = 43 kDa, pI 5.1), glyceraldehyde-3-phosphate dehydrogenase (MW = 36 kDa, pI 8.8), carbonic anhydrase (MW = 31 kDa, pI 5.9), trypsin inhibitor (MW = 21.5 kDa, pI 4.5) and myoglobin (MW = 17.5 kDa, pI 7.0) were loaded onto an IPG strip as described above.

2DE: SDS–PAGE and image analysis

Focused IPG strips were subjected to reduction/alkylation prior to second dimensional electrophoresis. Strips were incubated for 15 min in an equilibration buffer containing 50 mM Tris–HCl (pH 8.8), 6 M Urea, 2% (w/v) SDS and 1% (w/v) DTT. The incubation was repeated for another 20 min with the same solution substituting the DTT with 2.5% (w/v) iodoacetamide. 2DE was carried out on 12.5% gels using a Hoefer mini vertical electrophoresis system (miniVE) at 60 V for 30 min and then 120 V until the bromophenol-blue front reached the bottom of the gel. Thereafter, the gels were placed into a fixing solution containing 30% ethanol, 10% acetic acid, before staining. The spots were detected with Flamingo fluorescent stain (BioRad) according to the manufacturer’s instructions for quantitative analysis.

Each experimental condition was repeated four times. The 2D gels were scanned with a Pharos FX Plus Molecular imager (BIORAD). The four biological repeats were used to produce a master image for each of the three experimental conditions. Spots that were present in two or more of the gels were included in the respective master images. The master images obtained for the challenged hemolymph (aseptic injury and yeast-injected) were compared with the master image obtained for unchallenged hemolymph. Generation of the master images as well as statistical analysis of the modulated spots was performed using PDQuestTM software. Differentially expressed spots were analyzed using the Student’s t-test (p < 0.05).

In-gel tryptic digestion of proteins

Selected protein spots or bands were excised from gels for MS/MS analysis at the University of York (UK). Briefly, the gel pieces were washed twice with 50% (v:v) aqueous acetonitrile containing 25 mM ammonium bicarbonate, then once with acetonitrile and dried in a vacuum concentrator for 20 min. Sequencing-grade, modified porcine trypsin (Promega) was dissolved in the 50 mM acetic acid supplied by the manufacturer, then diluted fivefold by adding 25 mM ammonium bicarbonate to give a final trypsin concentration of 0.01 μg/μl. The gel pieces were rehydrated with 10 μl of trypsin solution and covered with 25 mM ammonium bicarbonate solution for 30 min. These digests were incubated overnight at 37°C.

Matrix-assisted laser desorption ionization- time-of-flight mass spectrometry (MALDI-TOF/TOF) and database searches

For the MS analysis 1 μl aliquot of each fraction was applied to the ground steel MALDI target plate. An equal volume of freshly prepared 5 mg/ml 4-hydroxy-α-cyano-cinnamic acid [Sigma, in 50% aqueous (v:v), acetonitrile containing 0.1% trifluoroacetic acid (v:v)] was then added to the fractions. A positive-ion MALDI-MS spectra was obtained (Bruker Ultraflex III in reflectron mode, equipped with a Nd:YAG smart beam laser) and the MS spectra were acquired over a m/z range of 800–4,000. The final mass spectra were externally calibrated against an adjacent spot containing six peptides (des-Arg1-bradykinin, 904.681; angiotensin I, 1,296.685; Glu1-fibrinopeptide B, 1,750.677; ACTH (1–17 clip), 2,093.086; ACTH (18–39 clip), 2,465.198; ACTH (7–38 clip), 3,657.929.). Monoisotopic masses were obtained using a SNAP averagine algorithm (C 4.9384, N 1.3577, O 1.4773, S 0.0417, H 7.7583) and a S/N threshold of 2.

