The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost.

The cattle fever tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae), is a one-host tick that infests primarily cattle in tropical and sub-tropical regions of the world. This species transmits deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have acquired resistance to many of the applied acaricides. Recent acaricide-resistant tick reintroductions detected in the U.S. underscore the need for novel tick control methods. The octopamine and tyramine/octopamine receptors, both G protein-coupled receptors (GPCR), are believed to be the main molecular targets of the acaricide amitraz. This provides the proof of principle that investigating tick GPCRs, especially those that are invertebrate-specific, may be a feasible strategy for discovering novel targets and subsequently new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as the myokinin- or kinin receptor, is such a GPCR. While the receptor was previously characterized in vitro, the function of the leucokinin signaling system in ticks remains unknown. In this work, the LKR was immunolocalized to the periphery of the female midgut and silenced through RNA interference (RNAi) in females. To optimize RNAi experiments, a dual-luciferase system was developed to determine the silencing efficiency of LKR-double stranded RNA (dsRNA) constructs prior to testing those in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with these two validated dsRNA constructs was verified by quantitative real time PCR (qRT-PCR) of female tick dissected tissues. Silencing was significant in midguts and carcasses. Silencing caused decreases in weights of egg masses and in the percentages of eggs hatched per egg mass, as well as delays in time to oviposition and egg hatching. A role of the kinin receptor in tick reproduction is apparent.

Molecular and expression analysis of a family of the Amblyomma americanum tick Lospins.

Serine proteinase inhibitors (serpins) are a family of structurally similar but functionally diverse proteins that regulate several important proteolytic cascades in most branches of life. We have characterized 17 Amblyomma americanum serpin cDNAs here named as ;Lospins' (L; an acronym for Lone Star tick serpin) that possess three beta-sheets, eight alpha-helices and a reactive center loop consistent with the consensus serpin superfamily secondary structures. Visual inspection of deduced amino acid sequences revealed two patterns of basic residues: (i) (86)DKSRVLKAYKRL(97) in L5 and L13-16 and (ii) (158)VRDKTRGKI(166) in all Lospins, which are similar to consensus glycosaminoglycan (GAG) binding sites (XBnXmBX, where X and B are non-basic and basic residues, n=1 or 2 and m=1, 2 or 3). On three-dimensional models, the two putative GAG binding sites mapped onto alpha-helices D and F, respectively, with calculation of electrostatic surface potentials revealing basic patches on L5 and L13-16 models that are comparable to the heparin-binding site on antithrombin. RT-PCR expression analysis of 15 selected genes showed that the majority (11/15) of the Lospins were ubiquitously expressed in the midgut, ovary and salivary glands. On a neighbor-joining phylogeny guide tree, 15 serpins from other ticks and 17 Lospins from this study, a total of 32 tick serpin sequences, segregated into five groups with Lospins in groups A and D being conserved across tick species. The discovery of Lospins in this study sets the framework for future studies to understand the role of serpins in tick physiology.