Effects of neonicotinoid and diamide-contaminated agricultural runoff on Lymnaea stagnalis: Insights into stress, neurotoxicity, and antioxidant response

Freshwater pollution from agrochemicals poses an increasing threat to aquatic ecosystems. Yet, the sublethal effects of such contaminants on cognition and neural function in non-target invertebrate species remain poorly understood. Using a field-collected source of pesticide-contaminated freshwater (Spirit Creek Pond water, SCW) and two Lymnaea stagnalis strains, one collected directly from Spirit Creek (SC strain) and another inbred laboratory strain (W strain), we address the core questions: (1) what are the transcriptional consequences of SCW exposure? (2) are these effects strain-specific? (3) does SCW impair higher-order configural learning (CL)? (4) and, if so, can cognitive function recover following a 14-day washout period in uncontaminated water? SCW exposure significantly altered the expression levels of stress-response and cholinergic receptors in a strain-dependent manner. In both strains, LymHSP70 and LymSOD1 were significantly upregulated during exposure and downregulated after washout, indicating acute stress with partial transcriptional recovery. In W-strain snails, LymGSR was uniquely and persistently upregulated post-washout, suggesting sustained oxidative imbalance. Moreover, W-strain snails exhibited significant upregulation of cholinergic receptor genes LymnAChR_G and LymnAChR_E during SCW exposure, followed by downregulation after washout, suggesting a reversible but lasting disruption in cholinergic signaling. Behaviorally, CL was abolished in both strains following SCW exposure, with no evidence of cognitive recovery after the washout period. These results demonstrate that exposure to environmentally relevant contaminants induces lasting neurobehavioral impairments and molecular dysregulation in L. stagnalis, with strain-specific differences in susceptibility and recovery. Our findings underscore the need for long-term ecological monitoring of freshwater neurotoxicants and their impacts on invertebrate neural health and cognition.