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  • RDL is of interest as

    2022-01-19

    RDL is of interest as it is the target of highly effective insecticides (Buckingham et al., 2005, Raymond-Delpech et al., 2005). Point mutations, particularly an alanine to serine or glycine mutation in TM2, underlie resistance to several insecticides, including dieldrin, picrotoxinin and fipronil (Ffrench-Constant et al., 1993, Hosie et al., 1995). This substitution of alanine is often used as a diagnostic marker for resistance and has been detected in varying species, ranging from crop pests (e.g. the planthopper Laodelphax striatellus (Nakao et al., 2011)), pests afflicting livestock (the horn fly Haematobia irritans (Domingues et al., 2013)), domesticated animals (the cat flea Ctenocephalides felis (Bass et al., 2004)) to disease vectors (the malaria vector mosquito Anopheles gambiae (Du et al., 2005)). Knowledge of mechanisms leading to insecticide insensitivity is crucial to detecting resistance in insect populations, which can then inform the use of alternative insecticide classes, as part of an effective resistance management program (Feyereisen et al., 2015). Recently, the cloning of Rdl from the miridbug, Cyrtorhinus lividipennis, revealed two isoforms, differing in subunit length by 31 amino acid residues, arising from differential splicing of the TM3-TM4 intracellular loop (Jiang et al., 2015). Two-electrode voltage-clamp electrophysiology applied to C. lividipennis RDL expressed in Xenopus laevis oocytes showed these two isoforms differed in their sensitivity to the antagonistic effects of fipronil, with the longer variant having greater insensitivity to this insecticide. It was thus suggested that C. lividipennis may take advantage of its RDL diversity to enhance tolerance to fipronil or other insecticides. Interestingly, the 31 amino acid insertion has so far been observed only in C. lividipennis, highlighting the presence of such TM3-TM4 splice variants as a possible species-specific mechanism for insecticide insensitivity. Since C. lividipennis plays an important predatory role in controlling the rice pest, Nilaparvata lugens, this is a useful example of a study assessing the effects of fipronil on its molecular target from a beneficial insect (Jiang et al., 2015). The honey bee, Apis mellifera, is another example of a beneficial insect, whose role in pollinating crop species is valued at over $200 billion worldwide (Fairbrother et al., 2014). In neuronal metabolism 2013, the European Union placed a moratorium of at least two years on the use of neonicotinoids, which target nicotinic neuronal metabolism receptors (nAChRs) (Matsuda et al., 2001), amidst fears that these insecticides are a contributing factor towards the alarming decline in bee numbers (Fairbrother et al., 2014). The use of fipronil was also restricted for the same reason (Official Journal of the European Union, 2013). To understand further the molecular target of fipronil in A. mellifera, we report here the cloning of the honey bee Rdl as well as the identification of variants arising from differential splicing of the TM3-TM4 intracellular loop. In addition, we present the first heterologous expression studies of an Apis RDL to assess the sensitivity of the different variants to fipronil. Previously, we showed that the neonicotinoid, imidacloprid, acts as an antagonist on heterologously expressed RDL of the mosquito, Anopheles gambiae (Taylor-Wells et al., 2015). We therefore used the X. laevis oocyte expression system to determine whether imidacloprid also acts directly on the honey bee GABA receptor.
    Materials and methods
    Results
    Discussion We report here the cloning and functional expression of the A. mellifera RDL GABA receptor. We identified three splice variants of the honey bee RDL arising from the differential use of the ag splice acceptor site (Fig. 2). This results in sequence variation and length in the large intracellular loop. Recently, two variants of C. lividipennis RDL were reported, the longer variant having a 31 amino acid insertion in the intracellular loop (Jiang et al., 2015). Interestingly, the site of insertion disrupts a TVR motif conserved in both A. mellifera and C. lividipennis RDL (Fig. 1). The TVR sequence is a protein kinase C phosphorylation consensus site (Sigrist et al., 2013). Along with putative casein kinase II phosphorylation sites also commonly found in the intracellular loop of RDL (Jones and Sattelle, 2006), these sites are predicted based on a database of protein families and domains from which signature sequences have been derived (Sigrist et al., 2013). A similar situation is evident in D. melanogaster RDL where one variant has the TVR motif (Accession NP_729462), which is disrupted by a 29 amino acid insertion in another variant (NP_523991). The 31 and 29 amino acid insertions in C. lividipennis and D. melanogaster RDL, respectively, are not similar to each other nor are they conserved in the A. mellifera genome, highlighting that the insertion sequences are species specific. Since phosphorylation of the intracellular loop of cysLGICs can modulate turnover, assembly, subcellular localization, desensitization, insecticide sensitivity and interactions with other proteins (Stokes et al., 2015, Talwar and Lynch, 2014, Thany et al., 2007, Bermudez and Moroni, 2006), the differential splicing has the potential to affect several receptor properties, assuming the TVR site is phosphorylated.