Introduction
The continuous increase in demand for fish puts pressure on the aquaculture industry to find alternatives to the limited fisheries-based dietary protein sources to avoid overexploitation of marine resources. The traditional fish meal is now being successfully replaced with more economically and environmentally sustainable ingredients. In this context, seaweeds and microalgae, the natural sources of nutrients and bioactive compounds, can improve fish growth and overall health. Furthermore, nutritional manipulation of the intestinal microbiota can have a positive impact on fish welfare and nutrition.
The macroalga Gracilaria gracilis and microalga Nannochloropsis oceanica have been recently explored by the feed industry, but the impact of the inclusion of such products on the gut microbiota of European seabass (Dicentrarchus labrax) is poorly understood. We evaluated the impact of seaweed G. gracilis and microalga N. oceanica, single or blended, on the composition of microbial community in the intestine of European seabass.
Materials and Methods
European seabass (30 g) in triplicate tanks were fed four diets for 106 days at CIIMAR facility (Matosinhos, Portugal): a commercial-based diet (CTRL) and three experimental diets with the inclusion of 8 % G. gracilis (GRA), 8 % N. oceanica (NAN), or a blend of 4 % of each alga (NANGRA), at the expense of fish meal and wheat meal. At the end of the trial, mucus from the posterior intestine was collected under sterile conditions. After DNA extraction, the V3-V4 region of the 16S rRNA was amplified and sequenced employing an Illumina® MiSeq platform. After quality filtering, taxonomic assignment of the representative bacterial ASVs was performed using the RDP classifier. The R packages “iNEXT” and “phyloseq” were used to calculate alpha diversity and then employing the functions in “ggplot2”, plots were generated for overall species richness, Shannon diversity and Simpson diversity. Kruskal-Wallis test followed by Dunn’s test was employed to detect significant differences. The package “microbiome” was employed to determine relative abundance of core taxa and then “DESeq2” was used to identify the OTUs that were differently abundant in the study groups.
Results and Discussion
Illumina sequencing reads were assigned to 4,371 ASVs. The inclusion of the seaweed G. gracilis and the microalga N. oceanica on diets for European seabass had a significant impact on the alpha diversity: species richness was significantly lower in fish that consumed the GRA diet compared to CTRL; while Shannon and the Simpson diversity measures were significantly reduced in fish fed both GRA and NAN diets compared to fish fed CTRL. Neverthless, when algae were included in a blend (NANGRA diet), the alpha diversity measures remained similar to those observed for fish that consumed the CTRL diet.
Core microbial taxa (prevalence and detection thresholds of 90 and 20 %, respectively) of the posterior intestine were composed of the genera Flavobacterium, Parcubacteria and Lactobacillus. The most abundant phyla in the posterior intestine were Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes. Compared to the CTRL fish, the inclusion of the tested algae, single or blended, led to a decrease or increase of specific groups of bacteria. The incorporation of the seaweed G. gracilis and the microalga N. oceanica, single or blended, led to a decrease in Acidobacteria (Gp4) and Firmicutes (Clostridium sensu stricto and Exiguobacterium); and an increase in Bacteroidetes (Kordia) and Proteobacteria (Acinetobacter). The diet GRA, in particular, led to a decrease in Acidobacteria (Gp6), Nitrospirae (Nitrospira), Proteobacteria (Rhizobium, Roseateles and Sphingobium) and Verrucomicrobia (Opitutus). The diet NAN was associated with a reduction in Bacteroidetes (Muricauda) and Proteobacteria (Ruminobacter); and an increase in Firmicutes (Clostridium XI) and Proteobacteria (Massilia). Concerning NANGRA, the decrease in the genera Opitutus, Muricauda and Gp6, and increase in Massilia reflected the differences observed with the inclusion of single algae G. gracilis or N. oceanica. The blend resulted in the decrease of only a single genus (Pseudomonas) that belongs to Proteobacteria, which is the predominant phyla in the gut microbiota of different marine fish species, including European seabass.
Conclusion
Overall, results indicate that the inclusion of seaweed G. Gracilis and microalga N. oceanica leads to a reduction of the gut microbial alpha diversity, while the blend attenuates these effects. Further clarification on the fuction of each specific group of bacteria affected by these dietary treatments will allow for a better undertanding on the impact of the tested algae, single or in combination, on the gut microbiota of European seabass.
Acknowledgements
This work was funded by the structured program of R&D&I ATLANTIDA - Platform for the monitoring of the North Atlantic Ocean and tools for the sustainable exploitation of the marine resources (reference NORTE-01-0145-FEDER-000040), supported by the North Portugal Regional Operational Programme (NORTE2020), through the European Regional Development Fund (ERDF). M. Ferreira acknowledge Fundação para a Ciência e a Tecnologia (FCT) for grant SFRH/BD/144843/2019 (FCT/FSE). The support received from Bisa Saraswathy, Researcher, Nord University, for data analyses is acknowledged.