Early life bacterial succession under different diet regime Atlantic salmon (Salmo salar L.)

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Eleni Nikouli, Konstantinos Kormas, Yang Jin, Yngvar Olsen, Ingrid Bakke, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-104073/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 May, 2021 Read the published version in Frontiers in Marine Science → Version 2 posted You are reading this latest preprint version Show more versions Abstract Backgound : The present study investigated the effect of different lipid source in the feed on the colonization and the bacterial succession in early life stages (fertilized eggs until 93 days post first feeding) of S . salar . The two diets used in this study, FD (fish oil based diet) and VD (vegetable oil based diet), were formulated to cover the fish nutritional requirements and except the lipid source the components were identical between them. Hindgut samples collected at 0, 35, 65 and 93 days post first feeding (dpff). Moreover, fertilized eggs, yolk sac larvae, rearing water and feed were also sampled in order to assess a possible contribution of their microbiota to the colonization of the gut. To analyze the composition of the bacterial communities, the Illumina MiSeq platform was used. Results: S . salar growth variables (mean wet weight and total length) did not differ significantly during the experiment (p> 0.05) across replicate tanks and between dietary treatments. The analysis of the 16S rDNA sequencing data revealed a total of 4548 unique OTUs, affiliated in 21 bacterial phyla. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the dominant bacterial phyla. 13 OTUs were shared among all S . salar samples independent of life stage and diet treatment. Similarity percentages analysis (SIMPER) based on Bray–Curtis distance, showed that the average dissimilarity among the groups of the same life stages was 76.0%, whereas the average dissimilarity within groups of the same dietary treatment was 78.5% (FD) and 83.6% (VD). Conclusion: Feeding on either fish oil or vegetable oil-based diets, did not result in significant differences in the intestinal microbiota. The composition of gut microbiota did not differ significantly between the two dietary treatments, but changed with age, and each stage was characterized by different dominant bacteria. These OTUs are related to species that provide different functions and have been isolated from a variety of environments. Finally, this study revealed the occurrence of a core microbiota independent of the studied life stages and diet during the early life stages of Atlantic salmon. Animal Science General Microbiology Salmo salar larvae gut microbiota fish oil replacement Figures Figure 1 Figure 2 Figure 3 Introduction Atlantic salmon ( Salmo salar L.) is a carnivorous fish species, with significant economic value in European aquaculture, and Norway is a main producer worldwide [ 1 ]. Atlantic salmon has a complex life cycle, with anadromous migrations pattern, that is associated with morphological and physiological changes [ 2 ]. Their intestinal microbiota is affected significant by these changes [ 3 , 4 ], and recent findings have revealed a stage-associated gut bacteria in S . salar individuals from the early life in freshwater to the adult stages in seawater[ 5 ]. Shifts in the intestinal microbial communities across development have also been reported for other fish species [ 4 , 6 – 9 ]. These findings suggest that gut microbiota are affected by environmental sources (feed and the rearing environment), with stage specific selection pressures inside the gut. Moreover, many studies have reported the crucial effect of the feed on the gut bacterial community composition in fish species (10, 11, 12). Fishmeal and fish oil have been the main ingredients in diets for carnivorous fish species, providing fed fish the necessary proteins and lipids for high growth performance and resulting in a nutritionally rich final product (13,1 4). Due to the declining availability of fishmeal and fish oil their contents in feed is reduced [ 15 ] and substituted by a variety of alternative feed ingredients. Consequently, it is important to evaluate the impact of these new diets with lower fish-meal and -oil contents on the composition of the gut microbial communities for reared fish species (for a review see 16). The effect of fish-meal and -oil replacement with alternative protein and lipid sources on the gut bacterial communities of S . salar have been evaluated previously [e.g. 17–21], and have in some cases revealed changes associated with intestinal disorders. These studies, however, have focused on juveniles and adult stages and on alternative protein sources. The effect on the gut microbiota during the very early stages of feeding, with diets without fish -meal/-oil remains unexplored. However, recent findings from a dietary experiment in diploid and triploid S . salar populations have revealed that early dietary interventions can improve the utilization of the new fish-ingredient free formulated diets [ 22 ]. The objective of the present study was to characterize the bacterial community assembly and succession in early stages of S . salar population fed diets with and without fish oil. We also characterized bacterial communities of the rearing environment to determine their contribution in the early colonization and the succession of the fish intestines. To the best of our knowledge, this is the first study reporting the presence of a core gut bacterial community in S . salar during its early life stages, independent of diet and taking into consideration the epibiotas of fertilized eggs and yolk sac larvae. Materials And Methods Experimental design and sampling The experiment was conducted at the Ervik hatchery (Frøya, Norway) as described previously in Jin et al. [ 23 ]. Briefly, a fast-growing S. salar aquaculture strain was cultivated from fertilized eggs until 93 days post first feeding (dpff). When yolk sac absorption was observed (D0), S . salar individuals were randomly distributed from the initial stock tank (ST) into four tanks and fed two diets with different dietary lipid sources (2 dietary treatments x 2 replicate tanks x 200 individuals). The two diets, FD (fish oil diet) and VD (vegetable oil diet), were formulated to cover the fish nutritional requirements and except the lipid source, the rest components were identical (See Supplementary file, Table S1). Hindgut and rearing water sampling was performed at 0, 35, 65 and 93 dpff from all tanks. Ten fish were sacrificed from each tank during sampling (130 individuals in total) by immersion in 40 mg/L Benzocaine (BENZOAK VET, ACD Pharmaceuticals AS, Oslo, Norway). Hindguts were removed by aseptic dissection, rinsed with ultra-pure water and stored at -80 °C until analysis. Moreover, ten whole fertilized eggs (EG) and whole yolk sac larvae (YS) were sampled in order to assess a possible contribution of their microbiota to early colonization of the gut. Duplicate samples of rearing water microbes (100 ml/tank) were filtered onto 0.2 µm filters (GTTP, Millipore, USA). Approximately 0.25 g of the diets were collected for microbiota analysis. DNA extraction and Sequencing Microbial DNA was isolated from all the three types of samples (hindgut, water and diets) by using the QIAGEN QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol "DNA Purification from Tissues". Bacterial communities were characterized by Illumina 16S rDNA amplicon sequencing. To reduce the number of samples in the amplicon library, DNA extracts from 5 individual guts were pooled, resulting in two pooled gut samples from each time point/fish tank. The DNA from the rearing water samples were also pooled, resulting in 1 water sample per tank (STW – initial stock tank, FW – rearing water FD treatment and VW – rearing water VD treatment). The primer pair S-D-Bact-0341-b-S-17 and S-D-Bact-115 0785-a-A-21 [ 24 ] was used to amplify the V3-V4 regions of the 16S rRNA gene. A total of 37 samples (representing 30 pooled fish samples, 5 pooled water samples and 2 feed samples) was used in the final amplicon library. Both sequencing and PCR amplifications were performed according to Dowd et al. [ 25 ] at the MRDNA Ltd. (Shallowater, TX, USA) sequencing facilities on a MiSeq Illumina instrument using paired end reads (2 × 300 bp). Briefly, one-step Polymerase Chain Reaction (PCR) of 30 cycles was applied using HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany). Reaction times and cycling conditions were 94 °C for 3 minutes, followed by 28 cycles of 94 °C for 30 seconds, 53 °C for 40 seconds and 72 °C for 1 minute, with a final elongation step at 72 °C for 5 minutes. After amplification, the resulted PCR products were checked in 2% agarose gel to verify the success of amplification and the relative intensity of bands. Then, the PCR products were pooled together in equal proportions based on their molecular weight and DNA concentrations and purified using calibrated Ampure XP beads. Subsequently, the pooled purified pcr product was used to generate the sequencing libraries by following Illumina TruSeq DNA library preparation protocol. Data analysis Sequencing raw data were processed with the MOTHUR platform (version 118 1.38) [ 26 , 27 ] and the operational taxonomic units (OTUs) were classified by the SILVA Incremental Aligner (SINA) [ 28 ] following the methodology described in Nikouli et al. [ 29 ]. Identification of closest relative of each OTU was performed with Blast search ( http://blast.ncbi.nlm.nih . gov). Raw sequence data from this study have been submitted to the Sequence Read Archive ( https://www.ncbi.nlm.nih.gov/sra/ ) with BioProject accession number PRJNA520982. Statistical analysis and graphical illustrations were performed using the Palaeontological STudies (PAST) software [ 30 ] and the R