Introduction
The sustainability of the aquafeed sector depends on an adequate and efficient response to the increasing demand for traditional fish-derived raw materials as aquaculture production increases. During the last decades, novel raw materials, including meals from terrestrial animal by-products, or oils from microalgae , have been proposed as alternatives to fishmeal (FM) and fish oil (FO). In parallel, great efforts have been spent for selective breeding to improve the productivity and sustainability of cultured fish species. Accordingly, the present study , in the frame of AquaIMPACT project (Horizon 2020), aimed to investigate in European sea bass (Dicentrarchus labrax ), the genetic- and nutritionally-mediated effects upon gut mucosal health and microbiota. D ifferent genetic stocks of fish fed with novel feed formulations completely devoid of FO and with low inclusion levels of FM , containing different additives, were tested.
Materials and methods
The feeding trials were carried out at the Parque Científíco-Tecnológico Marino of the University of Las Palmas de Gran Canaria (Telde, Canary Island, Spain) . Two batches of European sea bass populations: selected for high growth (HG), and wildtype (WT) , produced at MARBEC-IFREMER, were used. In the first trial, as described in details by Montero et al. (2023), 294 dph sea bass larvae (t=0) were randomly distributed in cylinder-conical tanks (10 tanks for each genotype and dietary group, 500 L or 1000 L) at an initial density of 50 fish per tank. F ish were fed to apparent satiation with either a control or a “future” diet (Table 1) , in which FO was totally replaced by a combination of poultry and DHA oil, and 50% of FM was replaced by poultry meal inclusion (Skretting ARC, Norway) . Fish were sampled at t=0, and at the end of the feeding trial (609 dph).
In the second trial, HG and WT sea bass were fed the “future” diet until achieving the initial experimental size of 16 g. Then, fish were randomly distributed in 24 tanks of 500 L (34 fish/tank ; 12 tanks for genotype ) and fed diets supplemented with 3 different functional additives (INVE, Belgium) as follows: 2 weeks at high dose followed by 10 weeks at low dose. The functional additives used were a probiotic mixture (PROB), organic acids mixture (ORG), and a phytogenic (PHYTO). In this case, fish were sampled for the scheduled analyses only at the end of trial ( after 12 weeks of feeding).
In both experimental trials, from each sampling point, 0.5 cm samples of proximal and distal intestine regions (n= 6 fish genotype/diet) were collected for GALT gene expression analysis by qPCR , whereas the mucosa associated microbiota was obtained by scraping the entire intestinal mucosa (n= 6 fish/ genotype/diet) with a sterile cotton swab . The high-throughput sequencing of 16S rRNA gene on MiSeq platform (Illumina) was performed to characterize the gut microbial communities as described in Rimoldi et al. (2019). The raw data obtained following sequencing were analyzed using the QIIME software. A Two -way ANOVA was applied to test for differences in gene expression and bacterial taxa relative abundances. Significance was set at p < 0.05.
Results and discussion
Results from the first feeding trial showed differences in the expression of GALT-related genes in distal intestine. In particular, the expression of cytokines il-1β , tnf-α , and il-10 was different, showing an interaction effect diet x genotype. With regard to gut microbiota, diet had a lower influence than genotype. Indeed, regardless of the diet, WT fish showed higher species richness than HG genotype . Furthermore, the gut microbiota of HG fish shared a reduced individual variability, indicating an enhanced capacity to cope with changes in diet composition. A significant genotype effect was found for specific bacterial taxa, such as Paracoccus genus and other genera belonging to Moraxellaceae family, which were more abundant in WT fish, regardless of the diet. Among them , the relative abundance of Paracoccus genus was positively correlated with the higher proinflammatory cytokine il-1β expression found in distal intestine of WT sea bass .
In terms of GALT-related gene expression , the second trial revealed a genotype x diet effect only for il-1β in distal intestine. I n terms of gut microbiota, discriminant analysis did not show a clear separation between fish fed the future diet and fish fed additive supplemented diets . Despite that, relative abundances of specific taxa varied among experimental groups. Specifically, fish fed ORG diet presented higher relative abundance of Streptococcus in both genotypes, whereas fish fed PHYTO had higher abundance of Lactobacillales . Interestingly , Bacillus -based PROB had bactericidal activity on Pseudomonas and Acinetobacter genera. This result confirmed, our previous evidence, that the Bacillus -based probiotics have the capacity to modulate gut microbiota in European sea bass despite of a lack of colonization of the host’s intestinal mucosa (Moroni et al., 2021).
Acknowledgements
The current study was supported by the EU Horizon 2020 AquaIMPACT (Genomic and nutritional innovations for genetically superior farmed fish to improve efficiency in European aquaculture) , number: 818367.
References
Montero, D., Carvalho, M., Terova, et al., 2023. Nutritional innovations in superior European sea bass (Dicentrarchus labrax ) genotypes: Implications on fish performance and feed utilization. Aquaculture 572, 739486.
Moroni, F., Naya-Català , F., Piazzon , M.C., et al., 2021. The Effects of Nisin -Producing Lactococcus lactis Strain Used as Probiotic on Gilthead Sea Bream (Sparus aurata ) Growth, Gut Microbiota, and Transcriptional Response. Front. Mar. Sci. 8, 364.
Rimoldi, S., Gini, E., Iannini, F., et al ., 2019 The effects of dietary insect meal from Hermetia illucens prepupae on autochthonous gut microbiota of rainbow trout (Oncorhynchus mykiss ). Animals , 9(4), 143.