Introduction
Novel functional feed-additives capable of improving fish health have gained attention in recent years. An increasing array of plant-derived compounds are being screened for their potential health safe-guarding characteristics. Recently halophytes, such as Salicornia ramosissima, are gaining traction as aquafeed supplements due to their potential immunomodulatory effects. Employing cells derived from Atlantic salmon (Salmo salar), the effects of several S. ramosissima extracts were assessed by conducting both functional and transcriptomic studies.
Materials and Methods
Head kidney primary cells were harvested from healthy Atlantic salmon, and adherent leukocytes (ASAL) were obtained following previously established protocols1. In addition, we employed the gill epithelial cell line ASG-10 that has been widely employed in in vitro studies2. These cells were exposed to different types of S. ramosissima extracts (labelled as EXT3, XAD and FERM), derived from distinct steps of the extraction cascade. The MTT (for ASAL) assay and propidium iodide staining-based analysis employing imaging flow cytometry (IFC; for ASG-10) determined the concentrations that are not toxic to the cell types. Suitable concentrations were then selected for the cell exposure studies.
Cells were exposed to each extract for either 3 or 24 h, followed by a challenge with inactivated pathogenic bacteria Tenacibaculum maritimum. Phagocytosis and reactive oxygen species (ROS) production were quantified employing IFC protocols1. Furthermore, a wound healing assay was performed on gill epithelial cells using an automated scratch system followed by live cell imaging. In parallel, a snapshot of the changes in the transcriptome of the two cell types was captured to reveal their molecular responses.
Results and Discussion
In the case of adherent leukocytes, only XAD elicited significant responses - an enhancement of phagocytosis and ROS production after 24 h exposure. These responses occurred after the transcriptional reprogramming toward immune activation, pathogen clearance and homeostasis, with early signs of molecular priming observed at the 3 h exposure timepoint.
As for the epithelial cells, at 24 h, XAD increased ROS production without affecting phagocytic activity. In contrast, EXT3 and FERM promoted a faster wound closure. These functional effects were, to a certain extent, supported by the information from transcriptomic analysis. XAD-treated epithelial cells were associated with an enrichment of oxidative stress and metabolic pathways, including glutathione metabolic process, cellular response to xenobiotic stimulus, and cellular iron ion homeostasis, suggesting a stress-adaptive response; while EXT3 exposure was associated with pathways linked to cell migration and tissue remodeling. These results indicate that S. ramosissima extracts promote a cell type-specific response, with XAD primarily enhancing immune activation in leukocytes and metabolic adaptation in epithelial cells, whereas EXT3 seems to favor epithelial repair processes.
Overall, the in vitro observations on S. ramosissima point to their potential as functional additives in aquafeeds, possibly capable of enhancing immune competence and enabling epithelial integrity of gill tissues of Atlantic salmon.
Acknowledgment
This study was co-funded by the European Union (Project: IGNITION - GA 101084651) and the UK Research and Innovation (UKRI). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union, the Research Executive Agency (REA) or the UKRI. Neither the European Union nor the granting authorities can be held responsible for them.
References
1.Park, Y. et al. Imaging flow cytometry protocols for examining phagocytosis of microplastics and bioparticles by immune cells of aquatic animals. Front. Immunol. 11, 203 (2020).
2.Slattery, O. et al. Functional and molecular characterization of the Atlantic salmon gill epithelium cell line ASG-10; a tool for in vitro gill research. Front. Mol. Biosci. 10, 1242879 (2023).