As the aquaculture sector continues to grow at a rapid pace, there is a need to discover effective feed supplements that enhance the growth and welfare of fish bred in intensive environments. The capacity of yeasts to transform low-value materials into valuable resources has garnered interest as a promising novel ingredient in aquaculture. Among these resources, polysaccharides (e.g. b-glucans, mannans) and pigments (carotenoids) are important ingredients and have attracted significant research efforts . Studies have shown that the dietary inclusion of yeasts could reduce the adverse effects of soybean meal inclusion in Atlantic salmon ( Agboola et al., 2021). Dietary hydrolyzed Rhodotorula mucilaginosa has improved the growth performance and antioxidant capacity of Nile Tilapia ( Oreochromis niloticus) (Chen 2019) . In the current study, the yeast R. mucilaginosa was used as a fish feed additive to investigate its effects o n juvenile seabream Sparus aurata individuals’ microbiome.
Materials and methods
Rhodotorula mucilaginosa strain ACA-DC 5340 were grown on Potato Dextrose Agar petri-dishes and freeze-dried into a dry powder. Four experimental isolipidic , isoenergetic and isoproteic diets containing lyophilized yeast at at 0%, 1%, 2%, and 3% inclusion levels, were formulated by the Aquaculture Laboratory of the University of Thessaly. One hundred twenty (120) fish were acclimatized to laboratory conditions . The experiment consisted of four experimental groups (control, R1, R2, and R3), and each group encompassed three replicate tanks . Fish were hand-fed twice daily at a rate of 1,2% BW. At the end of the experiment, three (3) fish from each tank (36 fish in total) were sacrificed and the distal intestine was dissected and stored at -20oC un til downstream applications. Bacterial DNA was extracted with DNA Nucleospin Tissue (Macherey – Nagel, Duren, Germany) according to manufacturer instructions. V3-V4 variable region of 16S rRNA was amplified with 341F/785R primers. The corresponding libraries were normalized, pooled, and sequenced (paired-end, 2×250, v2 chemistry) using Illumina MiSeq (Microbiome Core Facility, Technical University of Munich). N ormalization of OTU tables and downstream analysis was carried out with Rhea 1.1.6 software.
The results showed that a total of 1.1 million high-quality reads for the 36 samples accounted for 572 operational taxonomic units (OTUs). Corresponding OTUs were mainly classified into five (5) groups with Firmicutes being the most abundant followed by Bacteroidota , Proteobacteria, Actinobacteriota, and Verrucomicrobiota. Microbial richness, was not drastically affected in the fish provided with dried yeast. The alpha-diversity, the Shannon and Simpson indices as well as the effective diversity and effective richness were increased, but not at a statistically significant level, while the composition of their microbial communities was significantly differentiated. Significant differences among treatments were observed in the genera of Acinetobacter, Cellulosilyticum , Clostridium, Corynebacterium, Cutibacterium , Enhydrobacter, Lawsonella , Micrococcus, Paracoccus , Phascolarctobacterium και Staphylococcus. Nine (9) OTUs exhibited significant differences between control and Rhodotorula mucilaginosa enriched treatments.
The results of the study demonstrated the existence of two groups of dominant phylotypes for both control and treatment groups. One of the groups tended to significantly differentiate in terms of bacterial strains. The dominance of the phylum Firmicutes in the intestinal microbiota of Sparus aurata is in accordance with previous studies . Firmicutes, Proteobacteria and Actinobacteria are considered as important for nutrition, the immune system, and metabolic homeostasis (Panteli et al., 2021, Moroni et al., 2021). Furthermore, Clostridia reduction is in line with a previous study in gilthead seabream in which diets containing garlic, carvacrol, or thymol essential oils provided effective antibacterial characteristics against fish pathogens , enhancing immune responses (Firmino et al., 2021). B eneficial bacteria in the gut of fish modulate fish innate immune system, compete for nutrients and antagonize other bacteria for adhesion sites. Concluding, R. mucilaginosa addition in seabream diets did not reduce microbial biodiversity and richness; any significant species differentiation seems that positively affect the intestinal microbiome.
Professor E. Tsakalidou and Mrs. E. Manolopoulou from the Laboratory of Dairy Research of the Agricultural University of Athens are kindly acknowledged for providing the experimental yeast strain from the ACA-DC microorganisms collection. This study has been funded by the Operational Programme Maritime and Fisheries 2014-2020 and co-funded by the European Maritime and Fisheries Fund through the project “ Use of yeasts and fungi in gilthead seabream diets towards improving external coloration and immune enhancement – BRIGHTFISH (MIS 5074567)”.
Agboola , J.O., Øverland , M., Skrede, A., Hansen, J.Ø. 2021. Yeast as major protein-rich ingredient in aquafeeds: a review of the implications for aquaculture production. Reviews in Aquaculture, 13: 949–970
Chen, X.Q., Zhao, W., Xie , S.W, Xie , J.J., Zhang, Z.H., Tian, L.X., Liu, Y.J., Niu, J. 2019. Effects of dietary hydrolyzed yeast (Rhodotorula mucilaginosa) on growth performance, immune response, antioxidant capacity and histomorphology of juvenile Nile tilapia ( Oreochromis niloticus). Fish and Shellfish Immunology, 90: 30–39
Firmino, J. P., Vallejos-Vidal, E., Balebona, M., Ramayo-Caldas, Y., Cerezo, I. M., Salomón, R., Tort, L., Estévez, A., Moriñigo, M. Á., Reyes-López, F. E., & Gisbert, E. (2021). Diet, Immunity, and Microbiota Interactions: An Integrative Analysis of the Intestine Transcriptional Response and Microbiota Modulation in Gilthead Seabream (Sparus aurata) Fed an Essential Oils-Based Functional Diet. Frontiers in Immunology, 12.
Moroni, F., Naya-Català, F., Piazzon, M. C., Rimoldi, S., Calduch-Giner, J. A., Giardini, A., Martínez, I., Brambilla, F., Pérez-Sánchez, J., & Terova, G. (2021). The Effects of Nisin- Producing Lactococcus lactis Strain Used as Probiotic on Gilthead Sea Bream (Sparus aurata) Growth, Gut Microbiota, and Transcriptional Response. Frontiers in Marine Science, 8
Panteli , N., Mastoraki , M., Lazarina , M., Chatzifotis , S., Mente, E., Kormas , K. A., & Antonopoulou , E. (2021). Configuration of Gut Microbiota Structure and Potential Functionality in Two Teleosts under the Influence of Dietary Insect Meals. Microorganisms, 9(4), 699.