Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior

Abstract

Ticks evolved various mechanisms to modulate their host's hemostatic and immune defenses. Differences in the anti-hemostatic repertoires suggest that hard and soft ticks evolved anti-hemostatic mechanisms independently, but raise questions on the conservation of salivary gland proteins in the ancestral tick lineage. To address this issue, the sialome (salivary gland secretory proteome) from the soft tick, Argas monolakensis, was determined by proteomic analysis and cDNA library construction of salivary glands from fed and unfed adult female ticks. The sialome is composed of approximately 130 secretory proteins of which the most abundant protein folds are the lipocalin, BTSP, BPTI and metalloprotease families which also comprise the most abundant proteins found in the salivary glands. Comparative analysis indicates that the major protein families are conserved in hard and soft ticks. Phylogenetic analysis shows, however, that most gene duplications are lineage specific, indicating that the protein families analyzed possibly evolved most of their functions after divergence of the two major tick families. In conclusion, the ancestral tick may have possessed a simple (few members for each family), but diverse (many different protein families) salivary gland protein domain repertoire.

Fig. 1

Proteome analysis of SGE by Edman degradation. Salivary gland extract from A. monolakensis was heat-inactivated at 80°C, precipitated (P) and soluble (S) proteins were separated by centrifugation. The soluble and insoluble fractions were then fractionated by one-dimensional SDS-PAGE, blotted onto PVDF membranes and bands were cut-out for Edman sequencing. Edman sequences are indicated, as well as their corresponding sequences found in the cDNA library. The protein families and the number of transcripts are indicated in brackets.

Fig. 2

Two-dimensional electrophoresis of salivary gland extract from A. monolakensis. Approximately 50 μg total SGE was resolved on the gel. All prominent proteins spots (78 spots) were excised for peptide mass fingerprinting. Spots that could be matched to cDNA library transcripts are indicated. Protein families are shown in brackets.

Fig. 3

Two-dimensional liquid chromatography followed by peptide mass fingerprinting. Indicated is fractionation of SGE by anion exchange chromatography (AEC). The flow-through from the AEC was fractionated with cation exchange chromatography (CEC). Fractions (R1-R17) from AEC and CEC were fractionated using reversed phase chromatography (RPC). Peaks were collected and those identified by peptide mass fingerprinting are indicated.

Fig. 4

The BTSP, 7DBF and 18.7 kDa families belong to the thrombospondin repeat superfamily. A) Single domain BTSP members were aligned with TSP1 and TSP4 domains of f-spondin as well as Salp14, a representative BTSP from the genus Ixodes. B) Alignment of the 7DBF family with human TSP2 (Tan et al., 2002) at its N-terminal domain and TSP4 from f-spondin at its C-terminal domain (Pääkönen et al., 2006). C) Alignment of 18.7 kDa with TSP1 and TSP4 domains of f-spondin (Pääkönen et al., 2006). TSP1 was aligned with the N-terminal domain and TSP4 with the C-terminal domain. Indicated are the conserved cysteine patterns and predicted disulphide bonds as obtained from the structures of human TSP2 (Tan et al., 2002) and TSP1 and TSP4 from f-spondin (Pääkönen et al., 2006). Accession numbers for sequences are included in sequence name.

Fig. 5

Alignment of the 8kDa family from A. monolakensis and the ixodegrins from Ixodes. Indicated are the conserved cysteine pattern and RGD-motif. Accession numbers for sequences are included in sequence name.

Fig. 6

Phylogenetic analysis of the BTSP family. A neighbor-joining analysis shows nodes with more than 90% bootstrap support (10000 replicates) labeled with black dots. The gray dot indicates a single Ixodes sequence that grouped within the Argas clade with weak support.

Fig. 7

Phylogenetic analysis of the tick lipocalin family. A neighbor-joining analysis shows nodes with more than 60% bootstrap support (10000 replicates) labeled with black dots.