br Acknowledgements This work was supported in part by JSPS
Acknowledgements This work was supported in part by JSPS KAKENHI Grant Number 26292031.
Introduction The Cys-loop receptors constitute an important superfamily of ligand-gated ion Cy7 NHS ester receptor (LGICs) and mediate synaptic transmission in both invertebrate and vertebrate nervous systems (Jones et al., 2010; Jones and Sattelle, 2007). Among the members of Cys-loop LGICs, the glutamate-gated chloride channels (GluCls) have only been found in invertebrate and identified as the primary targets of insecticides such as abamectin and fipronil (Wolstenholme, 2012; Wolstenholme and Rogers, 2005; Janssen et al., 2007). While six GluCl subunits have been identified in the nematode Caenorhabditis elegans and the mite Tetranychus urticae (Jones and Sattelle, 2008; Dermauw et al., 2012), only one single orthologous gene encoding the α subunit was found in insects (Cully et al., 1996; Eguchi et al., 2006; El Hassani et al., 2012; Furutani et al., 2014). Similar to other LGICs, GluCls diversity was broadened by alternative splicing. In insects, three GluCl variants (GluCl A, GluCl B and GluCl C) generated by the alternative splicing of exon 3 were found in Musca domestica, Apis mellifera, Tribolium castaneum, Bombyx mori and Laodelphax striatellus (Jones and Sattelle, 2007; El Hassani et al., 2012; Furutani et al., 2014; Wu et al., 2017; Kita et al., 2014), and a truncated variant generated by the alternative splicing of exon 9 was identified in Drosophila melanogaster, B. mori, and L. striatellus (Furutani et al., 2014; Wu et al., 2017; Semenov and Pak, 1999). Notably, these GluCl variants showed different expression profiles and ligand binding capacities (El Hassani et al., 2012; Furutani et al., 2014; Wu et al., 2017). The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae) is one of the most damaging rice pests in China (He et al., 2013). The use of insecticides was the primary strategy employed to control C. suppressalis, however, due to the abuse of insecticides, C. suppressalis has developed resistance to conventional insecticides such as chlorantraniliprole, triazophos and abamectin (He et al., 2013; Sun et al., 2018; Qu et al., 2003; Li et al., 2007). While the role of target site insensitivity in chlorantraniliprole and triazophos resistance has been studied in C. suppressalis (Sun et al., 2018; Qu et al., 2003), the mechanism of abamectin resistance in C. suppressalis remains unclear. In the present study, we isolated a full-length GluCl cDNA (named as CsGluCl) from C. suppressalis. We report the expression patterns of three CsGluCl variants and document the role of CsGluCl in abamectin toxicity and C. suppressalis development. Our results are pivotal to the discovery of new chemicals targeting GluCls and resistance management of C. suppressalis.
Materials and methods
Discussion As a key posttranscriptional processing mechanism, alternative splicing results in a single gene coding for multiple processed mRNA, and generates remarkable protein diversity. Although only a single GluCl subunit was found in insect genomes, multiple variants generated by alternative splicing of exon 3 and 9 have been identified in insects (El Hassani et al., 2012; Furutani et al., 2014; Wu et al., 2017; Meng et al., 2018). The exon 3 encodes a part of the N-terminal domain and adjacently lies upstream of the agonist binding site Loop D. The alternative splicing of exon 3 was conserved in diverse insect species, and generates three GluCl variants (GluCl A, GluCl B, GluCl C) in insects (Furutani et al., 2014; Wu et al., 2017; Meng et al., 2018). Studies on the pharmacological properties of the three variants have revealed different binding capacities to ligands in M. domestica and B. mori (Furutani et al., 2014; Kita et al., 2014). In addition, a fourth variant completely lacking exon 3 was observed in B. mori (Furutani et al., 2014). Alternative splicing of exon 9 produced a truncated variant and has been found in D. melanogaster, B. mori, and L. striatellus (Furutani et al., 2014; Wu et al., 2017; Semenov and Pak, 1999). The alternative splicing site located in the large intracellular loop between transmembrane domains 3 and 4. While this region played important roles in modulating the receptor function and clusterization, channel expression and desensitization in other members of Cys-loop LGICs (McKinnon et al., 2012; Tsetlin et al., 2011), the truncated variant had similar EC50 and IC values for L-glutamate and channel blockers picrotoxinin and fipronil with the exon 9 complete variant in L. striatellus (Wu et al., 2017). In the present study, three CsGluCl variants (CsGluCl A, CsGluCl B, CsGluCl C) generated by the alternative splicing of exon 3 were found in C. suppressalis. The splicing of exon 9 was also observed in CsGluCl. The pharmacological properties of these CsGluCl variants need further study.