Production of the yolk protein precursor vitellogenin is mediated by target of rapamycin (TOR) in the soft tick Ornithodoros moubata (Acari: Argasidae).

Initiation of vitellogenesis by blood feeding is essential for egg maturation in ticks. Nutrients derived from the blood meal are utilized by female ticks to synthesize the yolk protein precursor vitellogenin (Vg). Engorged Ornithodoros moubata ticks can synthesize Vg whether mated or virgin, thus O. moubata is an excellent model for studying the relative roles of blood feeding and mating in tick vitellogenesis. Injection of rapamycin into engorged O. moubata resulted in a reduction of ovarian growth and yolk accumulation in the oocytes of mated females. OmVg expression in the midgut and fat body and protein concentrations in the hemolymph significantly decreased in mated ticks after injection with rapamycin, indicating that inhibition of the nutrient-sensing target of rapamycin (TOR) pathway disrupts egg maturation at the levels of Vg expression and synthesis. These results suggest that the TOR-signaling pathway induces vitellogenesis in response to nutritional stimulation after a blood meal in O. moubata and is functionally independent of the mating-induced pathway.

The sialotranscriptome of Antricola delacruzi female ticks is compatible with non-hematophagous behavior and an alternative source of food.

The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari toward haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL-domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, anti-microbials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacruzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL-domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively.

Function, mechanism and evolution of the moubatin-clade of soft tick lipocalins.

The "moubatin-clade" of soft tick lipocalins, although monophyletic, shows clear signs of paralogy as indicated by the various functions associated with this protein family. This includes anti-platelet (moubatin), anti-complement (OMCI) and toxic (TSGP2) activities in the vertebrate host. In order to understand the evolution of function and how it relates to the various paralogs in this clade, we characterized a number of different proteins in regard to undefined function and mechanism. By utilizing gain-of-function for TSGP2 and loss-of-function for TSGP3, we show that inhibition of collagen-induced platelet aggregation by moubatin and TSGP3 is due to scavenging of thromboxane A2. Moubatin, TSGP2 and TSGP3 are also able to bind leukotriene B4 with high affinity. TSGP2 and TSGP3, but not moubatin, binds complement C5, with kinetics that indicates that conformation change occurs during interaction. A conserved loop and histidine residue in the sequences of OMCI, TSGP2 and TSGP3 are implicated in the interaction with complement C5. The data presented suggest that the ancestral function evolved in this clade was aimed at inhibition of vasoconstriction, platelet aggregation and neutrophil aggregation, primarily by scavenging of thromboxane A2 and leukotriene B4. C5 complement targeting activity evolved within this clade, probably within the Old World Ornithodorinae. The moubatin-clade itself most probably derived from the related histamine and serotonin-binding lipocalin sub-family that is conserved within the Argasidae.

Structure, function, and evolution of biogenic amine-binding proteins in soft ticks.

Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to the lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.

Identification and expression analysis of an actin gene from the soft tick, Ornithodoros moubata (Acari: Argasidae).

Actin genes are found in all living organisms and highly conserved in various animals as shown by numerous studies on actin gene expression and function. Because of this ubiquitous nature of actin, it is often used as an internal control in gene expression studies. To clarify the suitability of actin gene as an internal control in soft ticks, isolation and expression analyses of an actin gene from Ornithodoros moubata was performed. An actin gene of Ornithodoros moubata (OmAct2, GenBank accession no. AB208021) with 1,131 bp and 376 amino acid residues was identified. The homology of OmAct2 with other arthropod actin genes was greater than 80% in nucleotides and 99% in amino acids. OmAct2 gene was classified as a cytoskeletal actin type by absence of muscle-specific amino acids commonly found in insects and ubiquitous expression in all stages and both sexes. Southern blot revealed that O. moubata has four to seven actin genes. In addition, actin expression analyzed by real-time PCR before and after blood feeding was not significantly different indicating OmAct2 is an appropriate internal control for the analysis of gene expression in these ticks.

A reassessment of argasid tick salivary gland ultrastructure from an immuno-cytochemical perspective.

Previous morphological and histochemical studies of argasid tick salivary glands indicated that they were less complex than ixodid salivary glands, with only three granular cell types. The present study shows that there exist at least four different granular cell types in the salivary glands of the argasid tick Ornithodoros savignyi, based on immuno-localization of the anti-hemostatic factors, apyrase and savignygrin. Both anti-hemostatic factors were localized to dense core granule type 'a' and to granule type 'b', that shares a similar homogenous morphology with non-labeled granule type 'd'. Furthermore, the major tick salivary gland proteins (TSGPs), previously implicated in granule biogenesis, were localized to all the granular cell types. This indicates that granular cell types with different morphologies can express the same proteins, while cell types that show similar morphologies may not express the same proteins. Argasid tick salivary glands seem to be more complex than previously thought and might not be amenable to morphological classification alone. Alternative classification methodologies that rely on physical expression patterns of the salivary gland proteome might be more reliable as markers for a specific granular cell type.

Existence of phenol oxidase in the argasid tick Ornithodoros moubata.

Phenol oxidase (PO, EC 1.10.3.1) activity was detected in the hemolymph of the fourth instar nymphs of the argasid tick, Ornithodoros moubata, with peak levels corresponding to the days before the majority of the nymphs had molted, suggestive of a protective role of PO during the ecdysial phase. Higher PO activity was detected in plasma relative to the hemolymph and was negligible in hemocytes. The concentration of the hemolymph and plasma assayed clearly influenced the level of PO activity, and was significantly reduced ( P<0.005) after treatment with 1-phenyl-2 thiourea, a specific PO inhibitor. This is the first report of the existence of PO in the hemolymph and plasma of a soft tick species. The regulation of PO activity and its precise role in soft tick immunity, particularly during the ecdysial phase, are interesting and need to be examined further.