Functional integrity of honeybee (Apis mellifera L.) resistant to dieldrin γ-aminobutyric acid receptor channels conjugated with three fluorescent proteins.

To generate an efficient tool used in Xenopus oocyte expression for in situ investigation of channel receptor expression, distribution and function, the C-terminus of the honeybee (Apis mellifera L.) resistant to dieldrin (RDL) subunit was fused with *FP, including monomeric red, enhanced yellow or enhanced green fluorescent protein (referred to as mRFP, EYFP and EGFP, respectively). In the present study, all fused *FP-AmRDLs could be visualized using fluorescence and laser confocal microscopy in cRNA-injected oocytes. Fluorescence was distributed isotropically in the cellular membrane. The potencies of the agonist γ-aminobutyric acid (GABA), but not ß-alanine, and the test antagonists (fipronil, flufiprole, dieldrin, α-endosulfan, bifenazate and avermectin B1a) in the *FP-AmRDL receptor did not significantly differ from that of the untagged receptor with two-electrode voltage clamp detection. The half maximal effective concentrations (EC50 s) of GABA in AmRDL, EGFP-AmRDL, EYFP-AmRDL and mRFP-AmRDL receptors were 11.98, 12.61, 18.92 and 22.11 µM, respectively, and those of ß-alanine were 651.6, 629.6, 1643.0 and 2146.0 µM, respectively. Inhibition percentages of test antagonists against *FP-AmRDL and AmRDL were not significantly different from each other. Overall, the consistency in functional properties between *FP-AmRDL and AmRDL receptors makes pGH19-*FP a promising tool for further in situ investigation of GABA receptors.

Ryanodine receptor genes of the rice stem borer, Chilo suppressalis: Molecular cloning, alternative splicing and expression profiling.

The ryanodine receptor (RyR) of the calcium release channel is the main target of anthranilic and phthalic diamide insecticides which have high selective insecticidal activity relative to mammalian toxicity. In this study, the full-length cDNA of Chilo suppressalis RyR (CsRyR) was isolated and characterized. The CsRyR mRNA has an open reading frame (ORF) of 15,387bp nucleotides, which encodes 5128 amino acids with GenBank ID: KR088972. Comparison of protein sequences showed that CsRyR shared high identities with other insects of 77-96% and lower identity to mammals and nematodes with only 42-45%. One alternative splicing site (KENLG) unique to Lepidoptera was found and two exclusive exons of CsRyR (I /II) were revealed. Spatial and temporal expression of CsRyR mRNA was at the highest relative level in 3rd instar larvae and head (including brain and muscle), and at the lowest expression level in egg and fat body. The expression levels of whole body CsRyR mRNA were increased remarkably after injection of 4th instar larvae with chlorantraniliprole at 0.004 to 0.4µg/g. This structural and functional information on CsRyR provides the basis for further understanding the selective action of chlorantraniliprole and possibly other diamide insecticides.