Diversity and biological activity of freshwater copepodamides during a spring bloom in the Římov reservoir

Copepodamides are kairomones released by copepods, one of the most dominant zooplankton groups in aquatic ecosystems, which trigger anti-grazing defences in phytoplankton. Whilst widespread in marine ecosystems, their prevalence, diversity, and ecological roles in freshwater ecosystems remain largely unexplored. In this study, we conducted a comprehensive analysis of copepodamides from the eutrophic Římov Reservoir (Czech Republic) during the spring bloom to evaluate their diversity, temporal dynamics, and bioactivity. Using untargeted mass spectrometry, we identified 18 distinct dihydrocopepodamides (dhCAs), including five previously unreported variants. Interestingly, the congener 20:1 dhCA was detected in freshwater for the first time. Temporal analysis revealed that whilst 20:1 dhCA was initially restricted to the macroplankton fraction (>50 μm), its concentration in the microplankton fraction (<50 μm) gradually increased from 0% to 13% of total copepodamides during bloom progression, indicating active release or cellular turnover. Bioassays using the marine diatom Skeletonema marinoi demonstrated that the freshwater zooplankton extract containing dhCAs induced a concentration-dependent reduction in chain length, confirming the conservation of anti-grazing activity across aquatic habitats. However, it remains unclear whether freshwater microalgal species possess the molecular machinery to sense and respond to copepodamides. Overall, future studies on the effect of these kairomones on native phytoplankton species are essential to assess the ecological relevance of these compounds. This study represents one of the first systematic reports of freshwater copepodamides and highlights their potential role in shaping planktonic interactions and evolutionary trajectories in freshwater ecosystems.