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%.