Studio platform [ 31 ]. Results Fish growth The initial mean weight (D0) was 0.23 ± 0.03 g (± SD) and the final mean weight (D93) was 4.58 ± 1.74 g for FD and 4.54 ± 1.78 g for VD treatments (Table S2). The initial total mean length (D0) was 29.9 ± 1.6 cm which increased to 76.0 ± 8.9 cm and 73.8 ± 9.2 cm for the diets FD and VD, respectively, at D93 (Table S2). At none of the sampling points the mean wet weight or total length of S . salar differed significantly across replicate tanks or between dietary treatments (FD & VD) (p > 0.05; Fig. S1). Bacterial diversity The analysis of the 16S rDNA sequencing data revealed a total of 4548 unique OTUs, with the rarefaction curves (Fig. S2) and the Chao1 index (Table S3) indicating a satisfactory sequencing depth for the majority of the samples. The diversity was considerably higher for rearing water (STW, FW, VW) than gut and diet samples, both in terms of OTU richness (Table 1 ) and evenness (Table S3). Table 1 Illumina results of 16S rRNA gene diversity reported in in all sample categories. Life stages of S. salar (EG, YS, D0, D35F, D35V, D65F, D65V, D93F, D93V), rearing water (VW, FW, STW) and feed (VD, FD) samples. N: Number of biological replicates analyzed. D: Day. OTUs: Operational Taxonomic Units Samples Reads Observed OTUs richness No. of the Most Dominant OTUs (Cumulative Relative Dominance ≥ 80%) Most Abundant OTU(% of total reads) and Closest Relative (≥ 97%) Code Type/treatment EG Fertilized eggs 22151 ± 7168.6 Ν = 2 172 ± 99.7 16 SOTU0011 (23,9%) - Methylotenera versatilis YS Yolk sac 14382 ± 3186.2 Ν = 2 87 ± 0.7 10 SOTU0013 (19,4%) - Delftia acidovorans D0 Hindgut 21081 ± 1712.6 Ν = 2 132 ± 26.2 14 SOUT0009 (32,3%) - Iodobacter fluviatilis D35F 7658 ± 5011.0 Ν = 4 121 ± 64.8 46 SOTU0017 (9,3%) - Pseudomonas viridiflava D65F 2735 ± 1660.5 Ν = 4 110 ± 29.9 56 SOTU0070 (7,9%) - Janthinobacterium agaricidamnosum D93F 2003 ± 637.1 Ν = 4 93 ± 6.4 51 SOTU0005 (11,5%) - Cloacibacterium normanense D35V 25175 ± 27875.9 Ν = 4 135 ± 46.2 33 SOTU0005 (10,4%) - Cloacibacterium normanense D65V 4812 ± 1975.0 Ν = 3 132 ± 11.7 37 SOTU0005 (11,1%) - Cloacibacterium normanense D93V 1170 ± 608.3 Ν = 4 79 ± 25.3 46 SOTU0004 (7,0%) - Weissella cibaria FD Fish oil diet 21022 Ν = 1 259 7 SOTU0004 (38,6%) - Weissella cibaria VD Vegetable oil diet 20699 Ν = 1 216 8 SOTU0004 (37,8%) - Weissella cibaria STW Initial stock tank 53280 Ν = 1 2422 259 SOTU0001 (9,4%) - Polynucleobacter necessarius FW Rearing water-Fish oil diet treatment 76806 ± 11852.5 Ν = 2 1683 ± 183.8 52 SOTU0001 (14,5%) - Polynucleobacter necessarius VW Rearing water-Vegetable oil diet treatment 53618 ± 8553.9 Ν = 2 1100 ± 137.2 35 SOTU0001 (20,8%) - Polynucleobacter necessarius Taxonomic classification showed the presence of 21 bacterial phyla (Fig. 1 , Fig. S4). OTUs that were not classified to known bacterial phyla were only 3.0% of the relative abundance and are assigned as "Bacteria_unclassified". Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the dominant bacterial phyla in the dataset. The remaining 18 phyla (Planctomycetes, Verrucomicrobia, Patescibacteria, Dependentiae, Acidobacteria, Gemmatimonadetes, Fusobacteria, Cyanobacteria, Deinococcus-Thermus, Fibrobacteres, Armatimonadetes, Nitrospirae, Spirochaetes, Elusimicrobia, Omnitrophicaeota, Tenericutes, Chloroflexi and Kiritimatiellaeota) were present with relative abundance ≤ 2%. Similarities between Microbial Communities Statistical analysis revealed no significant differences (Tukey's test, p > 0.05, Tab. S4) in the bacterial community composition of the S . salar samples between the first ontogenetic stages (EG, YS, D0). However, EG and D0 samples differed significantly from those taken during the feeding period (D35 - D93) in both dietary treatments, with stage D35V as the only exception. YS bacterial communities differed significantly (p < 0.05) with the bacterial communities only at D93 in both dietary treatments (FD & VD). The gut microbiota of the S . salar juveniles did not reveal significant differences between the two dietary treatments for the different stages (p > 0.05), again with sample D35V as the only exception (Tab. S4). Ordination of the bacterial community composition of S . salar guts, based on a Bray–Curtis distance matrix (Fig. 2 ), showed a clear separation between bacterial communities in gut and bacterial communities of the rearing environment (WST, VW, FW, FD, VD). Moreover, the bacterial communities of S . salar samples were more similar with respect to life stages than to the diet treatments (Fig. 2 , Fig. S4). Similarity percentages analysis (SIMPER) based on Bray–Curtis distance, showed that the average dissimilarity among the groups of the same life stages was 76.0%, whereas the average dissimilarity within groups of the same dietary treatment was 78.5% (FD) and 83.6%(VD). Common and Unique OTUs Overall, only 2.3% of the OTUs were found in all categories of samples (the rearing water, the pre-, after first feeding guts and the diets). 75.4% of OTUs occurred only in water samples (Fig. 3 ). From the 1004 OUTs detected in total in S . salar samples, 423 OTUs (9.3% of the OTUs) were unique in that type of samples. The majority of them (343 OUTs) were unique in S . salar samples at the active feeding stages, whereas 13 OTUs were shared among all samples independent of life stage and diet treatment. S . salar microbiota Comparing S . salar microbiota between ontogenetic stages, fertilized eggs (EG) had the highest observed and estimated (Chao1) OTU richness (172 ± 100 and 222 ± 114, respectively). At the yolk sac stage (YS), the OTU richness decreased to 87 ± 0.7 and increased again at first feeding (D0). After that, OTU richness was on the same level until D93 when it decreased (Table 1 , Fig. S5). Proteobacteria was the dominant bacterial phylum in S . salar samples, mainly due to γ - and β-Proteobacteria (Fig. S6). β -Proteobacteria was the dominant subphylum in S . salar samples before first feeding (EG, YS, D0), with representatives mainly from the Burkholderiaceae and Chitinibacteraceae families (Fig. S7). However, in fertilized eggs (EG), OTUs representing β -Proteobacteriales were classified only at class level (44.1% of the total reads). γ -Proteobacteria dominated the period with active feeding in both dietary treatments (D35F, D65F, D93F, D35V, D65V and D93V), with Pseudomonadaceae, Xanthomonadaceae, Vibrionaceae, Enterobacteriaceae, Moraxellaceae and Aeromonadaceae as the most abundant families. However, their relative abundances differed between the two dietary treatments (Fig. S8). Actinobacteria, the dominant bacterial phylum at the late stages (D35V and D65V) in vegetable oil dietary treatment, was due to the high relative abundance of mainly Propionibacteriales, Corynebacteriales and Micrococcales representatives. The presence of Firmicutes and Bacteroidetes in S . salar samples was due to the classes Bacilli and Bacteroidia. Microbial communities in Diets and rearing water The bacterial communities in feed samples (FD, VD), consisted almost exclusively of Firmicutes (relative abundance of 84.2 and 82.1% in FD and VD, respectively, Fig. 1 ). The Firmicutes were affiliated to the Lactobacillaceae (38.5 and 36.6% in FD and VD respectively) and Leuconostocaceae families (37.9 and 38.8% in FD and VD, respectively). The rearing water samples (VW, FW, WST) contained mainly Proteobacteria, Actinobacteria and Bacteroidetes species, with Burkholderiaceae ( β -Proteobacteria), Sporichthyaceae (Actinobacteria) and Chitinophagaceae (Bacteroidetes) as the most abundant families (Fig. 1 ). In contrast to the experimental diets, Firmicutes in water samples were detected in relative abundance ≤ 1%. Discussion In the present study, we characterize the gut bacterial communities of S . salar populations during early development (13 weeks of feeding) and fed two diets with different lipid source (FD, VD). Moreover, we characterized the bacterial communities from the rearing environment (rearing water and feeds) and the epibiotas of fertilized eggs and yolk sac larvae to determine their contribution in the bacterial colonization and succession of the gut. Previous studies suggest that the bacterial communities of the rearing environment, mainly from the rearing water and the feed, are important sources for community assembly of the intestinal microbiota of fish [ 12 , 32 – 37 ]. For example, Schmidt et al. [ 19 ], reported a significant effect on intestinal microbial communities in postsmolt S . salar following replacement of dietary fishmeal with plant ingredients. However, the results in the present study suggest that substitution of fish oil by vegetable oils did not significantly affect the composition of intestinal microbial communities in the same host species. The results of the present study indicate little relationship between the epibiotic, gut and water bacterial communities, whereas the life stage appeared to be the main factor affecting the structure of gut microbiota. These results are in agreement with previous findings from Llewellyn et al. [ 3 ], who studied 96 wild-caught individuals of S . salar with different age and habitats, and observed grouping of their intestinal bacterial communities based on the lifecycle stage. In addition, Lokesh et al. [ 5 ], reported stage specific microbial enrichment in intestinal mucosa of S . salar (samples from embryonic stages up to 80-week post hatch). Similar stage specific signatures have also been reported across development in Sparus aurata [ 38 ], Danio rerio [ 8 ] and Gadus morhua [ 7 ] supporting further that the life stage seems to be the primary force shaping gut microbiota in juveniles’ stages of fish. The change in microbiota with life stage can be due to both host-microbe (e.g. development in morphology and immune system) and microbe-microbe interactions (mutualism, competitions and antagonism). The significance of these factors are, however, still not known. