The amphipod ecdysone receptor as a complementary tool for environmental risk assessment: from functional analysis to proof of concept study

Endocrine-disrupting chemicals (EDCs) pose significant risks to aquatic wildlife by disrupting hormonal signaling pathways, notably those mediated by nuclear receptors (NRs) - key regulators of endocrine processes. For decades, in vitro bioassays based on the interaction between NRs and EDCs have been used to investigate EDC activity in chemical monitoring and risk assessment frameworks. Yet, current guidelines are mostly restricted to vertebrate species, failing to capture the diversity of chemical-induced NR-driven effects in aquatic animals - particularly arthropods, whose endocrine systems are highly divergent. Thus, this study aimed to develop an in vitro approach for the assessment of EDCs targeting ecdysone signaling, a key endocrine pathway regulating molting and development in arthropods. For this, we have examined the ecdysone receptor (EcR), a target of ecdysone-mimicking pesticides, and its heterodimeric partner, the retinoid X receptor (RXR), both crucial for ecdysone-mediated signaling, in key amphipod species for freshwater and coastal ecosystems, Gammarus fossarum and G. locusta. Following identification and phylogenetic validation, we established a luciferase-based reporter gene assay that allowed us to characterize EcR:RXR responsiveness to ecdysteroids (e.g., ponasterone A), as well as the detection of chemicals with EcR-disrupting activity, such as the insecticides methoxyfenozide and tebufenozide. The application of this tool was then extended to environmental water samples, for the screening of French river basins, providing the first proof of concept for the identification of sampling sites with putative EcR agonistic activity. These findings aim to offer a complementary approach to current environmental frameworks, in the scope of investigative monitoring, towards the inclusion of underrepresented invertebrate species.