The molecular basis of the Amblyomma americanum tick attachment phase

Towards discovery of molecular signaling cascades that trigger and/or facilitate the tick attachment and formation of its feeding lesion, suppressive subtractive hybridization, high throughput sequencing and validation of differential expression by cDNA dot blot hybridization were performed on Amblyomma americanum ticks that had attained appetence and were exposed to feeding stimuli. This approach allowed for identification of 40 genes that are up regulated before ticks begin to penetrate the host skin. Based on BLAST and secondary structure homology searches as well as motif scan analyses, provisional identification was assigned to similar to 38% (15/40) of the identified genes that have been classified into 6 groups: Ligand binding ( insulin-like growth-factor binding, lipocalin/ histamine binding), immune responsive (tumor necrosis receptor associated factor 6, Microplusin-like antimicrobial), stress response proteins ( Heat shock protein [HSP] 90, HSP40, 78 kDa glucose regulated protein [GRP78]), transporter polypeptides (ABC transporter and organic anion transporter polypeptide [contains Kazal-type serine proteinase inhibitor domain]) and enzymes/regulators (extracellular matrix mettaloprotease inducer, chitinase), extracellular matrix-like proteins (tropoelastin, flagelliform silk protein). Sixty- two percent (25/40) of genes that did not show similarity to known proteins are classified as orphans. BLASTN homology search against the tick EST database revealed that 50% (20/ 40) of candidate genes are conserved in other ticks suggesting that molecular events underlying the A. americanum tick attachment phase may be conserved in other tick species. Consistent with the general assumption that genes that are up regulated in ticks before they started to penetrate host skin represented the tick's molecular preparedness to evade host defense during the attachment phase, real time RT-PCR analyses data demonstrated that the majority of the tested genes (9/11) were highly expressed during the first 24 h of feeding. Identification of genes in this study provides the framework for future studies to elucidate molecular signaling cascades that regulate early molecular events during the tick attachment phase.