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the dominant bacterial phyla detected in S . salar samples for both dietary treatments in our study. This bacterial phylla seem to characterize the bacterial communities in individuals of Atlantic salmon ( S . salar ) at the freshwater life cycle stages [ 3 ]. These bacterial phyla are also commonly found in the gut bacterial communities of both saltwater and freshwater fish species [3 − 9, 16–21, 29, 32–42] Despite the fact that the two experimental feeds contained almost exclusively Firmicutes, the increase in relative abundance of Firmicutes in S . salar samples after the onset of feeding was not solely due to feed specific OTUs. It should also be noted that 26.4% of the bacterial representatives detected on fertilized eggs (EG) were not detected in the water of the incubation tank (WST). This support the view that the microbial communities of fish eggs may be vertically transmitted from their parents or horizontally from their breading tank [ 38 , 43 ]. In agreement with previous studies [ 5 , 19 , 38 ], the observed species richness in water samples was always an order of magnitude higher than the richness of the host samples. Bacterial communities in rearing water did not show major shifts during the experiment. OTU0001 dominated at all time points, with closest relative the bacterial species Polynucleobacter necessaries . This species is commonly found in freshwater samples and it can contribute to the catabolism of urea and reduction of nitrate [ 44 ]. The dominant bacterial species in S . salar samples are related with bacterial species from various habitats. The dominant OTU on fertilized eggs (OTU0011) was classified within the Methylotenera genus (β-Proteobacteria) and has previously been detected in fertilized salmon eggs by Lokesh et al. [ 5 ]. This genus consists of methylotrophic species that use methylamine as sole carbon, energy and nitrogen source [ 45 ]. The dominant OTU at the YS stage (OTU0013), seems to be related with Delftia acidovorans ( β -Proteobacteria). Species of the genus Delftia are obligate anaerobes, organotrophic and non-fermentative organisms (46). They have previously been detected in the gut of healthy individuals of Epinephelus coioides [ 47 ], Oncorhynchus mykiss [ 48 ] and Sparus aurata [ 29 , 40 ] and S . salar [ 49 ]. Just before onset on feeding (D0), the dominant OTU (OΤU0009) showed similarities with the species Iodobacter fluviatilis of the Chitinibacteraceae ( β -Proteobacteria) family. Species of this genus have been recorded mainly in sediment and water samples [ 50 – 52 ]. Their presence on fish skin ( Oncorhynchus mykiss and Salmo trutta ) has been associated with skin lesions [ 53 ]. However, the species has previously been detected in high relative abundance in healthy Coreius guichenoti individuals [ 54 ] whereas the present study reports the presence of this bacterial species in S . salar gut microbiota for the first time. After first feeding, although not statistically significant, differences were found between the bacterial communities in gut, each stage was characterized by different dominant OTUs. Moreover, gut bacterial communities differed also between dietary treatments regarding their dominant bacterial species (OTU). Chitinibacteraceae, the dominant bacterial family on D0, (with relative abundance 32.3%), was detected in ~ 50x lower relative abundance (≤ 0.6%) in the rest of the samples. At D35F and D65F, the dominant OTUs (OTU0017 and OTU0070, classified as Pseudomonas viridiflava and Janthinobacterium agaricidamnosum , respectively), are described as plant [ 55 – 58 ] and mushroom pathogens [ 59 – 60 ]. According to recent findings, Janthinobacterium lividum ( β -Proteobacteria) produce antimicrobial activity against multidrug resistant bacteria of clinical and environmental origin, such as Enterococci and Enterobacteriaceae [ 61 ]. Its presence in the gastrointestinal bacterial communities of S. salar, may have probiotic activity. At D35 and D65 samples from the VD dietary treatment were dominated by OTU0005, with closest relative Cloacibacterium normanense (Bacteroidetes). This OTU was also dominant at D93F. According to the literature, this species is frequently present in sewage treatment plants [ 62 , 63 ] where it contributes in decomposition of complex organic compounds [ 64 ]. Similar processes may take place in the intestinal system of S . salar at D35V, D65V and D93F. The dominant OTU at D93V (OTU0004), also dominant in the provided feed (FD, VD), was affiliated with Weissella cibaria (Firmicutes). This bacterial species belongs to the lactic acid bacteria, and has antimicrobial activity in the intestinal system of other fish species [ 65 ]. Other Weissella spp. have been found in gut of Oncorynchus mykiss [ 66 ] and S . salar [ 7 – 69 ]. It is worth noting that beside OTU0004, also OUT0013 and OTU0017 are associated with probiotic bacterial species (detected in all time points studied here, from EG to D93, independently of the dietary treatment, FD & VD). This observation suggests a co-evolutionary relationship of these bacterial species with the host studied here ( S . salar ), and a possible specialized function in the hosts intestinal system. Conclusions The present study investigated the effect of different lipid source in the feed on the colonization and the bacterial succession in early life stages of an aquaculture strain of S . salar , from fertilized eggs until 93 days dpff. We demonstrated that feeding on either fish oil or vegetable oil-based diets, did not result in differences in the intestinal microbiota. Our results complement those of other research groups [ 3 , 5 ] supporting that developmental stage and not the habitat and diet type determine the gut microbiota in S . salar . The composition of gut microbiota did not differ significantly between the two dietary treatments but changed with age, and each stage was characterized by different dominant bacteria. These OTUs are related to species that provide different functions and have been isolated from a variety of environments. Finally, this study revealed the occurrence of a core microbiota independent of the studied life stages and diet. Declarations Competing interests The authors declare that they have no competing interests. Author details 1 Department of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, Volos 384 46, Greece; EN: [email protected] ; KAK: [email protected] 2 Department of Biotechnology and Food Science, NTNU, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway; IB: [email protected] 3 Department of Biology, NTNU, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway; YJ: [email protected] ; YO: [email protected] Author’s contributions methodology, E.N., K.A.K., I.B., Y.O. and O.V.; formal analysis, E.N.; data curation, E.N. and K.A.K; writing—original draft preparation, E.N. and K.A.K.; writing—review and editing, E.N., K.A.K., Y.J., Y.O., I.B. and O.V.; supervision, O.V.; Acknowledgements We would like to thank Jostein Ervik for rearing the fish, AquaGen AS for providing the fish and Mari-Ann Østensen for assistance with the sampling and for her valuable suggestions during the planning and development of this study. Funding Eleni Nikouli was awarded with a visiting PhD student fellowship by State Scholarships Foundation/ IKY- Greece under the program ‘Scholarships of IKY in the Marine and Inland Management of Water Resources’ and was co-funded by EEA Grants – Financial Mechanism 2009- 2014 (85%) and the General Secretariat for Investments and Development (15%). Yang Jin: Internal IBI funding, China. Ethics approval The study was carried out within the Norwegian animal welfare act guidelines, in accordance with EU regulation (EC Directive 2010/63/ EU), approved by the Animal Ethics and Welfare Committee of the Norwegian University of Science and Technology (case number 16/10070). Consent for publication All authors approved the final version submitted and consent to its publication. Availability of data and materials The dataset supporting the conclusions of this article is available in the Sequence Read Archive repository, (https://www.ncbi.nlm.nih.gov/sra/) with BioProject accession number PRJNA520982 References FAO 2004-2019. Cultured Aquatic Species Information Programme. Salmo salar . Cultured Aquatic Species Information Programme. Text by Jones, M. In: FAO Fisheries and Aquaculture Department [online]. Rome. Updated 1 January 2004. McCormick, S.D., Sheehan, T.F., Björnsson, B.T., Lipsky, C., Kocik, JF., Regish, A.M.: Physiological and endocrine changes in Atlantic salmon smolts during hatchery rearing, downstream migration, and ocean entry. Canadian Journal of Fisheries and Aquatic Sciences 70(1), 105–18 (2013). Llewellyn, M.S., McGinnity, P., Dionne, M., Letourneau, J., Thonier, F., Carvalho, G.R., Creer, S., Derome, N.: The biogeography of the atlantic salmon ( Salmo salar ) gut microbiome. 