Receptors for the neuropeptides, myoinhibitory peptide and SIFamide, in control of the salivary glands of the blacklegged tick Ixodes scapularis
Graphical abstract
Highlights
► Receptors for two neuropeptides, MIP and SIFamide, were identified and characterized in the tick salivary glands. ► Immunohistochemistry of SIFamide receptor revealed three candidate target cell types of the SIFamide. ► The target cell types of the SIFamide are proposed to be myoepithelial cell, basal granular cells, and neck cells.
Introduction
A large number of G protein-coupled receptors (GPCRs) function as receptors for neuropeptides and protein hormones that control a vast majority of biological functions, such as reproduction, development, molting, behavior and feeding, in arthropods. In genomic and post-genomic analyses of many model arthropod species, deorphanization of neuropeptides and their receptors has led to the discovery of new peptidergic systems and functions. A combination of bioinformatics and advanced biotechnologies, such as peptidomic analyses and RNA interference (RNAi), has revealed more comprehensive lists of neuropeptides and their receptors and facilitated the investigation of their biological roles and evolutionary processes.
The blacklegged tick, Ixodes scapularis, is a well-known vector of Borrelia burgdorferi, which causes Lyme disease. An analysis of the I. scapularis genome (Hill and Wikel, 2005) has provided opportunities for the exploration of the biology of neuropeptides and their receptors in this unique hematophagous arthropod. A female tick must feed on a host for at least one week to achieve full engorgement. During feeding, the tick's body weight increases approximately 100-fold. The salivary secretion of the tick mediates pathogen transmission and promotes successful feeding by suppressing and modulating the host's immune system (Bowman et al., 1997; Bowman and Sauer, 2004; Ribeiro et al., 2006). In addition, salivary secretion facilitates the excretion of excessive water and waste during feeding (Kaufman and Phillips, 1973a,b; Sauer and Hair, 1971).
The mechanisms involved in the control of tick salivary secretion have long been under study because a better understanding of these mechanisms could lead to the development of tools to disrupt the saliva secretory process. Previous pharmacological studies suggested that pilocarpine, a muscarinic acetylcholine receptor agonist, activates the synganglion, the central nervous system of the tick, which generates the neural signals that lead to salivary secretion (Kaufman, 1978; McSwain et al., 1992). Dopamine, which is produced in the salivary glands (SGs), acts as a paracrine factor for the activation of SG acini for fluid secretion (Kaufman et al., 1999; Šimo et al., 2011a).
Tick SGs are a large pair of grape-like clusters that are located anterolaterally on the ventral part of the body cavity. Female tick SGs consist of three types of acini (I, II and III). Agranular acini type I are attached mainly to the anterior main salivary duct, while both granular acini types II and III are located more posteriorly and are attached to the main salivary duct and its branches. The types II and III acini, which are considered to be primarily involved in the production of bioactive components and the excretion of excess water and ions, respectively, are composed of several different cell types (Binnington, 1978). Given that there are a number of different bioactive salivary components that are produced and secreted from various cells in the different acini of the SGs at different feeding phases, tick salivation is likely orchestrated by a complex combination of neural and hormonal mechanisms.
The tick synganglion contains multiple neuronal cells of various sizes and shapes, which form a neuronal cortex on the surface of segmented lobes (Sonenshine, 1991). Aminergic and peptidergic neuronal cells were characterized by immunohistochemistry (IHC) (Hummel et al., 2007; Šimo et al., 2009a,b; 2011b). Our previous study utilized 15 different antibodies, which were originally raised against neuropeptides in many different species of insects and crustaceans, and found that the synganglion was rich in neuropeptides (Šimo et al., 2009a); these findings were confirmed by a peptidomic analysis (Neupert et al., 2009), as well as an analysis of homology-based neuropeptide gene prediction (Christie, 2008; Šimo and Park, unpublished data).
Šimo et al. (2009b) previously described a pair of protocerebral SG (PcSG) neurons with axonal projections that innervate the basal regions of the SG acini types II and III. The PcSG neurons and their projections contain both myoinhibitory peptide (MIP) and SIFamide, which were confirmed by matrix-assisted laser desorption/ionization (MALDI) analyses, in situ hybridization, and immunohistochemistry. In the present study, we identified and functionally characterized the receptors for MIP and SIFamide in the SGs of the blacklegged tick, I. scapularis. The temporal and spatial expression patterns of both ligands and their receptors were investigated using antibodies and quantitative PCR. This study highlights the unique neural control of tick SGs and suggests possible functions of the neuropeptidergic systems in tick salivary secretion.
Section snippets
Animals and tick feeding
Unfed adult I. scapularis ticks were obtained from a tick-rearing facility at Oklahoma State University. Approximately 30 individuals of I. scapularis, both male and female, were kept in a polypropylene tube (9 × 2.5 cm) with a small piece of filter paper (4 × 1 cm) in the vial. The tubes were kept in a dark, humid chamber at 4 °C. Tick feeding was performed on New Zealand White rabbits (Myrtle's Rabbitry, TN). Female and male ticks were placed at a 1:1 ratio in the feeding chamber that was
The genes encoding the MIP and SIFamide receptors
Blast searches for MIP and SIFamide receptors in the I. scapularis genome yielded two putative MIP receptors with highly conserved regions (mip-r1 and mip-r2) and one putative sifa-r (Fig. 1A–D). The sequences were further analyzed for predictions of gene structures and for primer designs. We obtained the full-length ORFs for mip-r1 and sifa-r, while mip-r2 lacked a 5′ translation initiation site. All receptor sequences in this study contained typical motifs for seven-transmembrane α-helical
Discussion
Multiple approaches in this study revealed authentic receptors for MIP and SIFamide and led to functional implications. Homology-based identification of the genes, followed by studies of temporal and spatial expression patterns and functional characterization of the receptors, all together support the MIP and SIFamide signaling systems as important neural factors controlling the SGs in ticks.
In the receptor functional assays, the pattern of calcium elevation, as measured by the luminescence of
Acknowledgments
We thank Dr. Richard Beeman and Joshua Urban for reviewing an earlier version of this manuscript. This paper is contribution no. 13-154-J from the Kansas Agricultural Experiment Station. The project described was supported by NIH Grant Numbers R01AI090062 and 1R21AI081136.
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2022, Ticks and Tick-borne DiseasesCitation Excerpt :In ticks and in many insects, myoinhibitory peptide (MIP), often called allatostatin B (Coast and Schooley, 2011), acts by inhibiting the contraction of hindgut and visceral muscles (Lange et al., 2012; Šimo and Park, 2014), while SIFamide stimulates their motility (Šimo and Park, 2014). Both neuropeptides innervate salivary gland acini (type II and type III) and were proposed to play a role in controlling the secretion of salivary components, which is analogous to the activity on the hindgut (Šimo et al., 2013a, 2009). Elevenin was also suggested to be involved in saliva secretion during rapid engorgement phase in I. scapularis females (Kim et al., 2018).