Expression Analysis and Target Validation of Genes Encoding Cement Proteins in Amblyomma americanum Tick Saliva

Abstract

Without feeding success, ticks can neither cause damage nor transmit disease agents to their hosts. From this perspective a deeper understanding of tick feeding physiology has been advocated. Hard ticks such as Amblyomma americanum are adapted to complete feeding over several days. A combination of key biological adaptations allows the tick to complete feeding without the host detecting the presence of the tick. One of these adaptations is the deposition of the tick cement cone, which glues tick mouthparts into the host’s skin tissue and prevents the host from grooming off the tick. Thus, understanding the molecular basis of how the tick cement cone is formed and deposited could lead to the discovery of important targets for development of methods against tick feeding and transmission of disease agents. One of the lines of research in the lab is to understand tick cement formation and deposition through discovery of the protein composition of the tick cement cone. Prior to this thesis research, a colleague in the lab utilized LC-MS/MS sequencing to identify 85 proteins in tick cement that was recovered from manually detached adult Amblyomma americanum ticks. This thesis determined the functional roles of glycine-rich proteins (n=13) of the 85 tick cement proteins in A. americanum tick feeding. This was motivated by published chemical analysis studies that demonstrated that the majority of proteins in the tick cement cone are characterized by high content of glycine amino acid residues. Data in this thesis research indicates that abundance of encoding mRNA of the glycine-rich proteins increase with blood meal feeding with majority of these proteins being associated with tick feeding events through five days feeding. Of significant interest, the transcription of some of the encoding mRNAs coincided with the developmental period when ticks attained appetence, implying that these could represent the tick's molecular preparation to start feeding. Additionally, RNAi silencing of some of the encoding mRNAs affected tick attachment onto host skin as indicated by apparent inflammation and subsequent bleeding around tick attachment sites. In conclusion, this thesis has contributed to our understanding of the molecular basis of tick cement physiology.