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MicrobiologyOpen, 8(4):e00672 (2019) Supplementary Files NikouliSalmonBMCsupplementary01.docx Cite Share Download PDF Status: Published Journal Publication published 19 May, 2021 Read the published version in Frontiers in Marine Science → Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-104073","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":20478346,"identity":"59573810-d1be-4c68-8362-30460f9656af","order_by":0,"name":"Eleni Nikouli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYDACdgaGgw0MDDL8DAxsRGphhmjhkWxgJkELI0iLwQFitfAz8x48OKPiMI/xjfxjDxh+3SOsRbKZL+HghjOHecxuJLMbMPYVE9ZicJjH4ODDNrAWNgnGngTCWuxhWoxnEKvFgBmoZSNQi4EEUAvDDyK0SIBsmXEmnUfizGMzicQGIrTwt/cYf+ypsJbjb098JvHhDxFaUEFiG6k6GBj+kK5lFIyCUTAKhj8AAJNnN2Bu7H7WAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2941-5053","institution":"University of Thessaly School of Agricultural Sciences: Panepistemio Thessalias Schole Geoponikon Epistemon","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Eleni","middleName":"","lastName":"Nikouli","suffix":""},{"id":20478347,"identity":"77c3f758-30ac-4637-8b5c-82dd7887134c","order_by":1,"name":"Konstantinos Kormas","email":"","orcid":"","institution":"University of Thessaly School of Agricultural Sciences: Panepistemio Thessalias Schole Geoponikon Epistemon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Konstantinos","middleName":"","lastName":"Kormas","suffix":""},{"id":20478348,"identity":"87a22828-d79e-4b15-92d9-6f346650138c","order_by":2,"name":"Yang Jin","email":"","orcid":"","institution":"Norwegian University of Science and Technology: Norges teknisk-naturvitenskapelige universitet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Jin","suffix":""},{"id":20478349,"identity":"83c82b3a-3588-4a7e-858f-a9041432e471","order_by":3,"name":"Yngvar Olsen","email":"","orcid":"","institution":"Norwegian University of Science and Technology: Norges teknisk-naturvitenskapelige universitet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yngvar","middleName":"","lastName":"Olsen","suffix":""},{"id":20478350,"identity":"382bc5ba-6111-4623-931e-96c22684a7e5","order_by":4,"name":"Ingrid Bakke","email":"","orcid":"","institution":"Norwegian University of Science and Technology: Norges teknisk-naturvitenskapelige universitet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ingrid","middleName":"","lastName":"Bakke","suffix":""},{"id":20478351,"identity":"29b7b16f-8788-4ebf-b460-8a0846b694a9","order_by":5,"name":"Olav Vadstein","email":"","orcid":"","institution":"Norwegian University of Science and Technology: Norges teknisk-naturvitenskapelige universitet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Olav","middleName":"","lastName":"Vadstein","suffix":""}],"badges":[],"createdAt":"2020-11-06 14:25:14","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-104073/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-104073/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3389/fmars.2021.665576","type":"published","date":"2021-05-20T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":7807998,"identity":"1a1ee888-9fa7-419a-bfb3-f296a32ebdd5","added_by":"auto","created_at":"2020-11-20 21:22:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":199499,"visible":true,"origin":"","legend":"Phylum composition of microbiota from rearing water (A), diets (B) and hindgut of S. salar samples in FD (C) and VD (D) dietary treatments, at D35, D65 and D93 post first feeding ","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-111428/v1/fe12dbe5fb192457786d865b.png"},{"id":7808001,"identity":"be62183b-4c38-4794-9317-b7dd3ce76436","added_by":"auto","created_at":"2020-11-20 21:22:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":743190,"visible":true,"origin":"","legend":"Non-metric multidimensional scaling (nMDS) plot for all the bacterial communities of all sample categories based on Bray-Curtis distances. D: day. Life stages of S. salar: EG, YS, D0, D35F, D35V, D65F, D65V, D93F and D93. Rearing water: VW, FW, STW. Feed: VD and FD","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-111428/v1/6c1885dd1b648cbad018f09d.png"},{"id":7808003,"identity":"9b238a9f-75d3-48a9-9802-e66928c5a5fd","added_by":"auto","created_at":"2020-11-20 21:22:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148084,"visible":true,"origin":"","legend":"Venn diagram demonstrating the number of the shared and unique operational taxonomic units (OTU) and their percentage of the total library, between S. salar samples (before the first-feeding stage, after the first-feeding stage), diets (FD \u0026 VD) and rearing water samples.","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-111428/v1/e95dbab228d8b17081303a6c.png"},{"id":56542365,"identity":"fbcf6794-ea24-4a0c-8f13-a9c12e9a0367","added_by":"auto","created_at":"2024-05-15 14:31:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1152750,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-104073/v2/748c8c3f-f8bf-427e-af43-5b451469bae1.pdf"},{"id":7807999,"identity":"3957c3bd-9254-4211-a79f-04ed46526923","added_by":"auto","created_at":"2020-11-20 21:22:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2616863,"visible":true,"origin":"","legend":"","description":"","filename":"NikouliSalmonBMCsupplementary01.docx","url":"https://assets-eu.researchsquare.com/files/rs-111428/v1/d04a4004b704becb1cf4c234.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEarly life bacterial succession under different diet regime Atlantic salmon (\u003cem\u003eSalmo salar \u003c/em\u003eL.)\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAtlantic salmon (\u003cem\u003eSalmo salar\u003c/em\u003e L.) is a carnivorous fish species, with significant economic value in European aquaculture, and Norway is a main producer worldwide [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Atlantic salmon has a complex life cycle, with anadromous migrations pattern, that is associated with morphological and physiological changes [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Their intestinal microbiota is affected significant by these changes [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e], and recent findings have revealed a stage-associated gut bacteria in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e individuals from the early life in freshwater to the adult stages in seawater[\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eShifts in the intestinal microbial communities across development have also been reported for other fish species [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. These findings suggest that gut microbiota are affected by environmental sources (feed and the rearing environment), with stage specific selection pressures inside the gut. Moreover, many studies have reported the crucial effect of the feed on the gut bacterial community composition in fish species (10, 11, 12).\u003c/p\u003e\n\u003cp\u003eFishmeal and fish oil have been the main ingredients in diets for carnivorous fish species, providing fed fish the necessary proteins and lipids for high growth performance and resulting in a nutritionally rich final product (13,1 4). Due to the declining availability of fishmeal and fish oil their contents in feed is reduced [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e] and substituted by a variety of alternative feed ingredients. Consequently, it is important to evaluate the impact of these new diets with lower fish-meal and -oil contents on the composition of the gut microbial communities for reared fish species (for a review see 16).\u003c/p\u003e\n\u003cp\u003eThe effect of fish-meal and -oil replacement with alternative protein and lipid sources on the gut bacterial communities of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e have been evaluated previously [e.g. 17\u0026ndash;21], and have in some cases revealed changes associated with intestinal disorders. These studies, however, have focused on juveniles and adult stages and on alternative protein sources. The effect on the gut microbiota during the very early stages of feeding, with diets without fish -meal/-oil remains unexplored. However, recent findings from a dietary experiment in diploid and triploid \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e populations have revealed that early dietary interventions can improve the utilization of the new fish-ingredient free formulated diets [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe objective of the present study was to characterize the bacterial community assembly and succession in early stages of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e population fed diets with and without fish oil. We also characterized bacterial communities of the rearing environment to determine their contribution in the early colonization and the succession of the fish intestines. To the best of our knowledge, this is the first study reporting the presence of a core gut bacterial community in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e during its early life stages, independent of diet and taking into consideration the epibiotas of fertilized eggs and yolk sac larvae.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design and sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted at the Ervik hatchery (Fr\u0026oslash;ya, Norway) as described previously in Jin et al. [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, a fast-growing S. salar aquaculture strain was cultivated from fertilized eggs until 93 days post first feeding (dpff). When yolk sac absorption was observed (D0), \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e individuals were randomly distributed from the initial stock tank (ST) into four tanks and fed two diets with different dietary lipid sources (2 dietary treatments x 2 replicate tanks x 200 individuals). The two diets, FD (fish oil diet) and VD (vegetable oil diet), were formulated to cover the fish nutritional requirements and except the lipid source, the rest components were identical (See Supplementary file, Table S1).