Receptors for the neuropeptides, myoinhibitory peptide and SIFamide, in control of the salivary glands of the blacklegged tick Ixodes scapularis

doi:10.1016/j.ibmb.2013.01.002

Insect Biochemistry and Molecular Biology

Volume 43, Issue 4, April 2013, Pages 376-387

https://doi.org/10.1016/j.ibmb.2013.01.002 Get rights and content

Abstract

Tick salivary glands are important organs that enable the hematophagous feeding of the tick. We previously described the innervation of the salivary gland acini types II and III by a pair of protocerebral salivary gland neurons that produce both myoinhibitory peptide (MIP) and SIFamide (Šimo et al., 2009b). In this study we identified authentic receptors expressed in the salivary glands for these neuropeptides. Homology-based searches for these receptors in the Ixodes scapularis genome sequence were followed by gene cloning and functional expression of the receptors. Both receptors were activated by low nanomolar concentrations of their respective ligands. The temporal expression patterns of the two ligands and their respective receptors suggest that the SIFamide signaling system pre-exists in unfed salivary glands, while the MIP system is activated upon initiation of feeding. Immunoreactivity for the SIFamide receptor in the salivary gland was detected in acini types II and III, surrounding the acinar valve and extending to the basal region of the acinar lumen. The location of the SIFamide receptor in the salivary glands suggests three potential target cell types and their probable functions: myoepithelial cell that may function in the contraction of the acini and/or the control of the valve; large, basally located dopaminergic granular cells for regulation of paracrine dopamine; and neck cells that may be involved in the control of the acinar duct and its valve.

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.

References (46)

K.,C. Binnington
Sequential changes in salivary gland structure during attachment and feeding of the cattle tick Boophilus microplus
Int. J. Parasitol.
(1978)
A.E. Christie
Neuropeptide discovery in Ixodoidea: an in silico investigation using publicly accessible expressed sequence tags
Gen. Comp. Endocrinol.
(2008)
L.B. Coons et al.
Evidence of a myoepithelial cell in tick salivary glands
Int. J. Parasitol.
(1994)
C.A. Hill et al.
The Ixodes scapularis Genome Project: an opportunity for advancing tick research
Trends Parasitol.
(2005)
Y.J. Hua et al.
Identification of a prothoracicostatic peptide in the larval brain of the silkworm, Bombyx mori
J. Biol. Chem.
(1999)
I. Janssen et al.
Isolation of NEB-LFamide, a novel myotropic neuropeptide from the grey fleshfly
Mol. Cell. Endocrinol.
(1996)
L.M. Jorgensen et al.
Molecular identification of the first SIFamide receptor
Biochem. Biophys. Res. Commun.
(2006)
K.J. Livak et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method
Methods
(2001)
M.W. Lorenz et al.
Identification of two allatostatins from the cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae): additional members of a family of neuropeptides inhibiting juvenile hormone biosynthesis
Regul. Pept.
(1995)
S. Neupert et al.
The neuropeptidomics of Ixodes scapularis synganglion
J. Proteomics
(2009)

Y. Park et al.

Two subtypes of ecdysis-triggering hormone receptor in Drosophila melanogaster

J. Biol. Chem.

(2003)

J.M. Ribeiro et al.

An annotated catalog of salivary gland transcripts from Ixodes scapularis ticks

Insect Biochem. Mol. Biol.

(2006)

L. Schoofs et al.

Isolation, identification and synthesis of locustamyoinhibiting peptide (LOM-MIP), a novel biologically active neuropeptide from Locusta migratoria

Regul. Pept.

(1991)

S. Terhzaz et al.

The neuropeptide SIFamide modulates sexual behavior in Drosophila

Biochem. Biophys. Res. Commun.

(2007)

G.E. Tusnady et al.

Principles governing amino acid composition of integral membrane proteins: application to topology prediction

J. Mol. Biol.

(1998)

A. Yasuda et al.

Identification of GYRKPPFNGSIFamide (crustacean-SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis

Gen. Comp. Endocrinol.

(2004)

A.S. Bowman et al.

Tick salivary glands: function, physiology and future

Parasitology

(2004)

A.S. Bowman et al.

Tick saliva: recent advances and implications for vector competence

Med. Vet. Entomol.

(1997)

L.B. Coons et al.

Fine structure of the salivary glands of unfed male Dermacentor variabilis (Say) (Ixodoidea: Ixodidae)

J. Parasitol.

(1973)

N.T. Davis et al.

Localization of myoinhibitory peptide immunoreactivity in Manduca sexta and Bombyx mori, with indications that the peptide has a role in molting and ecdysis

J. Exp. Biol.

(2003)

D.W. Fawcett et al.

The cell biology of the ixodid tick salivary gland

F. Hauser et al.

Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data

Brief. Funct. Genomic Proteomic

(2006)

N.A. Hummel et al.

Serotonin-like immunoreactivity in the central nervous system of two ixodid tick species

Exp. Appl. Acarol.

(2007)

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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).
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Receptors for the neuropeptides, myoinhibitory peptide and SIFamide, in control of the salivary glands of the blacklegged tick Ixodes scapularis

Abstract

Graphical abstract

Highlights

Introduction

Section snippets

Animals and tick feeding

The genes encoding the MIP and SIFamide receptors

Discussion

Acknowledgments

Int. J. Parasitol.

Gen. Comp. Endocrinol.

Int. J. Parasitol.

Trends Parasitol.

J. Biol. Chem.

Mol. Cell. Endocrinol.

Biochem. Biophys. Res. Commun.

Methods

Regul. Pept.

J. Proteomics

J. Biol. Chem.

Insect Biochem. Mol. Biol.

Regul. Pept.

Biochem. Biophys. Res. Commun.

J. Mol. Biol.

Gen. Comp. Endocrinol.

Tick salivary glands: function, physiology and future

Parasitology

Tick saliva: recent advances and implications for vector competence

Med. Vet. Entomol.

Fine structure of the salivary glands of unfed male Dermacentor variabilis (Say) (Ixodoidea: Ixodidae)

J. Parasitol.

Localization of myoinhibitory peptide immunoreactivity in Manduca sexta and Bombyx mori, with indications that the peptide has a role in molting and ecdysis

J. Exp. Biol.

The cell biology of the ixodid tick salivary gland

Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data

Brief. Funct. Genomic Proteomic

Serotonin-like immunoreactivity in the central nervous system of two ixodid tick species

Exp. Appl. Acarol.