The 10 strongest peaks of interest for each spot were then selected for MS/MS fragmentation (signal to noise ratio greater than 10). The fragmentation was performed without the introduction of a collision gas. A default calibration was used for the MS/MS spectra, which were first baseline-subtracted and smoothed (Savitsky-Golay, width 0.15 m/z, cycles 4); monoisotopic peak detection used a SNAP averagine algorithm (C 4.9384, N 1.3577, O 1.4773, S 0.0417, H 7.7583) with a minimum S/N of 3. Bruker FlexAnalysis software was used to perform the spectral processing and peak list generation for both the MS and MS/MS spectra.

MS/MS ion searches of the NCBInr database were performed using a locally-running copy of the Mascot program (Matrix Science Ltd., version 2.1), through the Bruker BioTools interface (version 3.1). Search criteria included: carbamidomethyl (C) as a fixed modification for all alkylated samples, enzyme, trypsin; variable modifications, oxidation (M); peptide tolerance, 200 ppm; MS/MS tolerance, 0.8 Da; instrument, MALDI-TOF-TOF.

For the de novo sequence interpretations from the tandem mass spectra PredictSequence routine (BioMultiview 1.4 software, MDS Sciex) was used. The complete and partial peptide sequences obtained were then searched with MS BLAST using PAM30 matrix and the nrdb95 database (NCBI). The search was performed with the WU-BLAST2 program (Gish W (1996–1999) http://blast.wustl.edu) on an EMBL server http://dove.embl-heidelberg.de/Blast2/.

Additionally de novo sequences were compared with virtual translations of the sequences deposited in the current tick EST databases at the Gen Index databases (http://compbio.dfci.harvard.edu/tgi/), namely, A. variegatum, R. microplus, Ixodus scapularis and Rhiphicephalus appendiculatis using the tBLASTn program.

Results and discussion

Comparative 2D-gel analysis of hemolymph proteins

Previous 1DE of hemolymph plasma proteins (results not shown) indicated that the most abundant proteins have molecular weights above 76 kDa, while fewer proteins were observed in the molecular weight range of 14–66 kDa. The fact that large (~200 kDa) proteins, hydrophobic proteins and basic (pI > 8) proteins can all precipitate during IEF and are poorly resolved by 2DE (Yarmush and Jayaraman 2002) may explain why protein spots >76 kDa were absent from 2D gels. In this study the 2D comparative analysis was thus restricted to proteins in the molecular weight range of 17.5–76 kDa.

To determine which proteins are differentially expressed in the hemolymph, the profile obtained for unchallenged hemolymph (UN) was compared to the profiles obtained for ticks injected with saline (S2) or heat killed C. albicans (Y2), respectively. Differentially expressed spots (P < 0.05) are indicated on the 2DE master images (Fig. 1) as determined by PDQuest. Two proteins were found to be down-regulated in the saline control, while four protein spots were up-regulated and one protein spot down-regulated in the hemolymph of ticks challended with yeast (Table. 1). The modulated proteins are all acidic proteins (pI 4.8–6.4) and their molecular weights range from 22 to 64 kDa. The proteins modulated by yeast challenge can be assigned to the immune response as they were not induced after aseptic injury.

Fig. 1
figure 1

Comparative 2D-gel analysis of hemolymph proteins. Comparison of the master image obtained for hemolypmh proteins from unchallenged ticks with (a) the image for proteins from saline-injected ticks and (b) the image for proteins from yeast injected ticks. Each experimental condition was repeated four times. The four biological repeats of each experimental condition were used to produce a master image and this was compared to the master image from the four biological repeats obtained from unchallenged ticks, respectively. Spots that are present in two or more of the gels are included in the master image. The numbered dark spots are differentially expressed (P < 0.05) as determined by PDQuest software