\u003c/p\u003e\n\u003cp\u003eHindgut and rearing water sampling was performed at 0, 35, 65 and 93 dpff from all tanks. Ten fish were sacrificed from each tank during sampling (130 individuals in total) by immersion in 40\u0026nbsp;mg/L Benzocaine (BENZOAK VET, ACD Pharmaceuticals AS, Oslo, Norway). Hindguts were removed by aseptic dissection, rinsed with ultra-pure water and stored at -80\u0026nbsp;\u0026deg;C until analysis. Moreover, ten whole fertilized eggs (EG) and whole yolk sac larvae (YS) were sampled in order to assess a possible contribution of their microbiota to early colonization of the gut. Duplicate samples of rearing water microbes (100\u0026nbsp;ml/tank) were filtered onto 0.2\u0026nbsp;\u0026micro;m filters (GTTP, Millipore, USA). Approximately 0.25\u0026nbsp;g of the diets were collected for microbiota analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction and Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003cp\u003eMicrobial DNA was isolated from all the three types of samples (hindgut, water and diets) by using the QIAGEN QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol \"DNA Purification from Tissues\". Bacterial communities were characterized by Illumina 16S rDNA amplicon sequencing. To reduce the number of samples in the amplicon library, DNA extracts from 5 individual guts were pooled, resulting in two pooled gut samples from each time point/fish tank. The DNA from the rearing water samples were also pooled, resulting in 1 water sample per tank (STW \u0026ndash; initial stock tank, FW \u0026ndash; rearing water FD treatment and VW \u0026ndash; rearing water VD treatment).\u003c/p\u003e\n\u003cp\u003eThe primer pair S-D-Bact-0341-b-S-17 and S-D-Bact-115 0785-a-A-21 [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] was used to amplify the V3-V4 regions of the 16S rRNA gene. A total of 37 samples (representing 30 pooled fish samples, 5 pooled water samples and 2 feed samples) was used in the final amplicon library. Both sequencing and PCR amplifications were performed according to Dowd et al. [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] at the MRDNA Ltd. (Shallowater, TX, USA) sequencing facilities on a MiSeq Illumina instrument using paired end reads (2\u0026thinsp;\u0026times;\u0026thinsp;300\u0026nbsp;bp). Briefly, one-step Polymerase Chain Reaction (PCR) of 30 cycles was applied using HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany). Reaction times and cycling conditions were 94\u0026nbsp;\u0026deg;C for 3 minutes, followed by 28 cycles of 94\u0026nbsp;\u0026deg;C for 30 seconds, 53\u0026nbsp;\u0026deg;C for 40 seconds and 72\u0026nbsp;\u0026deg;C for 1 minute, with a final elongation step at 72\u0026nbsp;\u0026deg;C for 5 minutes. After amplification, the resulted PCR products were checked in 2% agarose gel to verify the success of amplification and the relative intensity of bands. Then, the PCR products were pooled together in equal proportions based on their molecular weight and DNA concentrations and purified using calibrated Ampure XP beads. Subsequently, the pooled purified pcr product was used to generate the sequencing libraries by following Illumina TruSeq DNA library preparation protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003cp\u003eSequencing raw data were processed with the MOTHUR platform (version 118 1.38) [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] and the operational taxonomic units (OTUs) were classified by the SILVA Incremental Aligner (SINA) [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] following the methodology described in Nikouli et al. [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Identification of closest relative of each OTU was performed with Blast search (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nih\u003c/span\u003e\u003c/span\u003e. gov). Raw sequence data from this study have been submitted to the Sequence Read Archive (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sra/\u003c/span\u003e\u003c/span\u003e) with BioProject accession number PRJNA520982. Statistical analysis and graphical illustrations were performed using the Palaeontological STudies (PAST) software [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] and the R Studio platform [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eFish growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial mean weight (D0) was 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u0026nbsp;g (\u0026plusmn;\u0026thinsp;SD) and the final mean weight (D93) was 4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u0026nbsp;g for FD and 4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u0026nbsp;g for VD treatments (Table S2). The initial total mean length (D0) was 29.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u0026nbsp;cm which increased to 76.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u0026nbsp;cm and 73.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2\u0026nbsp;cm for the diets FD and VD, respectively, at D93 (Table S2). At none of the sampling points the mean wet weight or total length of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e differed significantly across replicate tanks or between dietary treatments (FD \u0026amp; VD) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. S1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of the 16S rDNA sequencing data revealed a total of 4548 unique OTUs, with the rarefaction curves (Fig. S2) and the Chao1 index (Table S3) indicating a satisfactory sequencing depth for the majority of the samples. The diversity was considerably higher for rearing water (STW, FW, VW) than gut and diet samples, both in terms of OTU richness (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and evenness (Table S3).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eIllumina results of 16S rRNA gene diversity reported in in all sample categories. Life stages of S. salar (EG, YS, D0, D35F, D35V, D65F, D65V, D93F, D93V), rearing water (VW, FW, STW) and feed (VD, FD) samples. N: Number of biological replicates analyzed. D: Day. OTUs: Operational Taxonomic Units\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSamples\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eReads\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eObserved OTUs richness\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNo. of the Most Dominant OTUs\u003c/p\u003e\n\u003cp\u003e(Cumulative Relative Dominance\u0026thinsp;\u0026ge;\u0026thinsp;80%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMost Abundant OTU(% of total reads) and Closest Relative (\u0026ge;\u0026thinsp;97%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCode\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eType/treatment\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFertilized eggs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22151\u0026thinsp;\u0026plusmn;\u0026thinsp;7168.6\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e172\u0026thinsp;\u0026plusmn;\u0026thinsp;99.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0011 (23,9%) - \u003cem\u003eMethylotenera versatilis\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYolk sac\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14382\u0026thinsp;\u0026plusmn;\u0026thinsp;3186.2\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0013 (19,4%) - \u003cem\u003eDelftia acidovorans\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"7\" align=\"left\"\u003e\n\u003cp\u003eHindgut\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21081\u0026thinsp;\u0026plusmn;\u0026thinsp;1712.6\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e132\u0026thinsp;\u0026plusmn;\u0026thinsp;26.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOUT0009 (32,3%) - \u003cem\u003eIodobacter fluviatilis\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD35F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7658\u0026thinsp;\u0026plusmn;\u0026thinsp;5011.0\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e121\u0026thinsp;\u0026plusmn;\u0026thinsp;64.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0017 (9,3%) - \u003cem\u003ePseudomonas viridiflava\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD65F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2735\u0026thinsp;\u0026plusmn;\u0026thinsp;1660.5\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;29.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0070 (7,9%) - \u003cem\u003eJanthinobacterium agaricidamnosum\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD93F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2003\u0026thinsp;\u0026plusmn;\u0026thinsp;637.1\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0005 (11,5%) - \u003cem\u003eCloacibacterium normanense\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD35V\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25175\u0026thinsp;\u0026plusmn;\u0026thinsp;27875.9\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e135\u0026thinsp;\u0026plusmn;\u0026thinsp;46.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0005 (10,4%) - \u003cem\u003eCloacibacterium normanense\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD65V\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4812\u0026thinsp;\u0026plusmn;\u0026thinsp;1975.0\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e132\u0026thinsp;\u0026plusmn;\u0026thinsp;11.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0005 (11,1%) - \u003cem\u003eCloacibacterium normanense\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD93V\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1170\u0026thinsp;\u0026plusmn;\u0026thinsp;608.3\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79\u0026thinsp;\u0026plusmn;\u0026thinsp;25.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0004 (7,0%) - \u003cem\u003eWeissella cibaria\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFish oil diet\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21022\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e259\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0004 (38,6%) - \u003cem\u003eWeissella cibaria\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVegetable oil diet\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20699\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e216\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0004 (37,8%) - \u003cem\u003eWeissella cibaria\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSTW\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInitial stock tank\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e53280\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2422\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e259\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0001 (9,4%) - \u003cem\u003ePolynucleobacter necessarius\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFW\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRearing water-Fish oil diet treatment\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76806\u0026thinsp;\u0026plusmn;\u0026thinsp;11852.5\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1683\u0026thinsp;\u0026plusmn;\u0026thinsp;183.