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

Abstract

Abstract This study demonstrates that Amblyomma americanum (Aam) constitutively and ubiquitously expresses the long (L) and short (S) putative acidic chitinases (Ach) that are distinguished by a 210 base pair (bp) deletion in AamAch-S. Full-length AamAch-L and -S cDNA are 1959 and 1718bp long containing 1332 and 1104bp open reading frames that code for 443 and 367 amino acid residues proteins with the former predicted to be extracellular and the latter intracellular. Both AamAch-L and -S mRNA are expressed in multiple organs as revealed by qualitative RT-PCR analysis. Furthermore, quantitative RT-PCR analysis revealed that AamAch-L mRNA was down regulated in the midgut, but was unchanged in the salivary gland and in other organs in response to feeding. Of significant interest, AamAch-L and/or AamAch-S functions are likely associated with formation and/or maintenance of stability of A. americanum tick cement cone. Dual RNAi silencing of AamAch-L and/or AamAch-S mRNA caused ticks to loosely attach onto host skin as suggested by bleeding around tick mouthparts and ticks detaching off host skin with a light touch. AamAch-L may apparently encode an inactive chitinase as indicated by Pichia pastoris-expressed recombinant (r) AamAch-L failing to hydrolyze chitinase substrates. Unpublished related work in our lab, and published work by others that found AamAch-L in tick saliva, suggest that native AamAch-L is a non-specific immunoglobulin binding tick saliva protein in that rAamAch-L non-specifically bound rabbit, bovine, and chicken non-immune sera. We discuss findings in this study with reference to advancing knowledge on tick feeding physiology.