Introduction
Seaweeds are an important source of nutrients and have been part of human nutrition worldwide. Their composition includes a large set of bioactive compounds that confer the algae antioxidant, immunostimulant, antibacterial and antiviral properties among others. In aquafeeds, algae inclusion as a main ingredient has been studied mainly as a fish meal replacement protein source, but more recently other properties have been highlighted pushing their usage as fish health boosters. The macroalgae Gracilaria gracilis has an interesting composition that includes biologically active phytochemicals and others that confer functional properties to be used in fish nutrition. Peixoto et al (2019) showed that a 5% inclusion of Gracilaria sp. biomass improved seabass resistance to a pathogen due to its immunostimulatory and antioxidant capacity. However, evidence have shown that algae can act as prebiotics, modulating gut microbial communities, stimulating the production of functional metabolites in the gut, and this might be one of the origins of the observed better physiological performances in fish.
The aim of this work was to evaluate the modulation of European seabass gut microbiota when fed with diets with Gracilaria gracilis biomass inclusion or its extract.
Material and Methods
Experimental diets were formulated to include seaweed dry powder at 2.5% and 5%, or 0.35% of the algae extract. The control diet was the base for all the diets, and these were provided to juvenile seabass (Dicentrarchus labrax) (17.49 ± 6.07 g; mean ± SD) ad libitum for 47 days.
By the end of the feeding trial, intestines were aseptically removed and a portion of anterior and another of posterior intestine were collected and stored at -80 °C until DNA extraction. DNA was extracted from the intestine mucosa and mucus and the V3 and V4 hypervariable region of the 16S rRNA gene was sequenced in a Miseq Illumina platform. Raw reads were processed in QIIME2, and as a first step, they were filtered for quality, merged and chimeras were removed with DADA2. Rarefaction was applied for alpha and beta diversity analysis. Taxonomical assignment of the obtained ASVs (amplicon sequence variant) was performed based on greengenes database and microbial community composition in both intestinal compartments of fish fed the four experimental diets was evaluated up to genus level (or species when available). Random forest analysis was performed to highlight the most important genera in the communities, and microbiome functionality was predicted with PICRUST2.
Results and Discussion
The community richness (Chao1 index) and diversity (Shannon index) were not altered by the diets, however, diets with algae biomass (both 2.5% and 5%) showed a tendency for higher richness and diversity. Both Weighted and Unweighted unifrac distances analysis indicated differences between anterior and posterior intestinal sections, however, the same analysis did not show differences in the microbial communities of each experimental group. The most abundant phyla were Proteobacteria, followed by Actinobacteria and Firmicutes, and in the anterior intestine groups fed with algae the last two phyla had higher abundance, whereas in posterior intestine abundances were more similar. Algae inclusion in diets significantly reduced the abundance of members of genus Photobacterium in both intestine sections, and inclusion of 2.5% algae biomass reduced the abundance of Staphylococcus genus’ members.
Based in a random forest analysis, the most important genera within the communities according to abundance shifts with diet were Photobacterium, Staphylococcus, Acinetobacter, Micrococcus and Sphingomonas, and their abundances were reduced when fish were fed diets with algae, but not always with algae extract.
Functional prediction of the intestinal microbiomes showed that in anterior intestine several pathways are modulated whereas in posterior modulation occurred in only few. These pathways were mainly related with metabolism and biosynthesis of protective compounds such as ectoine and were upregulated in fish fed diets supplemented with algae.
Conclusions
This study highlights that feeding fish with diets with Gracilaria gracilis biomass up to 5% did not change the gut microbial community structure. However, it produced an effect on specific taxa that might be related to disease states, and the reduction of abundance of these microorganisms in algae groups encourages to consider a possible association with reduced mortalities observed by Passos et al (2021), after a challenge with Photobacterium damselae subsp. piscicida. Despite the few changes in community composition, function of the microbiome was putatively modulated in a beneficial manner. This study brings light to the effects of algae as functional ingredient in fish gut microbiota and will serve as base for future studies.
References
Peixoto, M.J., Ferraz, R., Magnoni, L.J., Pereira, R., Gonçalves, J.F., Calduch-Giner, J., Pérez-Sánchez, J., Ozório, R.O.A., 2019b. Protective effects of seaweed supplemented diet on antioxidant and immune responses in European seabass (Dicentrarchus labrax) subjected to bacterial infection. Sci Rep 9. https://doi.org/10.1038/s41598-019- 52693-6.
Passos, R.; Correia, A.P.; Ferreira, I.; Pires, P.; Pires, D.; Gomes, E.; Carmo, B.; Santos, P.; Simões, M.; Afonso, C.; Baptista, T. (2021) Effect on health status and pathogen resistance of gilthead seabream (Sparus aurata) fed diets supplemented with Gracilaria gracilis. Aquaculture. 531:735888. https://doi.org/10.1016/j.aquaculture.2020.735888.
Aknowledgements
This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UIDB/04292/2020 granted to MARE, and the project MAR-02.01.01-FEAMP-0084- SeaWeedFeeds.