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0001 (14,5%) - \u003cem\u003ePolynucleobacter necessarius\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVW\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRearing water-Vegetable oil diet treatment\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e53618\u0026thinsp;\u0026plusmn;\u0026thinsp;8553.9\u003c/p\u003e\n\u003cp\u003e\u0026Nu;\u0026thinsp;=\u0026thinsp;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1100\u0026thinsp;\u0026plusmn;\u0026thinsp;137.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSOTU0001 (20,8%) - \u003cem\u003ePolynucleobacter necessarius\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaxonomic classification showed the presence of 21 bacterial phyla (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. S4). OTUs that were not classified to known bacterial phyla were only 3.0% of the relative abundance and are assigned as \"Bacteria_unclassified\". Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the dominant bacterial phyla in the dataset. The remaining 18 phyla (Planctomycetes, Verrucomicrobia, Patescibacteria, Dependentiae, Acidobacteria, Gemmatimonadetes, Fusobacteria, Cyanobacteria, Deinococcus-Thermus, Fibrobacteres, Armatimonadetes, Nitrospirae, Spirochaetes, Elusimicrobia, Omnitrophicaeota, Tenericutes, Chloroflexi and Kiritimatiellaeota) were present with relative abundance\u0026thinsp;\u0026le;\u0026thinsp;2%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSimilarities between Microbial Communities\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003cp\u003eStatistical analysis revealed no significant differences (Tukey's test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Tab. S4) in the bacterial community composition of the \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples between the first ontogenetic stages (EG, YS, D0). However, EG and D0 samples differed significantly from those taken during the feeding period (D35 - D93) in both dietary treatments, with stage D35V as the only exception. YS bacterial communities differed significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with the bacterial communities only at D93 in both dietary treatments (FD \u0026amp; VD). The gut microbiota of the \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e juveniles did not reveal significant differences between the two dietary treatments for the different stages (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), again with sample D35V as the only exception (Tab. S4).\u003c/p\u003e\n\u003cp\u003eOrdination of the bacterial community composition of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e guts, based on a Bray\u0026ndash;Curtis distance matrix (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), showed a clear separation between bacterial communities in gut and bacterial communities of the rearing environment (WST, VW, FW, FD, VD). Moreover, the bacterial communities of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples were more similar with respect to life stages than to the diet treatments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. S4).\u003c/p\u003e\n\u003cp\u003eSimilarity percentages analysis (SIMPER) based on Bray\u0026ndash;Curtis distance, showed that the average dissimilarity among the groups of the same life stages was 76.0%, whereas the average dissimilarity within groups of the same dietary treatment was 78.5% (FD) and 83.6%(VD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eCommon and Unique OTUs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall, only 2.3% of the OTUs were found in all categories of samples (the rearing water, the pre-, after first feeding guts and the diets). 75.4% of OTUs occurred only in water samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). From the 1004 OUTs detected in total in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples, 423 OTUs (9.3% of the OTUs) were unique in that type of samples. The majority of them (343 OUTs) were unique in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples at the active feeding stages, whereas 13 OTUs were shared among all samples independent of life stage and diet treatment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e\u003cspan class=\"BoldItalic\"\u003eS\u003c/span\u003e. \u003cspan class=\"BoldItalic\"\u003esalar\u003c/span\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emicrobiota\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparing \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e microbiota between ontogenetic stages, fertilized eggs (EG) had the highest observed and estimated (Chao1) OTU richness (172\u0026thinsp;\u0026plusmn;\u0026thinsp;100 and 222\u0026thinsp;\u0026plusmn;\u0026thinsp;114, respectively). At the yolk sac stage (YS), the OTU richness decreased to 87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 and increased again at first feeding (D0). After that, OTU richness was on the same level until D93 when it decreased (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. S5).\u003c/p\u003e\n\u003cp\u003eProteobacteria was the dominant bacterial phylum in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples, mainly due to \u003cem\u003e\u0026gamma;\u003c/em\u003e- and \u0026beta;-Proteobacteria (Fig. S6). \u003cem\u003e\u0026beta;\u003c/em\u003e-Proteobacteria was the dominant subphylum in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples before first feeding (EG, YS, D0), with representatives mainly from the Burkholderiaceae and Chitinibacteraceae families (Fig. S7). However, in fertilized eggs (EG), OTUs representing \u003cem\u003e\u0026beta;\u003c/em\u003e-Proteobacteriales were classified only at class level (44.1% of the total reads). \u003cem\u003e\u0026gamma;\u003c/em\u003e-Proteobacteria dominated the period with active feeding in both dietary treatments (D35F, D65F, D93F, D35V, D65V and D93V), with Pseudomonadaceae, Xanthomonadaceae, Vibrionaceae, Enterobacteriaceae, Moraxellaceae and Aeromonadaceae as the most abundant families. However, their relative abundances differed between the two dietary treatments (Fig. S8). Actinobacteria, the dominant bacterial phylum at the late stages (D35V and D65V) in vegetable oil dietary treatment, was due to the high relative abundance of mainly Propionibacteriales, Corynebacteriales and Micrococcales representatives. The presence of Firmicutes and Bacteroidetes in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples was due to the classes Bacilli and Bacteroidia.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial communities in Diets and rearing water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterial communities in feed samples (FD, VD), consisted almost exclusively of Firmicutes (relative abundance of 84.2 and 82.1% in FD and VD, respectively, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The Firmicutes were affiliated to the Lactobacillaceae (38.5 and 36.6% in FD and VD respectively) and Leuconostocaceae families (37.9 and 38.8% in FD and VD, respectively). The rearing water samples (VW, FW, WST) contained mainly Proteobacteria, Actinobacteria and Bacteroidetes species, with Burkholderiaceae (\u003cem\u003e\u0026beta;\u003c/em\u003e-Proteobacteria), Sporichthyaceae (Actinobacteria) and Chitinophagaceae (Bacteroidetes) as the most abundant families (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast to the experimental diets, Firmicutes in water samples were detected in relative abundance\u0026thinsp;\u0026le;\u0026thinsp;1%.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we characterize the gut bacterial communities of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e populations during early development (13 weeks of feeding) and fed two diets with different lipid source (FD, VD). Moreover, we characterized the bacterial communities from the rearing environment (rearing water and feeds) and the epibiotas of fertilized eggs and yolk sac larvae to determine their contribution in the bacterial colonization and succession of the gut. Previous studies suggest that the bacterial communities of the rearing environment, mainly from the rearing water and the feed, are important sources for community assembly of the intestinal microbiota of fish [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. For example, Schmidt et al. [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e], reported a significant effect on intestinal microbial communities in postsmolt \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e following replacement of dietary fishmeal with plant ingredients. However, the results in the present study suggest that substitution of fish oil by vegetable oils did not significantly affect the composition of intestinal microbial communities in the same host species.