Molecular cloning, expression and impact of ribosomal protein S-27 silencing in Haemaphysalis longicornis (Acari: Ixodidae)

Abstract

Ticks, obligatory blood-feeding arthropods, are a major pathogen vector in humans and animals worldwide. Anti-tick vaccines are an exciting alternative to chemical acaricides for controlling these disease-transmitting vectors. However, identification of protective antigens for anti-tick vaccine development is challenging. Different ribosomal proteins play multifunctional roles in tick survival and feeding. Here, we first report the cloning and molecular characterization of ribosomal protein S27 (RPS-27) from the hard tick Haemaphysalis longicornis. We identified a complete open reading frame (ORF) of RPS-27: a 255-bp (base pair) cDNA encoding a mature protein of 84 amino-acid residues with a 9.4-kDa predicted molecular mass. Amino-acid sequence analysis revealed that RPS-27 was highly conserved among different tick and vertebrate animals with identity ranges of 97–98% and 60–85%, respectively. Phylogenetic tree analysis showed that RPS-27 from different tick species clustered together. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the RPS-27 mRNA transcript was expressed in all life stages. At the tissue level, it was more highly expressed in the salivary gland than in the midgut for both the fed and unfed conditions, which indicates a role for RPS-27 in tick feeding. In vitro analysis showed that recombinant RPS-27 (10-RPS-27) was successfully expressed in a pGEMEX-2 vector with an estimated 45-kDa molecular mass. The functional importance of RPS-27 was determined by gene silencing through RNA interference (RNAi). RPS-27 silencing showed a significant (P < 0.05) reduction of feeding abilityand engorgement weight after the blood meal in both nymph and adult female ticks and also significantly (P < 0.05) reduced molting rate in nymph. In addition, RPS-27 silencing in eggs led to abnormalities in shape and hatching. Taken together, our results suggest that RPS-27 is an important molecule that plays multiple roles in the tick life cycle including in both feeding and reproduction. Therefore, RPS-27 is an exciting target for future tick control strategies.