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Table 1 Summary of differentially expressed spots
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It was previously observed that inoculation of O. savignyi ticks with either heat killed gram-positive or gram-negative bacteria increased the gram-positive antibacterial activity in the hemolymph up to 2 h after which the activity decreased (results not shown). Based on the above observation hemolymph was collected 2 h post challenge as hemolymph proteome changes would identify proteins involved in the tick’s immediate response to challenge before the induction of biosynthesis of defense peptides and proteins. The five modulated proteins analyzed in this study are most likely involved in the very first line of the tick’s defense against yeast. The four up-regulated proteins are probably released into the hemolymph plasma from storage sites in either hemocytes or from the fat body. The decrease in the level of the one protein suggests that due to its involvement in the immediate response it is rapidly depleted. In a differential proteomic analysis of Drosophila hemolymph, Levy et al. (2004) found that at least 70 proteins were up- or down-regulated after a bacterial or fungal challenge. These studies were performed on hemolymph collected 6 and 72 h, post bacterial and fungal challenge, respectively. Maximum alterations in protein expression occurred at these selected time points. However, similar to the results obtained in our study, Vierstraete et al. (2004) identified only 10 differentially expressed proteins in Drosophila hemolymph 25 min after challenge with LPS, suggesting that these proteins are involved in the very early defense response of the fruit fly.

A total of five spots (no 3–7, Fig. 1b) were subjected to MS/MS analysis. Peptide mass fingerprint searches were not performed, instead the MS/MS ion spectra data obtained for each protein were used to search for homologous proteins using MASCOT software. The results (Table 2) show that MS/MS ion searches failed to positively identify any of the five modulated proteins. For proteins not available in databases, it is necessary to determine partial or complete amino acid sequences (Shevchenko et al. 2001; Samyn et al. 2006). De novo sequencing based on MS/MS data provided amino acid sequence data for the proteins (Table 3). BLAST searching of the NCBInr database with these sequences also failed to identify these proteins. For instance, in the case of spot 3, each of the four derived amino acid sequences for this protein matched a different protein in the database. No significant alignments were observed between the deduced peptide sequences of the soft tick, O. savignyi, and the sequences in the tick EST databases. The fact that the proteins in this study do not match the sequences deposited in databases for salivary gland proteins of O. parkeri and O. coriaceus (Francischetti et al. 2008a, b), suggests that these proteins are most likely not expressed in salivary glands. The limited number of tick sequences available in databases is a serious drawback to the identification of tick proteins using proteomic approaches. Rachinsky et al. (2007) investigated the proteins that were differentially expressed in ovarian tissues collected from Babesia bovis-infected and uninfected R. microplus and could only identify 19 of the 43 differentially expressed proteins. Similarly, Oleaga et al. (2007) could only identify 27 spots out a total of 97 analyzed in the salivary glands of O. moubata and O. erraticus.

Table 2 MS/MS ion search results for proteins modulated by yeast challenge
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Table 3 De novo sequence results for proteins modulated by yeast challenge
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In this study 7-cm strips were employed because hemolymph samples were extremely limited in quantity. A higher protein load using longer strips would have resulted in more spots and could have improved protein identification. Another point worth mentioning is the presence of high- abundance proteins of high molecular weight in hemolymph samples which prevented optimal focusing and limited loading capacity of low-abundance proteins. In an attempt to remove high-abundance proteins initial hemolymph sample preparation included filtration of samples through a size-exclusion filter (cut-off, 100 kDa). 1DE analysis of the filtrate revealed that the filter excluded all proteins of molecular weight above 30 kDa (results not shown). This additional sample preparation step was thus omitted as it resulted in substantial protein loss and was unsuitable for enriching the proteins of interest.

Yeast-binding proteins from hemolymph

Due to the limitations associated with 2DE, alternative approaches are required to detect additional hemolymph proteins involved in the immune response. C. albicans cells were used as affinity beads to adsorb proteins from the hemolymph plasma. The strategy used in our study was based on the method described by Zhu et al. (2005) who were able to identify a functional homolog of vertebrate complement 3 in the hemolymph of the horseshoe crab, Carcinoscorpius rotundicauda, using gram-positive Staphylococcus aureus. Using a similar strategy Koizumi et al. (1997) isolated a protein involved in the clearance of E. coli from the larval hemolymph of the silkworm Bombyx mori.

