Introduction
Improving broodstock condition is a key goal in turbot (Scophthalmus maximus) aquaculture, as breeder status can influence reproductive performance and consequently offspring quality. Functional diets based on sustainable marine ingredients may help support broodstock robustness, but their effects remain poorly characterized, particularly through non-lethal approaches. This study delves into turbot breeders, a valuable flatfish for human consumption, predominantly farmed in Southern European countries, Iceland, and Norway.
Skin mucus is particularly relevant in this study, not only because it allows minimally invasive repeated sampling, but also because it represents the first line of defence in fish and contains a wide range of immune effectors involved in protection against external challenges, thereby reflecting mucosal immune status. This study aimed to evaluate whether dietary supplementation with a macro- and microalgae blend modulates systemic humoral responses and skin mucus immune/proteomic profiles in turbot breeders during the reproductive season.
Materials and Methods
A four-month feeding trial was conducted at Flatlantic fish farm using approximately 200 breeders distributed across four 16 m3 tanks (2 Control and 2 ALGAE). Fish were fed either a commercial semi-moist broodstock diet (Control) or an isoproteic, isoenergetic experimental diet (ALGAE) supplemented with an algae blend (Spirulina platensis and Laminaria digitata) and micronutrients (astaxanthin, vitamin C, and vitamin E). The ALGAE diet aimed to enhance the immune status of turbot breeders during the breeding season. Minimally invasive and non-lethal sampling techniques (8 fish/tank) were performed for the collection of blood and skin mucus to study humoral parameters and proteomics in skin mucus.
Results
Plasma responses were modest. Lysozyme activity tended to be higher in fish fed the ALGAE diet, particularly in females, whereas bactericidal activity was higher in control males. In contrast, skin mucus showed a more pronounced response to dietary supplementation. Fish fed the ALGAE diet displayed increased mucus IgM levels, with a stronger response in females, indicating modulation of mucosal immune status.
This pattern was supported by proteomic analysis, processed against the turbot reference proteome, which retained 876 and 877 proteins in the first and second samplings, respectively. The ALGAE diet altered the skin mucus proteome in both sexes, with 165 and 51 differentially abundant proteins in females and males at the first sampling, and 56 and 47 at the second sampling. No significant diet x sex interaction was detected, indicating that the dietary effect was largely shared between sexes, although generally more pronounced in females.
Functional analysis of proteins, showing stable diet-associated responses across both sampling points, further supported the biological relevance of the ALGAE effect. In males, the dietary response was mainly associated with epithelial barrier integrity, cytoskeletal organization, protein folding and energy metabolism. In females, it was more strongly linked to proteostasis, autophagy, antigen processing and immune-related pathways.
Together, the increased mucus IgM and the diet-associated proteomic shifts indicate that macro- and microalgae blend supplementation modulated mucosal condition in turbot breeders, supporting skin mucus as a useful non-lethal matrix for nutritional assessment under production/farming conditions.
Discussion
The results indicate that supplementation with a macro- and microalgae blend modulated mucosal immune condition in turbot breeders, with skin mucus IgM and proteomic profiles responding more clearly than plasma humoral parameters. Notably, one of the most affected humoral parameters by the ALGAE diet was the skin mucus IgM. This finding is of particular interest, as IgM in fish has been shown to possess neutralizing and agglutinating activities against viral and bacterial pathogens. Additionally, it activates the classical pathway of complement upon recognizing pathogens (Salinas, et al., 2021). Given the prevalence of IgM in coating a substantial portion of the microbiota on different mucosal surfaces, including the skin, it is plausible that IgM plays a relevant role in maintaining microbiota homeostasis at these sites
Although systemic humoral responses were limited, the mucus proteome revealed diet-associated changes in pathways related to epithelial integrity, protein homeostasis and immune function. These findings suggest that functional broodstock diets can influence mucosal condition under commercial farming conditions and highlight skin mucus proteomics as a useful tool for nutritional assessment in aquaculture.
Overall, this study supports the potential of algae-based dietary supplementation to modulate mucosal status in turbot breeders and reinforces the value of non-lethal approaches for monitoring broodstock condition during the reproductive season.
Acknowledgment
This work was part of the BREEDFLAT project, funded by the EEA Grants Blue Growth Programme, project reference EEA.BG.CALL4.019.2020.
References
Salinas, I., Fernandez-Montero, A., Ding, Y., Sunyer, J.O., 2021. Mucosal immunoglobulins of teleost fish: A decade of advances. Dev Comp Immunol. 121, 104079. https://doi.org/10.1016/j.dci.2021.104079.