Introduction
Europe is a major contributor of mussels by supplying over a third of the world total production. It is known that inland hatchery cultures can support the extensive farming of this bivalve, by ensuring known quantity and quality of spats each year. However, these systems are still not economically viable , due to the high costs of conventional live microalgae feeds. For this reason, new alternative microencapsulated inert diets have been developed
. The aim of the present study was to test microencapsulated feeds containing Schizochytrium sp. as alternatives or supplement to a live microalgal diet and evaluate their effect on Mytilus galloprovincialis spat and adult microbiota , with the subsequent link of the microbiota structure of mussel with growth rate, proximal composition and gametogenesis .
Material and Methods
Mytilus galloprovincialis spats and adults were cultured at hatchery semi-industrial scale for 8 and 6 weeks, respectively, with different feeding conditions: 1) NC (Negative Control: no food supplied) ; 2) A (commercial microalgae: 100% ShellfishReed); 3) B (BioBullets as alternative singular diet : 100% Schyzochytrium ); 4) ABL (A lternative m ixed diet: 40% A + 60% B; 5) ABM (A lternative mixed diet: 20%A + 80%B) . To investigate their effect on gut microbiota, spat and adult mussel bacterial DNA from the digestive gland was sequenced (MiSeq Illumina) by targeting the V3-V4 16S rRNA gene. Sequences were processed with MOTHUR v1.44.0
and taxonomically affiliated by BLAST against SILVA database (Release 138.1). Alfa diversity estimators were calculated using Past 4.05 software
. Beta diversity analyses including h ierarchical clustering analysis (based on Bray-Curtis dissimilarity coefficients ), non-metric multidimensional scaling (NMDS) and permutational analysis of variance (PERMANOVA) w ere performed with R software using the “vegan” package
in order to investigate significant (p-value < 0.05) differences of gut microbial community structure between different treatments and over time. Finally, indicator species and metabolic predictions of microbiota were investigated for each diet using LEfSe and PiCRUST programs.
Results and conclusions
Microbial diversity of adult and spat gut microbiota were composed of 11682 and 5083 OTUs (after quality control and data normalization), respectively . Overall, mussel gut microbiota was dominated by phyla Proteobacteria (representing 63 % of the reads) and Bacteroidetes (25 %), followed by other significant phyla such as Firmicutes (4 %), Campylobacteria (1.5 %), Planctomycetes (1.3 %) and Verrucomicrobia (1.1 %) . As expected, differences were observed between adult and spat taxa composition, and different feeding conditions resulted in disparate proportions of bacterial community composition . Alpha diversity (Shannon, Simpson, Chao1 and Berger-Parker ) for gut microbiota revealed no statistical significances (p-value > 0.05) between diet treatments. Hierarchical cluster analysis, based on Bray-Curtis dissimilarity, showed that samples were primarily clustered according to the diet and then according to time of exposure. However, mixed diets (ABL and AMB) were clustered together according to time. These results correlate with the NMDS, where significant differences (p =0.00099 ) were recorded across microbial communities from different feeding conditions and time exposure. Next steps will be to search for indicator species and perform metabolic predictions of microbiota using LEfSe and PiCRUST programs . The whole analysis would help to link of the microbiota structure of mussel with growth rate, proximal composition and gametogenesis.
Acknowledgments
This project was supported by EIT Food Grant MIDSA (Grant number 20293). The authors want to acknowledge PI E-UPV/EHU staff to get access to their facilities to Camilla Campanati and AZTI’s staff to their facilities through funding from the European Union’s Horizon 2020 research and innovation program under grant agreement Nº 730984, ASSEMBLE Plus project. Ane del Rio is the recipient of a PhD grant from the Education Department of the Basque Government .
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