Introduction
In the last decades, the aquaculture industry has mainly directed its efforts towards the replacement of fishmeal with alternative protein sources, as a consequence of its sustainability and economic issues. Vegetable proteins have already been successfully included into commercial diets for rainbow trout, but novel feed ingredients – such as insects and microalgae – have also gained increasing attention (Sheikhzadeh et al., 2019; Biasato et al., 2022). In particular, Hermetia illucens (HI) meal has been reported to positively modulate the gut microbiota of rainbow trout in terms of selection of short-chain fatty acids (SCFAs)-producing bacteria and reduction of foodborne disease-causing pathogens (Biasato et al., 2022), while Spirulina (SP) seems to be able to promote gut microbiota diversity and increase beneficial bacteria in different fish species (Ren et al., 2022; Zhang et al., 2024). However, these two alternative protein sources have always been tested individually. Therefore, the present study aims to evaluate the impact of HI and SP meals – both individually and in different mixture percentages – on the gut microbiota of rainbow trout.
Materials and Methods
Seven isonitrogenous, isolipidic, and isoenergetic diets were formulated: a control diet (CTRL) containing 20% of fishmeal, and six diets where the fish meal was 100% replaced with HI meal (HI100), 100% SP meal (SP100), 75% HI and 25% SP (MIX1), 66.6% HI and 33.3% SP (MIX2), 33.3% HI and 66.6% SP (MIX3), and 25% HI and 75% SP (MIX4). A total of 441 fish (initial body weight of 200.5 ± 2.6 g) were randomly allotted to 21 400L-tanks (3 replicates/diet, 21 fish/tank). At the end of the trial (88 days), 6 fish per tank were slaughtered and the posterior content was collected for the characterization of the gut microbiota by means of 16S rRNA sequencing analysis. The bioinformatics analysis was performed using QIIME 2 vr. 2022.2.0 (https://docs.qiime2.org/) importing the raw reads for quality and chimera filtering step by dada2 denoise-paired script (sampling depth 7,500). The amplicon-sequence variants (ASVs) were used for taxonomic assignment by comparison with the Greengenes2 v2022.10 databases obtaining taxa abundances. The R package microeco (vr. 1.16.0) was used in RStudio (R vr. 4.4.2; RStudio 2024-10-31 ucrt) to perform statistical analysis (P<0.05).
Results
The 16S rDNA amplicon sequencing analysis allowed the detection of 329 bacterial genera. Observed, Fisher and Chao1 alfa diversity indices were lower in the CTRL, HI and MIX4 diets when compared to MIX3, while the latter also was also characterized by a higher Simpson and Shannon indices than the CTRL, and CTRL, HI, MIX1 and MIX4 groups, respectively (One-way ANOVA, P<0.05). Inverted Simpson and Pielou indexes also resulted higher in the MIX3-fed fish in comparison with the CTRL, MIX1 and MIX4 diets (One-way ANOVA, P<0.05). Differently, the beta-diversity analysis did not show a clear separation among the diets (Bray-Curtis's distances; Principal Coordinates Analysis [PCoA], P>0.05). However, Permutational Multivariate Analysis of Variance (PERMANOVA) demonstrated differences in gut microbiota composition between the CTRL and HI100 diet (F = 4.06, R2 = 0.30, P<0.05). Part of the taxa resulted differentially abundant among the groups, with Weissella spp. resulting less abundant in the CTRL and SP100 groups when compared to HI100-fed fish, and Streptococcus spp. being lower in the SP100 diet than the HI100 group (Dunn's Test of Multiple Comparisons, P adj.<0.05). Lastly, each dietary treatment displayed a distinct set of unique bacterial genera, thus indicating a strong selective pressure on the gut microbiota of the fish. In particular, the SP100 diet showed the highest number of diet-exclusive taxa (including Holdemanella, Paraeggerthella and Gallalistipes), followed by MIX3 and MIX4 groups (mainly characterized by Cellulomonas, Trichococcus and Nocardioides).
Discussion
The results herein obtained suggest that the diets containing the highest percentages of inclusion of SP meal (SP100, MIX3 and MIX4) were able to exert a most pronounced effect on the gut microbiota of the rainbow trout, as a consequence of its highly degradable polysaccharides and fermentable oligosaccharides (which led to the selection of unique bacterial genera associated with carbohydrate degradation and fiber fermentation) (ibrahim et al., 2026). However, the highest alpha diversity overall identified for the MIX3 diet interestingly suggests a potential synergy between SP and HI. Indeed, the concomitant presence of chitin (cellulose-like, insoluble fiber) – which reasonably explains the selection of cellulose-degrading (Weissella) (Biasato et al., 2022) and degraded chitin-using (Streptococcus) (Yildirim-Aksoy et al., 2019) ASVs in the HI100 group – and fermentable oligosaccharides (soluble fibers) may offer more diversified nutrients for the gut microbiota.
Acknowledgment
Financial support has been provided by PRIMA, a program supported by the European Union, under grant agreement No 2231, project CIPROMED (PRIMA Call 2022 Section 1 Agri-food IA).
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