Non-invasive functional indicators of early development in Solea senegalensis: Ontogeny of the light-dark response and endogenous autofluorescence

The light–dark test (DLT) is a useful paradigm to assess light-dependent modulation of locomotor activity during development. Here, we characterized the ontogeny of the DLT response in Senegalese sole ( Solea senegalensis ) from 72 to 192 hpf and compared it with the dynamics of an endogenous autofluorescence signal observed at early stages. Analysis of integrated activity (actinteg, a pixel-based measure of detected movement accumulated per time bin) suggested an ontogenetic trajectory in which activity peaked at 96 hpf and then decreased towards 192 hpf. In parallel, phase-dependent organisation became more stereotyped towards late stages under the light–dark stimulus used here. Because early stages were represented by a single independent recording (R = 1), these early-stage patterns should be considered exploratory. Autofluorescence observable without exogenous staining was stronger at 96–168 hpf and decreased by 192 hpf; spectral or multi-filter controls are needed to confirm fluorophore identity, assess overlap with common green/red channels, and exclude exogenous/background contributions. Overall, under fixed detection settings, we observed a descriptive transition from higher detected activity at early stages to lower detected activity at 192 hpf, alongside stronger light-dependent structuring. Since the DLT quantifies a locomotor response driven by illumination, these findings should be interpreted as non-invasive functional indicators of larval development and light–locomotion coupling, rather than as a direct measure of neurosensory/visual maturation.