\u003c/p\u003e\n\u003cp\u003eThe results of the present study indicate little relationship between the epibiotic, gut and water bacterial communities, whereas the life stage appeared to be the main factor affecting the structure of gut microbiota. These results are in agreement with previous findings from Llewellyn et al. [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e], who studied 96 wild-caught individuals of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e with different age and habitats, and observed grouping of their intestinal bacterial communities based on the lifecycle stage. In addition, Lokesh et al. [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e], reported stage specific microbial enrichment in intestinal mucosa of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e (samples from embryonic stages up to 80-week post hatch). Similar stage specific signatures have also been reported across development in \u003cem\u003eSparus aurata\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e], \u003cem\u003eDanio rerio\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e] and \u003cem\u003eGadus morhua\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e] supporting further that the life stage seems to be the primary force shaping gut microbiota in juveniles\u0026rsquo; stages of fish. The change in microbiota with life stage can be due to both host-microbe (e.g. development in morphology and immune system) and microbe-microbe interactions (mutualism, competitions and antagonism). The significance of these factors are, however, still not known.\u003c/p\u003e\n\u003cp\u003eProteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the dominant bacterial phyla detected in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples for both dietary treatments in our study. This bacterial phylla seem to characterize the bacterial communities in individuals of Atlantic salmon (\u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e) at the freshwater life cycle stages [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. These bacterial phyla are also commonly found in the gut bacterial communities of both saltwater and freshwater fish species [3 \u0026minus;\u0026thinsp;9, 16\u0026ndash;21, 29, 32\u0026ndash;42]\u003c/p\u003e\n\u003cp\u003eDespite the fact that the two experimental feeds contained almost exclusively Firmicutes, the increase in relative abundance of Firmicutes in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples after the onset of feeding was not solely due to feed specific OTUs. It should also be noted that 26.4% of the bacterial representatives detected on fertilized eggs (EG) were not detected in the water of the incubation tank (WST). This support the view that the microbial communities of fish eggs may be vertically transmitted from their parents or horizontally from their breading tank [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn agreement with previous studies [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e], the observed species richness in water samples was always an order of magnitude higher than the richness of the host samples. Bacterial communities in rearing water did not show major shifts during the experiment. OTU0001 dominated at all time points, with closest relative the bacterial species \u003cem\u003ePolynucleobacter necessaries\u003c/em\u003e. This species is commonly found in freshwater samples and it can contribute to the catabolism of urea and reduction of nitrate [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. The dominant bacterial species in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples are related with bacterial species from various habitats. The dominant OTU on fertilized eggs (OTU0011) was classified within the \u003cem\u003eMethylotenera\u003c/em\u003e genus (\u0026beta;-Proteobacteria) and has previously been detected in fertilized salmon eggs by Lokesh et al. [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. This genus consists of methylotrophic species that use methylamine as sole carbon, energy and nitrogen source [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. The dominant OTU at the YS stage (OTU0013), seems to be related with \u003cem\u003eDelftia acidovorans\u003c/em\u003e (\u003cem\u003e\u0026beta;\u003c/em\u003e-Proteobacteria). Species of the genus \u003cem\u003eDelftia\u003c/em\u003e are obligate anaerobes, organotrophic and non-fermentative organisms (46). They have previously been detected in the gut of healthy individuals of \u003cem\u003eEpinephelus coioides\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e], \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e] and \u003cem\u003eSparus aurata\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eJust before onset on feeding (D0), the dominant OTU (O\u0026Tau;U0009) showed similarities with the species \u003cem\u003eIodobacter fluviatilis\u003c/em\u003e of the Chitinibacteraceae (\u003cem\u003e\u0026beta;\u003c/em\u003e-Proteobacteria) family. Species of this genus have been recorded mainly in sediment and water samples [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]. Their presence on fish skin (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e and \u003cem\u003eSalmo trutta\u003c/em\u003e) has been associated with skin lesions [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, the species has previously been detected in high relative abundance in healthy \u003cem\u003eCoreius guichenoti\u003c/em\u003e individuals [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e] whereas the present study reports the presence of this bacterial species in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e gut microbiota for the first time.\u003c/p\u003e\n\u003cp\u003eAfter first feeding, although not statistically significant, differences were found between the bacterial communities in gut, each stage was characterized by different dominant OTUs. Moreover, gut bacterial communities differed also between dietary treatments regarding their dominant bacterial species (OTU). Chitinibacteraceae, the dominant bacterial family on D0, (with relative abundance 32.3%), was detected in ~\u0026thinsp;50x lower relative abundance (\u0026le;\u0026thinsp;0.6%) in the rest of the samples. At D35F and D65F, the dominant OTUs (OTU0017 and OTU0070, classified as \u003cem\u003ePseudomonas viridiflava\u003c/em\u003e and \u003cem\u003eJanthinobacterium agaricidamnosum\u003c/em\u003e, respectively), are described as plant [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e] and mushroom pathogens [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. According to recent findings, \u003cem\u003eJanthinobacterium lividum\u003c/em\u003e (\u003cem\u003e\u0026beta;\u003c/em\u003e-Proteobacteria) produce antimicrobial activity against multidrug resistant bacteria of clinical and environmental origin, such as Enterococci and Enterobacteriaceae [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]. Its presence in the gastrointestinal bacterial communities of S. salar, may have probiotic activity.\u003c/p\u003e\n\u003cp\u003eAt D35 and D65 samples from the VD dietary treatment were dominated by OTU0005, with closest relative \u003cem\u003eCloacibacterium normanense\u003c/em\u003e (Bacteroidetes). This OTU was also dominant at D93F. According to the literature, this species is frequently present in sewage treatment plants [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e] where it contributes in decomposition of complex organic compounds [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]. Similar processes may take place in the intestinal system of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e at D35V, D65V and D93F. The dominant OTU at D93V (OTU0004), also dominant in the provided feed (FD, VD), was affiliated with \u003cem\u003eWeissella cibaria\u003c/em\u003e (Firmicutes). This bacterial species belongs to the lactic acid bacteria, and has antimicrobial activity in the intestinal system of other fish species [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e]. Other \u003cem\u003eWeissella\u003c/em\u003e spp. have been found in gut of \u003cem\u003eOncorynchus mykiss\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e] and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e]. It is worth noting that beside OTU0004, also OUT0013 and OTU0017 are associated with probiotic bacterial species (detected in all time points studied here, from EG to D93, independently of the dietary treatment, FD \u0026amp; VD). This observation suggests a co-evolutionary relationship of these bacterial species with the host studied here (\u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e), and a possible specialized function in the hosts intestinal system.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study investigated the effect of different lipid source in the feed on the colonization and the bacterial succession in early life stages of an aquaculture strain of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e, from fertilized eggs until 93 days dpff. We demonstrated that feeding on either fish oil or vegetable oil-based diets, did not result in differences in the intestinal microbiota. Our results complement those of other research groups [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e] supporting that developmental stage and not the habitat and diet type determine the gut microbiota in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e. The composition of gut microbiota did not differ significantly between the two dietary treatments but changed with age, and each stage was characterized by different dominant bacteria. These OTUs are related to species that provide different functions and have been isolated from a variety of environments. Finally, this study revealed the occurrence of a core microbiota independent of the studied life stages and diet.