Quantitative evaluation of the molting hormone activity in coleopteran cells established from the Colorado potato beetle, Leptinotarsa decemlineata

Abstract

A novel reporter gene assay system using BCIRL-Lepd-SL1, a cell line from the coleopteran Colorado potato beetle (Leptinotarsa decemlineata), was established. Cells were transiently transfected with the reporter plasmid that was composed of a firefly luciferase gene with upstream ecdysone response elements, and an internal control plasmid that constitutively produces Renilla reniformis luciferase. Transfected cells were incubated with various molting hormone agonists, and the activity of these agonists was quantitatively determined by measuring luminescence emission. Transcription-inducing activity for ecdysone, 20-hydroxyecdysone and ponasterone A in terms of EC50 (50% effective concentration) were determined to be 1μM (pEC50=5.99), 68nM (pEC50=7.17) and 1.3nM (pEC50=8.88), respectively. Among tested diacylhydrazine (DAH)-type compounds, 11 compounds were active (pEC50=3.56∼6.41), but two compounds were inactive. The EC50 values were linearly correlated to their binding affinity except for one compound. While several ecdysone reporter systems were developed before that employ dipteran and lepidopteron cell lines, the assay system described here is only the second one that employs a coleopteran cell line. This reporter system will allow screening in high-throughput format for new and more potent molting accelerating compounds that specifically target coleopteran pests.