Acute effects of cadmium and zinc on crayfish (Procambarus clarkii): insights from deep learning-based behavioral analysis and physiological assessments.

While the biochemical impacts of heavy metals on aquatic organisms are well-documented, quantitative behavioral analyses remain limited. This study investigated the effects of sublethal cadmium (Cd2+, 10 mg/L) and zinc (Zn2+, 5 mg/L) on the behavior, physiology, biochemistry, and gut microbiota of crayfish (Procambarus clarkii). DeepLabCut (DLC) was employed for high-precision pose estimation to quantify behavioral responses. Cd exposure significantly reduced key locomotor metrics, such as velocity, acceleration, and interaction intensity (P < 0.05), resulting in a shift toward conservative behavioral patterns. Consistently, severe tissue damage was observed in the hepatopancreas, intestine, and gills. Cd exposure severely impaired the immune function (e.g., decreased lysozyme) of the crayfish, significantly increased the production of reactive oxygen species (ROS) and malondialdehyde (MDA), and enhanced antioxidant enzyme activities (P < 0.05). Furthermore, Cd exposure induced a significant proliferation of pathogenic bacteria within the gut microbiota (P < 0.05). Conversely, Zn supplementation effectively mitigated these toxicities. Zn restored motor and interaction intensities, alleviated tissue injury, significantly suppressed excessive ROS and MDA generation, normalized antioxidant enzyme activities (P < 0.05), and promoted beneficial gut bacteria colonization. By utilizing DLC to precisely quantify subtle locomotor shifts, these results demonstrate that deep learning-based tracking serves as a highly sensitive tool with promising potential for broader applications in aquatic toxicology and ecological risk assessment.