Cloning and functional expression of intracellular loop variants of the honey bee (Apis mellifera) RDL GABA receptor

Abstract

The insect GABA receptor, RDL (resistance to dieldrin), plays central roles in neuronal signalling and is the target of several classes of insecticides. To study the GABA receptor from an important pollinator species, we cloned Rdl cDNA from the honey bee, Apis mellifera. Three Rdl variants were identified, arising from differential use of splice acceptor sites in the large intracellular loop between transmembrane regions 3 and 4. These variants were renamed from previously, as Amel_RDLvar1, Amel_RDLvar2 and Amel_RDLvar3. When expressed in Xenopus laevis oocytes, the three variants showed no difference in sensitivity to the agonist, GABA, with ECs of 29μM, 20μM and 29μM respectively. Also, the potencies of the antagonists, fipronil and imidacloprid, were similar on all three variants. Fipronil IC values were 0.18μM, 0.31μM and 0.20μM whereas 100μM imidacloprid reduced the GABA response by 17%, 24% and 31%. The possibility that differential splicing of the RDL intracellular loop may represent a species-specific mechanism leading to insensitivity to insecticides is discussed.

Cloning and Functional Characterisation of the Duplicated RDL Subunits from the Pea Aphid, Acyrthosiphon pisum

“…All the eight full length clones for Apisum Rdl2 encoded for the exon3b variant; however, four of these clones had open reading frames of 1674 bp, whilst the other four had 1677 bp, encoding 557 and 558 amino acids, respectively. The difference in the open reading frame lengths is due to the presence of either a TVR or TEVR peptide motif in the TM3-TM4 intracellular domain, which were previously found in A. mellifera RDL, and were denoted variants 1 or 2, respectively [31]. No potential A-to-I RNA editing was observed in the twenty clones analysed.…”

“…In line with this, more recent studies have shown that 100 μM imidacloprid acted as an antagonist of An. gambiae and A. mellifera RDL expressed in Xenopus oocytes [12,31]. We show here that imidacloprid at 100 μM has no antagonistic actions on Apisum RDL1 or Apisum RDL2, highlighting the fact that RDL can respond to neonicotinoids in a species-dependent manner.…”

“…Unlike for An. gambiae and A. mellifera RDLs [12,31], imidacloprid at 100 μM had no detectable effect on responses of Apisum RDL1 ad or Apisum RDL bd induced by GABA at EC 50 concentration (Figure 7a) or at 1 mM. We therefore tested to see whether other neonicotinoids showed any actions on the aphid RDLs.…”

“…Here, an insertion of a single amino acid (TVR to TEVR) was identified. We did not functionally characterise these variants, as we have already shown that they have similar responses to GABA and fipronil in A. mellifera RDL [31]. With no potential A-to-I RNA editing isoforms detected, the extent of functional diversification of aphid RDL is less than that of other insects such as D. melanogaster and An.…”

“…The heterologous expression of both aphid RDLs may provide a useful screening tool for the discovery of novel insecticidal compounds. This, in addition to screening against RDLs that have been cloned from other species such as C. suppressalis (a crop pest) [20], C. lividipennis (a predator of crop pests) [33] and A. mellifera (a pollinator) [31], may facilitate the identification of compounds which are selective for insect pests but benign for beneficial species. Furthermore, using expressed RDL with the 2′ mutation [15,35] in these screens can highlight novel compounds that are still active on insects with the TM2 mutation as an important step in managing resistance.…”