Antimicrobial peptides represent an essential alternative first line of defense. Naturally occurring molecules associated with the innate immune system in disease-bearing vectors such as mosquito, tick could be the target for searching more potent and effective agents to combat against the pathogens resistant to conventionally used antibiotics. Recently, we explored expression of a defensinlike peptide, longicin from the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analog (P4) displayed antimicrobial/fungicidal/parasiticidal activity and, therefore, proposed to be a chemotherapeutic compounds against tick-borne disease organisms. Structural characterization of antimicrobial peptides is very important to understand the peptide activity. In addition, harmful side effects such as lysis of red blood cells or cytotoxicity towards mammalian host cells commonly associated with antimicrobial peptides as potential therapeutic agent should also be elucidated. In this study, we analyzed some structural features using bioinformatics tool, CD Spectroscopy, and also determined cytolytic activity of P4 peptide. According to the chemicophysical characteristics, the P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of β-sheet which was also observed in modelled tertiary structure. CD spectroscopy results also revealed the existence of a β-sheet and changes of helical content in the presence of membrane-mimic condition. These structural observations on P4 suggest that the antimicrobial activity could be due to the well developed β-sheet. In addition, sequence homology search showed that antimicrobial molecule identified in other ticks and in organisms have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved antimicrobial domain. The peptide P4 also showed less cytolytic activity against various cell lines or erythrocytes of various species. The data presented here strongly suggests that the peptide P4 could be developed as future therapeutic agent against tick-borne microorganisms.

The life cycle of ticks is characterized by alternate off-host and on-host conditions. The life span is estimated at several years and most ixodid ticks spend more than 95% of their life off the host. They seem to have a unique strategy to endure the off-host state for a long period. By electron microscopy, isolation membrane-, autophagosome- and autolysosome-like structures were found in the midgut epithelial cells of unfed ticks. Therefore, we focused on autophagy which is well-conserved from yeast to higher eukaryotes and induced by starvation. We have identified homologues of autophagy-related (ATG) genes (ATG3, ATG4 and ATG8) from cDNA libraries of the 3-host tick Haemaphysalis longicornis. Each expression profile of H. longicornis ATG (HlATG) genes and HlAtg proteins at the stages of nymph and adult were examined by real-time PCR and immunoblotting. Moreover, autophagy is known to be induced by inactivation of target of rapamycin (TOR), a phosphatidylinositol kinase. To examine the effect of TOR function on the expression of HlAtg protein(s), rapamycin, a specific inhibitor of the signal transduction mediated by TOR, was injected to unfed adults. It was revealed that the expression of HlAtg protein(s) was enhanced in response to the rapamycin. This result indicates that tick have the nutrient-sensitive TOR signaling pathway which regulate autophagy.