1. You report significant decreases in caspase 3/7 activity (to 36–58% of control) after PS and PS-NH₂ exposure (Fig 4H). Most nanoplastic toxicity studies show caspase activation and increased apoptosis. Do you interpret this as an anti-apoptotic effect, and if so, what biological mechanism would explain reduced caspase activity? Could this be a dialysis-related artifact or a hormetic response?
2. In Fig 2H–I, you compare locomotor activity between larvae with inflated vs. uninflated swim bladders. With only 2–20% of larvae showing uninflated bladders (Fig 2G), the sample size for uninflated groups is extremely small (e.g., ~1–3 larvae per 16). Did you perform a power analysis, and how do you rule out Type II errors in the “no significant effect” conclusion for uninflated larvae?
3. You list “circadian rhythm” and “circadian rhythm fly” as two distinct enriched pathways (Fig 3B). These are functionally identical (ko04710 vs. ko04711 in KEGG). Why present them separately, and does this duplication artificially inflate pathway enrichment significance?
4. You used red-fluorescent labeled NPs for 2D/3D imaging but unlabeled NPs for toxicity assays. How did you verify that the red fluorescence in yolk/intestine (Fig 1L–R) represents intact NPs rather than leached free dye? Did you perform dye-leakage validation (e.g., dialysis of free dye controls) as done for sodium azide?