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1 \u003c/sup\u003eDepartment of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, Volos 384 46, Greece; EN: \u003ca href=\"mailto:[email protected]\"\[email protected]\u003c/a\u003e ; KAK: \u003ca href=\"mailto:[email protected]\"\[email protected]\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2 \u003c/sup\u003eDepartment of Biotechnology and Food Science, NTNU, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway; IB: \u0026nbsp;\u0026nbsp;\u0026nbsp;\u003ca href=\"mailto:[email protected]\"\[email protected]\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDepartment of Biology, NTNU, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway; YJ: \u003ca href=\"mailto:[email protected]\"\[email protected]\u003c/a\u003e ; YO: \u003ca href=\"mailto:[email protected]\"\[email protected]\u003c/a\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emethodology, E.N., K.A.K., I.B., Y.O. and O.V.; formal analysis, E.N.; data curation, E.N. and K.A.K; writing\u0026mdash;original draft preparation, E.N. and K.A.K.; writing\u0026mdash;review and editing, E.N., K.A.K., Y.J., Y.O., I.B. and O.V.; supervision, O.V.;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Jostein Ervik for rearing the fish, AquaGen AS for providing the fish and Mari-Ann \u0026Oslash;stensen for assistance with the sampling and for her valuable suggestions during the planning and development of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEleni Nikouli was awarded with a visiting PhD student fellowship by State Scholarships Foundation/ IKY- Greece under the program \u0026lsquo;Scholarships of IKY in the Marine and Inland Management of Water Resources\u0026rsquo; and was co-funded by EEA Grants \u0026ndash; Financial Mechanism 2009- 2014 (85%) and the General Secretariat for Investments and Development (15%). Yang Jin: Internal IBI funding, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was carried out within the Norwegian animal welfare act guidelines, in accordance with EU regulation (EC Directive 2010/63/ EU), approved by the Animal Ethics and Welfare Committee of the Norwegian University of Science and Technology (case number 16/10070).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved the final version submitted and consent to its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article is available in the Sequence Read Archive repository, (https://www.ncbi.nlm.nih.gov/sra/) with BioProject accession number PRJNA520982\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFAO 2004-2019. 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Australasian Plant Disease Notes. 6(1):28\u0026ndash;9 (2011)\u003c/li\u003e\n\u003cli\u003eSarris, P.F., Trantas, E.A., Mpalantinaki, E., Ververidis, F., Goumas, D.E.: \u003cem\u003ePseudomonas viridiflava\u003c/em\u003e, a Multi Host Plant Pathogen with Significant Genetic Variation at the Molecular Level. PLOS ONE, 7(4):e36090 (2012)\u003c/li\u003e\n\u003cli\u003eLincoln, S.P., Fermor, T.R., Tindall, B.J.: \u003cem\u003eJanthinobacterium agaricidamnosum\u003c/em\u003e sp. nov., a soft rot pathogen of \u003cem\u003eAgaricus bisporus\u003c/em\u003e. International Journal of Systematic and Evolutionary Microbiology, 49(4):1577\u0026ndash;89 (1999)\u003c/li\u003e\n\u003cli\u003eGraupner, K., Lackner, G., Hertweck, C.: Genome Sequence of Mushroom Soft-Rot Pathogen \u003cem\u003eJanthinobacterium agaricidamnosum\u003c/em\u003e. Genome Announcements, 3(2):e00277-15. doi: 10.1128/genomeA.00277-15 (2015)\u003c/li\u003e\n\u003cli\u003eBaricz, A., Teban, A., Chiriac, C.M., Szekeres, E., Farkas, A., Nica, M., Dascălu, A., Lavin, P., Teban, A., Chiriac, C.M., Oprișan, C.: Investigating the potential use of an Antarctic variant of \u003cem\u003eJanthinobacterium lividum \u003c/em\u003efor tackling antimicrobial resistance in a One Health approach. Scientific Reports, 8(1):15272 (2018)\u003c/li\u003e\n\u003cli\u003eBenedict, R.G., Carlson, D.A.: Aerobic heterotrophic bacteria in activated sludge. Water Research, 5(11):1023\u0026ndash;30 (1971)\u003c/li\u003e\n\u003cli\u003eG\u0026uuml;de ,H.: Occurrence of Cytophagas in Sewage Plants. Applied Environmental Microbiology, 39(4):756\u0026ndash;63 (1980)\u003c/li\u003e\n\u003cli\u003eBernardet, J-F., Nakagawa, Y., Holmes, B., Subcommittee On The Taxonomy Of Flavobacterium And Cytophaga-Like Bacteria Of The International Committee On Systematics Of Prokaryotes: Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. International Journal of Systematic and Evolutionary Microbiology, 52(Pt 3):1049\u0026ndash;70 (2002)\u003c/li\u003e\n\u003cli\u003eMouri\u0026ntilde;o, J.L.P., Pereira, G., Vieira, F., Jatob\u0026aacute; ,A.B., Ushizima, T.T., Silva, B.C., Seiffert, W., Jesus, G.F.A., Martins, M.L.: Isolation of probiotic bacteria from the hybrid South American catfish \u003cem\u003ePseudoplatystoma reticulatum\u003c/em\u003e \u003cem\u003e\u0026times; Pseudoplatystoma corruscans\u003c/em\u003e (Siluriformes: Pimelodidae): A haematological approach. Aquaculture Reports, 3:166\u0026ndash;71 (2016)\u003c/li\u003e\n\u003cli\u003eLyons, P.P., Turnbull, J.F., Dawson, K.A., Crumlish, M.: Effects of low-level dietary microalgae supplementation on the distal intestinal microbiome of farmed rainbow trout \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e (Walbaum). Aquaculture Research, 48(5):2438\u0026ndash;52 (2017)\u003c/li\u003e\n\u003cli\u003eReveco, F.E., \u0026Oslash;verland, M., Romarheim, O.H., Mydland, L.T.: Intestinal bacterial community structure differs between healthy and inflamed intestines in Atlantic salmon (\u003cem\u003eSalmo salar\u003c/em\u003e L.). Aquaculture, 420\u0026ndash;421:262\u0026ndash;9 (2014)\u003c/li\u003e\n\u003cli\u003eGodoy, F.A., Miranda, C.D., Wittwer, G.D., Aranda, C.P., Calder\u0026oacute;n, R.: High variability of levels of \u003cem\u003eAliivibrio\u003c/em\u003e and lactic acid bacteria in the intestinal microbiota of farmed Atlantic salmon \u003cem\u003eSalmo salar \u003c/em\u003eL. Annals of Microbiology, 65(4):2343\u0026ndash;53 (2015)\u003c/li\u003e\n\u003cli\u003eLokesh, J., Kiron, V., Sipkema, D., Fernandes, J.M.O., Moum, T.: Succession of embryonic and the intestinal bacterial communities of Atlantic salmon (\u003cem\u003eSalmo salar\u003c/em\u003e) reveals stage-specific microbial signatures. MicrobiologyOpen, 8(4):e00672 (2019)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Salmo salar, larvae, gut microbiota, fish oil replacement","lastPublishedDoi":"10.21203/rs.3.rs-104073/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-104073/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackgound\u003c/strong\u003e: The present study investigated the effect of different lipid source in the feed on the colonization and the bacterial succession in early life stages (fertilized eggs until 93 days post first feeding) of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e. The two diets used in this study, FD (fish oil based diet) and VD (vegetable oil based diet), were formulated to cover the fish nutritional requirements and except the lipid source the components were identical between them.\u003c/p\u003e\u003cp\u003eHindgut samples collected\u0026nbsp;at 0, 35, 65 and 93 days post first feeding (dpff). Moreover, fertilized eggs, yolk sac larvae, rearing water and feed were also sampled in order to assess a possible contribution of their microbiota to the colonization of the gut. To analyze the composition of the bacterial communities, the Illumina MiSeq platform was used. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e growth variables (mean wet weight and total length) did not differ significantly during the experiment (p\u0026gt; 0.05) across replicate tanks and between dietary treatments. The analysis of the 16S rDNA sequencing data revealed a total of 4548 unique OTUs, affiliated in 21 bacterial phyla. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the dominant bacterial phyla. 13 OTUs were shared among all \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalar\u003c/em\u003e samples independent of life stage and diet treatment. Similarity percentages analysis (SIMPER) based on Bray–Curtis distance, showed that the average dissimilarity among the groups of the same life stages was 76.0%, whereas the average dissimilarity within groups of the same dietary treatment was 78.5% (FD) and 83.6% (VD). \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Feeding on either fish oil or vegetable oil-based diets, did not result in significant differences in the intestinal microbiota. The composition of gut microbiota did not differ significantly between the two dietary treatments, but changed with age, and each stage was characterized by different dominant bacteria. These OTUs are related to species that provide different functions and have been isolated from a variety of environments. Finally, this study revealed the occurrence of a core microbiota independent of the studied life stages and diet\u0026nbsp;during the early life stages of Atlantic salmon.\u003c/p\u003e","manuscriptTitle":"Early life bacterial succession under different diet regime Atlantic salmon (Salmo salar L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-11-20 21:22:00","doi":"10.21203/rs.3.rs-104073/v2","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-11-11 22:11:24","doi":"10.21203/rs.3.rs-104073/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"61d336c7-5e38-4ba2-87da-e0f78a8deb00","owner":[],"postedDate":"November 20th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":3514389,"name":"Animal Science"},{"id":3514390,"name":"General Microbiology"}],"tags":[],"updatedAt":"2024-05-15T14:31:21+00:00","versionOfRecord":{"articleIdentity":"rs-104073","link":"https://doi.org/10.3389/fmars.2021.665576","journal":{"identity":"frontiers-in-marine-science","isVorOnly":true,"title":"Frontiers in Marine Science"},"publishedOn":"2021-05-20 00:00:00","publishedOnDateReadable":"May 20th, 2021"},"versionCreatedAt":"2020-11-20 21:22:00","video":"","vorDoi":"10.3389/fmars.2021.665576","vorDoiUrl":"https://doi.org/10.3389/fmars.2021.665576","workflowStages":[]},"version":"v2","identity":"rs-104073","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-104073","identity":"rs-104073","version":["v2"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}