Transcriptome Analysis of the Central and Peripheral Nervous Systems of the Spider Cupiennius salei Reveals Multiple Putative Cys-Loop Ligand Gated Ion Channel Subunits and an Acetylcholine Binding Protein

Abstract

Invertebrates possess a diverse collection of pentameric Cys-loop ligand gated ion channel (LGIC) receptors whose molecular structures, evolution and relationships to mammalian counterparts have been intensely investigated in several clinically and agriculturally important species. These receptors are targets for a variety of control agents that may also harm beneficial species. However, little is known about Cys-loop receptors in spiders, which are important natural predators of insects. We assembled de novo transcriptomes from the central and peripheral nervous systems of the Central American wandering spider Cupiennius salei, a model species for neurophysiological, behavioral and developmental studies. We found 15 Cys-loop receptor subunits that are expected to form anion or cation permeable channels, plus a putative acetylcholine binding protein (AChBP) that has only previously been reported in molluscs and one annelid. We used phylogenetic and sequence analysis to compare the spider subunits to homologous receptors in other species and predicted the 3D structures of each protein using the I-Tasser server. The quality of homology models improved with increasing sequence identity to the available high-resolution templates. We found that C. salei has orthologous γ-aminobutyric acid (GABA), GluCl, pHCl, HisCl and nAChα LGIC subunits to other arthropods, but some subgroups are specific to arachnids, or only to spiders. C. salei sequences were phylogenetically closest to gene fragments from the social spider, Stegodyphus mimosarum, indicating high conservation within the Araneomorphae suborder of spiders. C. salei sequences had similar ligand binding and transmembrane regions to other invertebrate and vertebrate LGICs. They also had motifs associated with high sensitivity to insecticides and antiparasitic agents such as fipronil, dieldrin and ivermectin. Development of truly selective control agents for pest species will require information about the molecular structure and pharmacology of Cys-loop receptors in beneficial species.

The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei

“…Over 90% of somata in similar clusters were previously found to be immunoreactive to GABA, and many also to glutamate (Barth 2002; Fabian-Fine et al 2002). Spider mechanosensory neurons and some efferent neurons have GABA, glutamate and ACh receptors and the neurons respond to these transmitters in electrophysiological experiments (Panek et al 2002; Panek and Torkkeli 2005; Widmer et al 2006; Torkkeli et al 2012; 2015). An attractive hypothesis is that neurons in the ANC may be efferents, containing one or more of these three transmitters, each being released in different behavioral situations.…”

“…Two genes homologous to mammalian and arthropod choline acetyltransferase gene ( ChAT ) were identified from the C. salei transcriptome by searching the databases at low stringency against amino acid sequences of published arthropod genes using the transcriptome walking method described previously in detail (French 2012; Torkkeli et al 2015). Custom designed primers (Table 1) were purchased from Integrated DNA Technologies (Coralville, IA, USA).…”

Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine‐Binding Protein as a Structural Surrogate

“…The acetylcholine‐binding protein (AChBP) is a soluble protein that was first discovered in the freshwater snail Lymnaea stagnalis ( Ls ) in 2001 and has been thoroughly characterized structurally in the following years. After this finding, AChBPs from the sea snail Aplysia californica ( Ac ) , the freshwater snail Bulinus truncates ( Bt ) , the abalone Haliotis discus hannai ( Hdh ) , the marine polychaete Capitella teleta ( Ct ) , the planorbid snail Biomphalaria glabrata ( Bg ) and the spider Cupiennius salei ( Cs ) have also been characterized. AChBP is a naturally occurring homologue of the ligand‐binding domain of nAChRs, and like some nAChRs, AChBP assembles as a homopentamer.…”

Expression of Cys‐loop receptor subunits and acetylcholine binding protein in the mechanosensory neurons, glial cells, and muscle tissue of the spider Cupiennius salei

“…Recently, we assembled and characterized 16 Cys‐loop subunits from the C. salei central and peripheral nervous system transcriptomes (Torkkeli et al, ). These sequences include 12 subunits that are predicted to form anion‐permeable channels, plus three cation‐conducting nACh receptor subunits and a putative acetylcholine‐binding protein (AChBP) that lacks the transmembrane region.…”

“…The C. salei AChBP is the first homologous protein described for any arthropod species, but orthologous genes in other spiders can be found in GenBank. The C. salei AChBP has higher similarity to vertebrate nicotinic AChα subunits than the molluscan AChBPs (Torkkeli et al, ). Snail AChBP was initially proposed to modulate synaptic transmission but was later found only in extrasynaptic locations (Smit et al, ; Banks et al, ).…”