Cloning and functional analysis of the ecdysteroid receptor complex in the opossum shrimp Neomysis integer (Leach, 1814)

► We cloned the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR) of the opposum shrimp Neomysis integer. ► We developed a chimeric Gal4 cell reporter system to investigate the heterodimerization of NiEcR-CrcRXR in vitro. ► PonA facilitated dimerization, while the insecticide tebufenozide with endocrine disruptive activities had no effect. ► In silico binding of tebufenozide with NiEcR revealed steric hindrance, while PonA fitted perfectly. ► Neomysis integer juveniles exposed to tebufenozide, in vivo, showed no molting disturbances. In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. Major results were: (i) cDNAs encoding the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likelihood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacostracan clade (bootstrap percentage=75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the common shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein–protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to teb