DIETARY MICROENCAPSULATED BOTANICALS AND ORGANIC ACIDS PROMOTES SYSTEMIC THERMAL RESILIENCE IN EUROPEAN SEABASS Dicentrarchus labrax

Introduction

Mediterranean aquaculture is being threatened by yearly summer heatwaves phenomena, exposing fish to prolonged thermal stress with negative effects on health, immune function, and performance, especially for temperature-sensitive species (Martínez et al. 2023). Chronic heat stress (HS) in fish triggers a cascade of physiological responses, including upregulation of heat shock proteins and induction of systemic inflammation, ultimately compromising gut integrity, nutrient absorption, and overall health (Ma et al. 2025). Among the nutritional strategies investigated to mitigate HS effects, dietary botanical compounds and organic acids have shown promising results to enhance fish resilience both under homeostatic and stressful conditions (He et al. 2017; Caruso et al. 2026; Busti et al. 2020). This study investigated the systemic effects of a microencapsulated blend of botanicals and organic acids (AviPlus®Aqua) on the molecular stress response of European seabass (Dicentrarchus labrax) subjected to chronic HS, with a multi-tissue gene expression approach.

Materials and Methods

A total of 720 European seabass (initial body weight ~10 g) was placed into 12 tanks of 300 L in a marine RAS and allocated into 4 experimental groups (3 tanks/treatment, 60 fish/tank): thermoneutral control (CTR), thermoneutral treated (TRT), heat stress control (CTR-HS), and heat stress treated (TRT-HS). Treated groups received dietary supplementation of AviPlus®Aqua at 2 kg/MT of feed. Fish were acclimated on the study diets for one week at 25°C, after which water temperature was gradually increased by 1°C/day up to 30°C (5 days) and maintained for 23 additional days. The start of the acclimation period was considered as day 0, day 7 is the start of the thermal challenge and day 35 is the end of the challenge. Fish were weighed on d0 and d35 to evaluate growth performance. At the end of the trial, 9 fish per group (n=3 per tank) were sacrificed for qPCR-based gene expression analysis on intestine, liver and gill. A panel of markers related to inflammation (il-1β, tnf-α, and il-10) and stress-adaptation (hsp70, hsp90, gr) was assessed. Relative gene expression of selected markers was normalized using two housekeeping genes (β-actin and ef1α) following 2^-ΔΔCt method. Two-way ANOVA was used to evaluate the effects of dietary treatment and HS and their interaction, with significance set at p ≤ 0.05, using GraphPad Prism v.11.0.1 (GraphPad Software, Inc., Massachusetts, USA).

Results and Discussion

Growth performance at d35 was significantly affected by both HS and dietary treatment, with treated fish showing consistently better results (+5% of average weight and 0.14%/day of specific growth rate) than controls, particularly under HS. Concerning gene expression (Figure 1), two-way ANOVA revealed a significant effect of thermal challenge across all three target tissues, confirming the systemic molecular impact of chronic HS. Also, a significant treatment × temperature interaction was detected in most of the statistical comparisons across markers and tissues, indicating a stronger supplement effect under HS conditions. In particular, HS significantly upregulated the expression of il-1β, tnf-α, hsp70, hsp90, and gr (glucocorticoid receptor, a key mediator of cortisol-driven stress response) across the target tissues compared to thermoneutral conditions, while il-10 was reduced. This molecular signature is consistent with a sustained inflammatory and stress response. The dietary treatment attenuated the HS-dependent upregulation of hsp70, il-1β, and tnf-α in intestine, and hsp70, hsp90, gr, il-1β, and tnf-α in both gill and liver in TRT-HS fish compared to CTR-HS, with expression levels being restored to values comparable to those of CTR. Regarding il-10, the treatment produced a tissue-specific response, with a significant increase in gill and liver of TRT-HS fish compared to CTR-HS (restoring to value comparable with thermoneutral conditions), while an upregulation was also observed in the intestine, collectively suggesting a promotion of anti-inflammatory tone across all examined tissues.

Figure 1. mRNA levels of hsp70 and il-1β in (a) intestine, (b) liver, (c) and gill. Data are means (n = 5) ± SEM as vertical bars. Different letters indicate statistical significance with p  ≤ 0.05.

Figure 1. mRNA levels of hsp70 and il-1β in (a) intestine, (b) liver, (c) and gill. Data are means (n = 5) ± SEM as vertical bars. Different letters indicate statistical significance with p  ≤ 0.05.

Conclusions

This multi-tissue approach highlights the potential of targeted nutritional interventions to improve the adaptive capacity of fish under climate-driven heatwaves, supporting the sustainability of marine aquaculture.

References

Busti S., Rossi B., Volpe E., et al. 2020. «Effects of Dietary Organic Acids and Nature Identical Compounds on Growth, Immune Parameters and Gut Microbiota of European Sea Bass». Scientific Reports 10 (1): 21321. https://doi.org/10.1038/s41598-020-78441-9.

Caruso F., Toschi A., García-Beltrán J.M., et al. 2026. «Microencapsulated Botanicals and Organic Acids Improve Immune Status and Growth in Gilthead Seabream (Sparus Aurata L.)». Aquaculture Nutrition 2026 (1): 4213038. https://doi.org/10.1155/anu/4213038.

He W., Rahimnejad S., Wang L., Song K., et al. 2017. «Effects of Organic Acids and Essential Oils Blend on Growth, Gut Microbiota, Immune Response and Disease Resistance of Pacific White Shrimp (Litopenaeus Vannamei) against Vibrio Parahaemolyticus». Fish & Shellfish Immunology 70: 164–73. https://doi.org/10.1016/j.fsi.2017.09.007.

Ma S., Lv Y., Hou L., et al. 2025. «Effect of acute temperature stress on energy metabolism, immune performance and gut microbiome of largemouth bass (Micropterus salmoides)». Aquaculture and Fisheries 10 (2): 260–70. https://doi.org/10.1016/j.aaf.2023.10.001.

Martínez J., Leonelli F.E., García-Ladona E., et al. 2023. «Evolution of Marine Heatwaves in Warming Seas: The Mediterranean Sea Case Study». Frontiers in Marine Science 10: 1193164. https://doi.org/10.3389/fmars.2023.1193164.