Silencing of three Amblyomma americanum (L.) insulin-like growth factor binding protein-related proteins prevents ticks from feeding to repletion.

The insulin-like growth factor (IGF) binding proteins (IGFBP) family is the regulatory arm of the IGF signaling system that control mitogenic and anabolic actions of IGF peptide hormones. This study describes cloning and biological characterization of three Amblyomma americanum (L.) (Aam) proteins that show amino-terminal sequence and secondary structure similarity to the IGFBP superfamily. The three molecules here provisionally identified as AamIGFBP-rP1 and short (S) and long (L) AamIGFBP-rP6 are expressed in multiple tick organs and are responsive to tick feeding activity with the former being upregulated and the latter being downregulated. We show that they regulate tick physiological functions that may be related to A. americanum tick feeding success as revealed by RNAi-mediated dual silencing of AamIGFBP-rP6S and AamIGFBP-rP6L or AamIGFBP-rP1 alone, which caused a reduction in blood meal size compared to the controls. Additionally, in the case of AamIGFBP-rP1 silencing, 47% of ticks died while attempting to feed and those that did survive and spontaneously detached from the host failed to lay eggs. Although AamIGFBP-rP6S and AamIGFBP-rP6L show overall identities of 49% and 59%, respectively, to Rhipicephalus microplus C protein, the identity level jumps to ~84% when the comparison is restricted to first 70 amino acids of the mature protein. Similarly, the AamIGFBP-rP1 mature protein is ~72%, 87%, 88% and 92% identical to that of Ixodes scapularis S, R. microplus, R. appendiculatus N and A. variegatum F, respectively. The observed across-tick-species conservation suggests that the three molecules (AamIGFBP-rP1, AamIGFBP-rP6S and AamIGFBP-rP6L) represent target for development of vaccines to protect animals against multiple tick species. The data are discussed with reference to advances in tick molecular biology and the potential of the three proteins as targets for immunizing animals against tick feeding.