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

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 AamAch-S cDNA are 1959 and 1718 bp long, containing 1332 and 1104 bp 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 AamAch-S mRNA are expressed in multiple organs as revealed by qualitative RT-PCR analysis. Furthermore, quantitative reverse transcription polymerase chain reaction analysis revealed that AamAch-L mRNA was downregulated in the mid-gut, 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 probably associated with formation and/or maintenance of stability of A. americanum tick cement cone. Dual RNA interference 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 AamAch-L failing to hydrolyse chitinase substrates. Unpublished related work in our laboratory, and published work by others that found AamAch-L in tick saliva, suggest that native AamAch-L is a non-specific immunoglobulin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 nonspecifically bound rabbit, bovine and chicken non-immune sera. We discuss findings in this study with reference to advancing knowledge on tick feeding physiology.

Tick attachment cement – reviewing the mysteries of a biological skin plug system

“…This sealing prevents loss of fluids and increases the effectiveness of the muscular pharynx, which is responsible for blood uptake (Saito & Ohara, 1961; Coons & Alberti, 1999). Intense bleeding at the feeding sites of Amblyomma americanum (Linnaeus, 1758) after silencing presumed cement compounds (Kim et al ., 2014) corroborates this assumption.…”

“…To address this problem, temporary expression patterns of the salivary glands were analysed (Radulovic et al ., 2014; Kim et al ., 2016; Bullard, Williams & Karim, 2016), showing which salivary products were produced at certain points during feeding. These subsets of proteins were then tested for their functions by specific inactivation in living ticks and verification in feeding trials (Kim, Curran & Mulenga, 2014). However, some proteins of salivary glands have very similar nucleotide sequences, hampering efforts at specific silencing (Bullard et al ., 2016).…”

“…Their inactivation (silencing by RNA interference) impaired cement deposition leading to a significant decrease in attachment; treated ticks could be detached with a light touch. Furthermore, their feeding sites often showed extensive bleeding (Kim et al ., 2014). Both proteins lack putative chitin‐binding domains and are also expressed in other parts of the tick body (Kim et al ., 2014).…”

“…Furthermore, their feeding sites often showed extensive bleeding (Kim et al ., 2014). Both proteins lack putative chitin‐binding domains and are also expressed in other parts of the tick body (Kim et al ., 2014). Recently, two chitinases have been identified from the saliva of Ixodes scapularis Say, 1821, one of them inactive and with 64–65% similarity to the chitinase‐like proteins from Amblyomma americanum .…”

Ixodes scapularistick saliva proteins sequentially secreted every 24h during blood feeding

“…It is interesting to note that both the active and inactive forms of chitinase were identified in the first 48 h. The former is highlighted by a peritrophin-A chitin-binding domain, which is involved in remodeling the chitinous tick exoskeleton, particularly the mouthpart [144,145]. The latter is highly identical to A. americanum tick feeding stimuli responsive acidic chitinase [134], which when silenced by RNAi caused ticks to loosely attach onto host skin [146]. Blast2seq alignments revealed that the two I. scapularis inactive chitinases (EEC01936.1 and JAB70416.1) identified in both 24 and 48 h saliva are respectively 64 and 65% identical to A. americanum inactive chitinase (AIR95100.1).…”