Amblyomma americanum (L.) (Acari: Ixodidae) tick salivary gland serine protease inhibitor (serpin) 6 is secreted into tick saliva during tick feeding

Abstract

SUMMARYIn order to successfully feed and transmit disease agents, ticks are thought to inject serine protease inhibitors (serpins) into the host to modulate host defense responses to tick feeding, such as inflammation, the complement activation pathway and blood coagulation. In this study, we show that Amblyomma americanum (Aam) serpin (S) 6 is putatively injected into the host during tick feeding, in that the antibody to recombinant (r) AamS6 specifically reacted with the expected ~43/45kDa AamS6 protein band on western blots of pilocarpine-induced tick saliva. Additionally, antibodies to tick saliva proteins that were generated by repeated 48h infestations of rabbits with adult A. americanum specifically reacted with rAamS6. We speculate that AamS6 is associated with regulating events at the start of the tick feeding process, as temporal and spatial RT-PCR and western blot analyses revealed that both AamS6 mRNA and protein are strongly expressed during the first 24-72h of feeding time before starting to fade from 96h. The AamS6 protein has an apparently slow turnover rate in that, although the injection of AamS6 dsRNA into unfed ticks triggered complete disruption of the AamS6 mRNA by the 48h feeding time point, western blot analysis of protein extracts of the same animals showed that the AamS6 protein that may have been expressed prior to disruption of the AamS6 mRNA was not depleted. We speculate that the presence of the AamS6 protein in ticks despite the complete disruption of the AamS6 mRNA explains the observation that RNAi-mediated silencing of the AamS6 mRNA did not affect the ability of A. americanum ticks to attach onto host skin, successfully feed and lay eggs. These findings are discussed in regards to advances in the molecular biology of ticks.

Gene expression profiling of adult female tissues in feeding Rhipicephalus microplus cattle ticks

“…1B, Table 2, Supplementary Table S3). These include putative protease inhibitors such as serine protease inhibitors or serpins (Contig 1520, Contig 6586, CK178656 and Contig 688) that are similar to those described in other tick species (Anderson et al, 2008;Francischetti et al, 2010;Chalaire et al, 2011).…”

Dual silencing of long and short Amblyomma americanum acidic chitinase forms weakens the tick cement cone stability

“…Antibodies used here were produced by repeated tick infestation of rabbits every 48 h (Chalaire et al, 2011) and repeatedly allowing ticks to feed to repletion (Mulenga et al, 2013). Our preliminary analysis showed that rAamAch-L non-specifically reacted with both pre-immune (PI) serum and immune sera to tick saliva proteins.…”

“…Western blotting analysis of rAamAch-L using antibodies to 48 h (Chalaire et al, 2011) and replete fed (Mulenga et al, 2013) A. americanum tick saliva proteins was carried out to investigate the possibility of native AamAch-L being injected into the host during tick feeding. The expectation is that if native AamAch-L was immunogenic and injected into the host during tick feeding, rAamAch-L would specifically bind antibodies to 48 h and/or replete fed A. americanum tick saliva proteins.…”

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control

“…The attachment site of longistatin-silenced H. longicornis ticks did not show pathological changes, such as hemorrhagic lesions corresponding to the blood pool, while the attachment site of ticks simultaneously silenced acidic chitinases exhibited blood leakage and these ticks can be easily removed. Various protease inhibitors that have roles in anti-hemostatic, anti-inflammatory, and immunomodulatory mechanism have been also characterized using RNAi, including a cystatin, sialostatine L [81], a Kunitz type protease inhibitor, rhipilin [82], and serine protease inhibitors (serpin) [83,84].…”