Introduction
Functional feeds are a promising strategy to improve disease resistance in aquaculture. In this context, plant extracts are rich in bioactives, making them an interesting source of compounds capable of modulating fish health and resilience. Particularly interesting are halophytes such as Salicornia ramosissima, which are salt tolerant plants capable of growing in unproductive soils. However, there is limited knowledge about the mechanisms of action of such products, specifically their interactions with the gut microbiota of fish and aquatic animals. Hence, in the present study, we evaluated the effects of diets supplemented with halophytes extracts on survival and gut microbiota of European seabass (Dicentrarchus labrax).
Materials and Methods
European seabass of 23.56 ± 0.71 g were fed five experimental diets (CTRL; XAD 0.1%; XAD 0.5%; EXT3 0.1%; EXT3 0.5%) for four weeks. At the end of the feeding trial, fish were challenged with Photobacterium damselae subsp. piscicida (intraperitoneal injection). Mortality was recorded over eight days, and gut microbiota samples were collected 72 hours post-challenge.
Gut microbiota was analysed using 16S rRNA gene amplicon full-length sequencing. Sequences were processed to generate amplicon sequence variants (ASVs) following quality filtering, denoising, and chimera removal, and were taxonomically assigned using the GTDB reference database. Alpha diversity (Shannon index) and beta diversity (Bray-Curtis dissimilarity) were used to assess community structure across treatments. Differential abundance analysis was performed with metagenomeSeq to identify taxa affected by the diets.
Results and Discussion
Fish fed diets supplemented with XAD 0.1% and EXT3 0.1% had a significantly lower mortality, which indicates that halophyte extracts at lower inclusion levels provide a protective effect against Photobacterium infection. Microbiota analysis showed no significant differences in alpha or beta diversity between the treatments. However, despite the lack of clear clustering by diet on Bray-Curtis-based ordination, some differences in dispersion were observed, particularly in the XAD 0.1% and Ext3 0.1% groups.
In contrast, differential abundance analysis revealed some significant differences. Fish fed diets supplemented with halophyte extracts showed a reduction in the relative abundance of Photobacterium, with the strongest decrease in the XAD 0.1% group. The genus Vibrio, which includes several fish pathogens, was also significantly reduced, but only in fish fed the diets with the lower extracts inclusion (0.1%). In contrast, both XAD diets showed increased abundance of Acinetobacter, while both extracts promoted taxa such as Rhodococcus gordoniae and Sphingomonas, which are often considered beneficial or environmentally relevant.
These results suggest that the protective effects of halophyte extracts might be connected with shifts in specific microbial groups, including reductions in potentially pathogenic genera, rather than large changes in overall microbiota diversity. To clarify the biological relevance of these changes, key ASVs are currently being analysed to understand their closest known relatives using BLAST and phylogenetic analysis.
Moreover, these extracts are being tested as both prophylactic and therapeutic strategies in other aquaculture species, including Atlantic salmon, rainbow trout, and whiteleg shrimp under chronic stress conditions, to understand their broad potential application in the aquaculture industry.
Acknowledgment
This study was co-funded by the European Union (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.