Exon encodes a section of the extracellular ligand
Exon 3 encodes a section of the extracellular ligand-binding domain and two variants in the 5′ region were first identified in Drosophila melanogaster (Semenov and Pak, 1999). These variants were initially termed modules 1 and 2 and correspond to exons 3a and 3b, respectively. To date, exon 3 alternative splice variants have been reported for the following insect species: Apis. mellifera (Jones and Sattelle, 2006) and Nasonia vitripennis (Jones et al., 2010) have two alternatives whereas Tribolium castaneum (Jones and Sattelle, 2007), Musca domestica (Kita et al., 2014), Bombyx mori (Furutani et al., 2014), Anopheles gambiae (Meyers et al., 2015), Plutella xylostella (Wang et al., 2016a) and Laodelphax striatellus (Wu et al., 2017) have three alternatives. In addition, a fourth variant that lacks exon 3 was observed in B. mori (Furutani et al., 2014) and P. xylostella (our unpublished data).
Exon 9 encodes a section of the intracellular TM3-TM4 linker and, as with exon 3, variants in this region were also identified first in the D. melanogaster GluCl: a 12-bp stretch immediately downstream of an intron ending in AG results in four additional CP 154526 receptor inserted before the predicted TM4 segment (Semenov and Pak, 1999). Similarly, a 21-bp stretch is found in the L. striatellus GluCl (Wu et al., 2017) and a 65-bp insertion is added downstream of the P. xylostella GluCl (PxGluCl) exon 9 (Liu et al., 2014). In addition, a 33-bp insertion that was previously predicted to be part of intron 9 was found in A. gambiae GluCl (Meyers et al., 2015) (which the author defined as an additional exon 10 but we suggest is an exon 9 insertion following multi-sequences alignment analysis). In our previous study, alternative splicing of PxGluCl mRNA was detected in exon 9, which have three variants (exon 9a, 9b, 9c) as shown in Fig. 1 (Wang et al., 2016a). A 36-bp deletion variant (exon 9c in our study) was observed in an abamectin-resistant P. xylostella strain (Liu et al., 2014) and a partial deletion of exon 9 termed 9pΔ (also corresponding to exon 9c in our study) was found in B. mori GluCl (Furutani et al., 2014). Additionally, one exon skipping variant in the C-terminal amino acids sequences of B. mori GluCl was also detected in the larva brain (Furutani et al., 2014), which lacks the tenth exon.
Exon 3 splice variants have been well characterized in terms of GluCl function, pharmacology and spatiotemporal expression patterns (Furutani et al., 2014; Kita et al., 2014; Meyers et al., 2015; Wu et al., 2017). In contrast, little is known about the physiological roles and pharmacology of GluCls with exon 9 splice variants in the nervous system of any invertebrate. Therefore, in the present study we cloned and constructed the GluCl 9a, 9b and 9c splice variants from the diamondback moth P. xylostella, a notorious pest of cruciferous vegetables worldwide. The three variants were expressed in Xenopus oocytes (both individually and in combination) and their electrophysiological responses examined with the endogenous agonist glutamate, the allosteric modulator abamectin (avermectin B1a) and the channel blocker fipronil. Our results demonstrate that functionally indistinguishable but pharmacologically distinct channels can form from alternative splice variants and we suggest the constitutive expression of these variants might affect insecticide toxicity in the field.
Materials and methods
Discussion Field populations of the P. xylostella pest have evolved various levels of resistance to 95 active ingredients (APRD, 2018), including abamectin (Pu et al., 2010) and fipronil (Wang et al., 2016b) pesticides that act on GluCls. We previously identified three mutually exclusive splice variants of P. xylostella GluCl exon 3 (Wang et al., 2016a) and in this current study we focused on exon 9 splice variants. We show that 9a, 9b and 9c variants expressed individually or co-expressed in oocytes produce robust inward currents in a dose-dependent pattern in response to glutamate. In addition, abamectin or fipronil either induced or inhibited inward currents, respectively, and there was a significant difference in GluCl sensitivity to these compounds depending on the expressed exon 9 variant.