Introduction
Early life stages are critical periods marked by high vulnerability to stressors and constituting a critical bottleneck in marine aquaculture. In Senegalese sole (Solea senegalensis), unavoidable handling events and bacterial challenges often elicite acute stress responses that disrupt immune regulation and metabolic homeostasis, compromising health and survival. Enhancing larval resilience under these conditions is therefore essential to improve health management and production efficiency. Functional diets have gained attention as a strategy to modulate larval stress responses by supplying bioactive compounds that act on immune, antioxidant and stress���related pathways rather than directly promoting growth. Among functional ingredients, micro��� and macroalgae represent particularly attractive candidates due to their complex biochemical composition. Collectively, these components may support mucosal integrity, reduce oxidative damage and promote coordinated systemic responses during acute stress exposure. In this context, the present study investigated whether a short functional feeding period with an algae���based blend modulates stress resilience in sole larvae exposed to combined transport and bacterial challenge. By integrating survival outcomes with humoral, oxidative and gut–liver transcriptomic responses, this work aims to characterize the impact of algaeenriched microdiets on the biological response to stress during early development of sole.
Methodology
The experimental trial was conducted in SPAROS experimental facilities (Olh��o, Portugal). Sole larvae were reared in a RAS system, in triplicate tanks under controlled zootechnical conditions, and fed the experimental diets from 67 to 72 DAH: a commercial-like control diet (CTRL) formulated with high quality ingredients for a high performance, a negative control (CTRL-) formulated with high quality ingredients for a medium performance and a functional diet containing Ulva and Tetraselmis biomasses (ULVTET). After the feeding period, larvae were transported to a second facility (CIIMAR, Porto, Portugal) and exposed by bath to Tenacibaculum maritimum (1×104 CFU/mL), followed by a 14���day mortality follow���up. Two sampling points were considered: Pre���stress and Post���stress (18 h after pathogen exposure). Survival was analysed using Kaplan–Meier curves and a Cox hazard model. Humoral indicators and antioxidant enzymes activity were measured to assess the immune response and oxidative stress. Gut and liver RNA���seq was conducted (differentially expression analysis with FDR < 0.01) to assess larvae transcriptome response.
Results and Discussion
Transport followed by bacterial challenge induced a pronounced stress response in Senegalese sole larvae across all dietary groups, reflected by coordinated molecular, physiological and survival outcomes. Independent of diet, the gut transcriptomic response after challenge was characterized by a marked downregulation of cell cycle progression, DNA replication and repair, and proteasome���related pathways, indicating a conserved shift away from proliferative functions. Concomitantly, immune��� and stress���responsive signaling pathways were activated, although their biological organization differed substantially between diets. In larvae fed the CTRL diet, the post���stress gut response was dominated by strong upregulation of cytokine and interleukin signaling, chemokine���mediated pathways and MAPK/AKT signaling, suggesting a robust inflammatory activation. This was associated with extensive liver remodeling, with enrichment of ER–Golgi trafficking, ribosome biogenesis and RNA processing, consistent with a broad systemic metabolic and secretory reprogramming. CTRL- fed larvae exhibited a similarly extensive transcriptional response but with gut enrichment additionally involving developmental and structural pathways, including ciliary assembly and morphogenetic processes. This molecular profile, together with less favorable oxidative stress balance and significantly increased mortality risk during the infection follow���up, indicates a reduced capacity to cope with the combined stressors. In contrast, larvae fed the ULVTET algae blend displayed a distinct resilience���associated signature. Although gut immune activation was maintained, with enrichment of cytokine–cytokine receptor interactions, chemokine signaling, eicosanoid/prostanoid receptors and ion homeostasis, hepatic transcriptomic disruption was markedly attenuated. Liver responses were more targeted, involving upregulation of protein processing, membrane trafficking and innate immune recognition pathways, together with unfolded protein response mechanisms. This coordinated gut–liver response aligned with improved oxidative stress regulation. While survival probability after challenge of ULVTETfed larvae was not statistically higher than that of the CTRL diet (p = 0.9400), Cox regression analysis revealed similarly lower mortality risk, in contrast with the CTRL- group, which was significantly higher than CTRL (p = 0.0019). Overall, the integrated data indicate that short���term functional feeding supports stress adaption and promotes a more regulated immune���metabolic response, limiting systemic destabilization and enhancing stress resilience in sole larvae.
Acknowledgment
This work was financially supported by "Pacto da Bioeconomia Azul" (Project No. C644915664-00000026) within the WP5 Algae Vertical, funded by Next Generation EU European Fund and the Portuguese Recovery and Resilience Plan (PRR), under the scope of the incentive line "Agendas for Business Innovation" through the funding scheme C5 – Capitalization and Business Innovation. This study received Portuguese national funds from FCT - Foundation for Science and Technology through contracts UID/04326/2025 (https://doi.org/10.54499/UID/04326/2025), UID/PRR/04326/2025 (https://doi.org/10.54499/UID/PRR/04326/2025) and LA/P/0101/2020 (DOI:10.54499/LA/P/0101/2020) to CCMAR.