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(1) Background: Nanoplastics are emerging environmental pollutants with potential toxic effects on aquatic organisms. This study investigates the toxicity of NPs in Biomphalaria glabrata, a freshwater snail species widely used as a bioindicator species in ecotoxicology studies.; (2) Methods: We exposed three generations (F0–F2) of B. glabrata snail embryos to different sizes of polystyrene nanoparticles and assessed responses.; (3) Results: We observed severe effects on F0 to F2 B. glabrata embryos, including size-dependent (30 to 500 nm) increases in mortality rates, size and dosage-dependent (1 to 100 ppm) effects on hatching rates with concentration-dependent toxicity in the 30 nm exposure group. The F2 generation embryos appear to be most responsive to detoxification (CYP450) and pollutant metabolism (HSP70) at 48-h-post-treatment (HPT), while our developmental marker (MATN1) was highly upregulated at 96-HPT. We also report a particle-size-dependent correlation in HSP70 and CYP450 mRNA expression, as well as enhanced upregulation in the offspring of exposed snails. We also observed significant reductions in hatching rates for F2.; (4) Conclusions: These findings indicate that F2 generation embryos appear to exhibit increased stress from toxic substances inherited from their parents and grandparents (F1 and F0). This study provides valuable insights into the impact of plastic particulate pollution on multiple generations and highlights the importance of monitoring and mitigating plastic waste. 

Calcium waves and oscillation during embryonic development are key elements in the intricate process of molluscan shell formation. However, understanding calcium dynamics in the early embryonic shell formation in gastropod development is still insufficient. The present study explores the role of calcium flux in early shell formation within the embryo of gastropod Biomphalaria glabrata. We hypothesized that the role of calcium is not only in providing a critical element for shell formation but also in serving as a signaling molecule for the genetic regulation of calcification. The calcium flux was visualized using the Fura-2 and Fluo-4 calcium indicators through the trochophore (72 hours) and veliger (120 hours) stages of B. glabrata development. The dynamics of calcium signals were correlated to the rapid transition from motile trochophore to veliger, marked by cilia-mediated movement and premature shell and foot development. According to our observation, the intracellular calcium signals were attenuated from 72 to 120 hours of embryo development. The expression profiles of genes encoding calmodulin and related protein kinase following the calcium flux in embryos suggested a critical role of the calcium-binding proteins in the early shell development of gastropods. Although the embryonic calcium dynamics and the related signaling pathway of shell formation are under further observation and analysis, the role of calcium in the singling pathway of shell formation has been demonstrated by this preliminary study.