Abstract
The success of the acaricide amitraz, a ligand of the tick tyramine/octopamine receptor (a G protein-coupled receptor; GPCR), stimulated interest on arthropod-specific GPCRs as targets to control tick populations. This search advances tick physiology because little is known about the pharmacology of tick GPCRs, their endogenous ligands or their physiological functions. Here we explored the tick kinin receptor, a neuropeptide GPCR, and its ligands. Kinins are pleiotropic insect neuropeptides but their function in ticks is unknown. The endogenous tick kinins are unknown and their cDNAs have not been cloned in any species. In contrast, more than 271 insect kinin sequences are available in the DINeR database. To fill this gap, we cloned the kinin cDNA from the cattle fever tick, Rhipicephalus microplus, which encodes 17 predicted kinins, and verified the kinin gene structure. We predicted the kinin precursor sequences from additional seven tick species, including Ixodes scapularis. All species showed an expansion of kinin paracopies. The “kinin core” (minimal active sequence) of tick kinins FX1X2WGamide is similar to those in insects. Pro was predominant at the X2 position in tick kinins. Toward accelerating the discovery of kinin function in ticks we searched for novel synthetic receptor ligands. We developed a dual-addition assay for functional screens of small molecules and/or peptidomimetics that uses a fluorescent calcium reporter. A commercial library of fourteen small molecules antagonists of mammalian neurokinin (NK) receptors was screened using this endpoint assay. One acted as full antagonist (TKSM02) with inhibitory concentration fifty (IC50) of ∼45 μM, and three were partial antagonists. A subsequent calcium bioluminescence assay tested these four antagonists through kinetic curves and confirmed TKSM02 as full antagonist and one as partial antagonist (TKSM14). Antagonists of NK receptors displayed selectivity (>10,000-fold) on the tick kinin receptor. Three peptidomimetic ligands of the mammalian NK receptors (hemokinin 1, antagonist G, and spantide I) were tested in the bioluminescence assay but none were active. Forward approaches may accelerate discovery of kinin ligands, either as reagents for tick physiological research or as lead molecules for acaricide development, and they demonstrate that selectivity is achievable between mammalian and tick neuropeptide systems.
Highlights
The cattle fever tick or southern cattle tick, Rhipicephalus microplus (Canestrini), and the diseases it transmits cause significant losses to the livestock industry in tropical and subtropical regions of the world (Pérez de León et al, 2012)
We focused on processes on the left of the figure, by using transcriptomics and genomic data to clone the kinin cDNA, followed by forward pharmacological approaches toward the identification of novel ligands for the kinin receptor of the southern cattle tick, R. microplus
We listed the sequence of the 19 paracopies of putative bioactive kinins from Ix. scapularis (Table 1). Using this curated precursor protein as query, the tBLASTn results against whole genome shotgun contig (WGS) assembly and transcriptome shotgun assembly (TSA) of R. microplus identified a genomic DNA fragment of accession number LYUQ01126194.1 and four transcripts with high similarities
Summary
The cattle fever tick or southern cattle tick, Rhipicephalus microplus (Canestrini), and the diseases it transmits cause significant losses to the livestock industry in tropical and subtropical regions of the world (Pérez de León et al, 2012). The current available pesticides will fail to control populations of these ticks as many exhibit multiple mechanisms of resistance with apparently no fitness cost. We describe a model study using a forward pharmacological approach to investigate a tick neuropeptide G protein-coupled receptor (GPCR) as potential target for tick control (Figure 1). This receptor, known as leucokinin-like peptide receptor (LKR) (accession AF228521), or myokinin receptor (Holmes et al, 2000, 2003) has been suggested as a promising novel target for pest control (Lees et al, 2010; Audsley and Down, 2015; Guerrero et al, 2016; Pietrantonio et al, 2018). A kinin peptidomimetic is antifeedant and lethal to the pea aphid (Smagghe et al, 2010), prevents the blood feeding to repletion in the kissing bug, Rhodnius prolixus, decreasing the chance of a successful molt (Lange et al, 2016) and triggers avoidance behavior in the mosquito Aedes aegypti when given in a sucrose solution (Kwon et al, 2016)
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