High-resolution in situ imaging reveals size-specific moonlight responses in zooplankton diel vertical migration.

Light is the primary cue driving zooplankton diel vertical migration (DVM), a strategy that balances predation risk with resource access. However, DVM is often oversimplified, with limited consideration of how light-driven risks and resource needs vary across taxa and life stages. This simplification is partly due to constraints on collecting high-resolution, size-resolved data -especially at night, when subtle shifts in illumination reshape nocturnal risk landscapes. To overcome these limitations, we deployed a high-resolution in situ modular Deep-focus Plankton Imager and an image-recognition approach to quantify fine scale DVM and body sizes of Cladocerans and Copepods in Lake Stechlin, Germany. Data was collected from day into night and across moonrise and was compared with environmental data from vertical profiling sondes. Typical DVM patterns emerged, with deeper daytime distributions, however, moonlight introduced additional behavioural complexity: larger individuals avoided illuminated layers, likely managing predation risk, while smaller individuals moved into these layers, possibly exploiting foraging opportunities and reduced risk. These light-mediated shifts were further shaped by ecological conditions; copepods tracked food-rich layers regardless of light levels at night, while cladocerans showed light-dependent responses to both temperature and food, such that light caused them to avoid otherwise favourable (warm, food-rich) layers. Our approach provides new insight into how zooplankton navigate nocturnal lightscapes, revealing size- and taxon-specific strategies. By establishing size-dependent responses to natural moonlight, this work provides a crucial baseline for predicting how artificial light at night may restructure zooplankton communities and destabilize freshwater food webs.