In these experiments, yeast cells were incubated with UN and with hemolymph collected from ticks 2 h after injection with saline (S2) or β-1,3-glucan (B2) or heat killed yeast (Y2). In each case the bound proteins were eluted from the yeast cells with triethylamine, pH 11.5, and analyzed with 1DE (Fig. 2). The results showed that several proteins present in unchallenged hemolymph as well as hemolymph collected from both injured and yeast-challenged ticks bound to the yeast cells. These proteins were not present when yeast cells were incubated with buffer alone, confirming that the eluted proteins were of hemolymph origin. The 97 and 88 kDa proteins correspond to high-abundance proteins present in the hemolymph plasma control and may be binding to yeast non-specifically. However, the 26 kDa protein showed a clear differential pattern and appeared mainly in the hemolymph fraction collected after β-1,3-glucan injection. For these experiments, hemolymph was collected directly into TBB followed by centrifugation to remove the hemocytes. It can thus be assumed that the plasma samples prepared in this manner represent mainly plasma proteins as lysis of hemocytes was prevented. One can speculate that these proteins may play an important role in the recognition and removal of yeast cells in tick plasma. They are possibly pattern-recognition proteins that specifically bind to β-1,3-glucans, conserved surface determinants of fungi. Similar experiments using gram-negative E. coli as affinity beads resulted in the analysis of two different high molecular weight bacteria-binding proteins from tick hemolymph (results not shown). However, these gram-negative binding proteins were only detected in hemolymph after either aseptic or bacterial challenge. These preliminary findings suggest that tick hemolymph plasma contains different proteins involved in pathogen recognition, some being permanently present while others are released following either aseptic or septic activation of the immune response.

Fig. 2
figure 2

1D-gel analysis of hemolymph proteins bound to Candida albicans. Yeast cells were incubated with only buffer (C), unchallenged hemolymph (UN) or hemolymph collected 2 h after injecting ticks with either saline (S2), β-1,3-glucan (B2) or heat-killed yeast (Y2). Eluted proteins were precipitated with acetone, re-suspended and analyzed on a 12.5% gel. In each case 10 μl of protein was loaded and visualized by silver staining. Controls representing total hemolymph plasma proteins (HL) and total yeast proteins (YC) were included. The molecular masses of the standard proteins are on the left

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For their identification, the experiment was repeated and the three proteins (97, 88 and 26 kDa) were excised from a colloidal Coomassie stained 1DE gel and subjected to MS/MS analysis. MASCOT searches using the MS/MS ion spectra (Table 4) as well as BLAST searches using derived de novo sequences (Table 5), failed to positively identify any of the C. albicans binding proteins.

Table 4 MS/MS ion search results for Candida albicans binding proteins
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Table 5 De novo sequence results for Candida albicans binding proteins
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Conclusion

This study enabled the analysis of hemolymph proteins involved in the immediate immune response of the tick when challenged with C. albicans. The proteins analyzed by differential expression are released from their storage sites and are most likely involved in the very first line of the tick’s defense against yeast. These proteins can be assigned to the immune response as they were not induced after aseptic injury. Using the yeast cells as affinity beads allowed the analysis of additional hemolymph proteins involved in the yeast-response, not detected with differential expression analysis. For these investigations a combination of strategies is therefore necessary in order to obtain the maximum amount of information on the proteins involved in a process as complex as innate immunity.

None of the proteins analyzed in this study could be identified. The limited number of tick protein sequences available in databases is a drawback to the identification of tick hemolymph proteins using a proteomics approach. However, the number of sequence information is rapidly growing and the MS/MS data as well as de novo sequences obtained for these proteins may be archived and used to search databases in the future. Until then, de novo peptide sequences may be used for the design of degenerate primers for cloning of the genes of the corresponding proteins by a PCR-based approach. RNA interference studies can be performed to investigate the functional role of these proteins in tick innate immunity. Such investigations are in progress.

In conclusion, results obtained from these and similar studies will lead to a better understanding of tick innate immunity. Moreover, information obtained from structural and functional characterization of tick proteins involved in pathogen defence may be applied in future for the design of novel anti-infective agents.