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  • Firefly Luciferase mRNA Ionotropic aminobutyric acid GABA re

    2021-09-27

    Ionotropic γ-aminobutyric Firefly Luciferase mRNA (GABA) receptors (GABARs) belong to the Cys-loop ligand-gated ion channel (LGIC) superfamily and play an important role in the rapid inhibitory synaptic transmission in nervous systems of both vertebrate and invertebrate (Buckingham et al., 2005). Insect ionotropic GABAR subunits consist of resistance to dieldrin (RDL) (ffrench-Constant et al., 1991), ligand-gated chloride channel homolog 3 (LCCH3) (Henderson et al., 1993), and GABA/glycine-like receptor of Drosophila (GRD) subunit (Harvey et al., 1994). Insect GABARs are an important target of insecticides, such as ethiprole, fipronil (Wei et al., 2017a), avermectin (Sanches et al., 2017), dieldrin and fluralaner (Sheng et al., 2017). LGICs are composed of five subunits and how the insect GABAR subunits assemble to form receptors in vivo still remains to be elucidated (Jones, 2018). As is known, only the RDL subunit is considered to be responsible for insecticidal resistance of insects (Fujii et al., 2012; Novelli et al., 2016; ffrench-Constant et al., 1993; Nakao et al., 2011). The pharmacological characteristics of both GRD and LCCH3 subunits from D. melanogaster Meigen co-expressed in Xenopus laevis oocytes differed from those of native GABARs (Gisselmann et al., 2004). Thus, the role, if any, of GRD and LCCH3 in insecticide resistance is unclear. Hence, as a major target of insecticides and acaricides, insect GABARs still need to be further studied regarding the control of agricultural pests (Casida, 1993; Casida and Durkin, 2015). We have recently characterized the RDL subunits (RDL1 and RDL2) from C. suppressalis by heterologous expression in X. laevis oocytes (Sheng et al., 2018) and by RNA interference (RNAi) (Meng et al., 2018). We report here the identification of the LCCH3 subunit in C. suppressalis. In addition, another possible GABAR-like subunit homologous to 8916, first found in D. melanogaster (Knipple and Soderlund, 2010) and successively in Apis mellifera Linnaeus (Jones and Sattelle, 2006), Tribolium castaneum (Herbst) (Jones and Sattelle, 2007), Nasonia vitripennis (Walker) (Jones et al., 2010), and Laodelphax striatellus (Fallén) (Wei et al., 2017b), was investigated in the current study, improving our understanding about the ionotropic GABARs of C. suppressalis.
    Materials and methods
    Results
    Discussion GABARs are important inhibitory neurotransmitter receptors that include ionotropic and metabotropic types (Miller and Aricescu, 2014; Ozoe, 2013). Nineteen ionotropic GABAR subunit genes, including 6 alpha, 3 beta, 3 gamma, 3 rho, 1 delta, 1 epsilon, 1 theta, and 1 pi, respectively, have been identified in the human genomic database (Olsen and Sieghart, 2008). However only three subunits in insects, RDL, LCCH3 and GRD, are known to contribute to GABA-gated ion channels with a possible additional subunit of 8916 (ffrench-Constant et al., 1991; Henderson et al., 1993; Harvey et al., 1994; Jones, 2018; Jones and Sattelle, 2006; Wei et al., 2017b). In combination with our previous study (Sheng et al., 2018), four complete GABAR subunit cDNA sequences (RDL1, RDL2, LCCH3 and 8916) have now been identified from C. suppressalis. According to the phylogenetic tree (Fig. 2), the four GABAR subunits were clustered into RDL, LCCH3 and 8916 clans, respectively. Most insects analyzed to date also possess a GRD subunit (Jones, 2018). However, a putative CsGRD subunit has yet to be identified. The Cs8916 subunit exhibited very close sequence relationship with BmGRD, which was also classified into the 8916 clan. We therefore speculate this to be Bm8916, the B. mori orthologue of Drosophila CG8916 (Wei et al., 2017b). It is interesting to note that there are no clear orthologues of CG8916 and GRD in the aphid Acyrthosiphon pisum (del Villar and Jones, 2018). It is thus of interest to see whether Lepidoptera also lack an obvious GRD counterpart or whether CsGRD and BmGRD remain to be identified. Unexpectedly, except the four conserved typical transmembrane domains, Cys-loop, six ligand binding loops (loops A-F) and three N-glycosylation sites identified in the CsLCCH3 and Cs8916 subunits, an additional fifth transmembrane domain (named as TM0) was found at the N-terminal region of Cs8916 (Fig. 1). Similar analysis did not find a predicted TM0 in BmGRD (GenBank ID: NP_001182633.1), Dm8916 (GenBank ID: NP_001162770.1), Am8916 (GenBank ID: ABG75745.1), Tc8916 (GenBank ID: ABU63605.1), Ls8916 (GenBank ID: AOO87784.1) and Nv8916 (GenBank ID: XP_008203400.1) (data not shown). No other study about the TM0 has been reported apart from in the Danio rerio (Hamilton) γ2 subunit (Huang et al., 2018). Therefore, it would be of interest to determine whether TM0 performs a particular function.