2.1. Ethical Statement

The methods and procedures employed in this study adhered to the guidelines outlined in Directive 2010/63/EU of the European Parliament and the Council dated 22 September 2010 concerning the protection of animals used for scientific purposes. Additionally, they were in accordance with the Polish legislation outlined in the law of 15 January 2015 concerning the protection of animals used for scientific purposes (Polish Journal of Laws 2015, Item 266). The study also followed the ethical standards and recommendations set forth by both the National Ethics Committee for Animal Experiments and the Local Ethics Committee for Animal Experiments at Poznań University of Life Sciences. The experimentation took place in facilities certified for animal research by the National Ethics Committee for Animal Experiments (authorized by the Ministry of Science and Higher Education, approved unit no. 0091), specifically, the Laboratory of Inland Fisheries and Aquaculture within the Faculty of Veterinary Medicine and Animal Science at Poznań University of Life Sciences.

2.2. Full-Fat Insect Meal and Experimental Diets

H. illucens biomass was sourced from HiProMine S.A. in Robakowo, Poland. BSF larvae were fed a blend of plant by-products, standardized in dry matter (DM) content through the inclusion of wheat middlings [21]. The insect biomass underwent freezing at −20°C followed by drying initially at 130°C for 1 h and then at 80°C for 23 h until reaching a constant weight, using a chamber air dryer from HiProMine S.A. in Robakowo, Poland. Subsequently, the prepared insects were homogenized to a particle size of less than 0.1 mm using a beater mill to produce a full-fat meal.

The formulation of the diet is guided by the ratio of amino acids to the total protein content, utilizing the N × 6.25 conversion method [22–24]. The presence of chitin in insect biomass may lead to the overestimation of crude protein. Therefore, a calculation based on the analysis of BSFM used for the present experiment was performed. All the experimental diets were isonitrogenous, isoenergetic and isolipidic (Table 1).

The experimental diets were formulated and processed at the Experimental Station of Feed Production Technology and Aquaculture in Muchocin, Poland, utilizing a single-screw extruder (Metalchem S-60, Gliwice, Poland). Extrusion was conducted at 90°C in the cylinder and 110°C in the head, with a screw speed of 52 rpm and a 2 mm matrix size. Following drying and removal of dust particles through screening, the pellets were coated with fish oil at 40°C according to the specified recipe. Finally, the formulated diets were packed into plastic bags and stored at –18°C for preservation.

Five experimental feeds were prepared: a control diet (CON) without the addition of BSFM (crude protein 41.2%, crude fat 16.2%) and with 30% FM (crude protein 61.8%); BSFM5, with 5% addition of full-fat BSFM and 27.1% FM; BSFM10, with 10% full-fat BSFM and 24.3% FM; BSFM15, with 15% addition of full-fat BSFM and 21.3% FM; and BSFM20, with 20% addition of full-fat BSFM and 18.6% FM. The ingredient composition of the presmolt diets is presented in Table 2.

Ingredients and experimental diets were analysed according to the AOAC [25] methodology for crude protein (976.05), ether extract (920.39), crude ash (920.153) and crude fibre (985.29). The DM content was analysed according to the ISO 6496 method. The gross energy content was analysed according to the ISO 9831 method using an adiabatic bomb calorimeter (KL 12Mn, Precyzja-Bit PPHU, Poland) standardized with benzoic acid. To determine the number of macroelements (C and P), the methodology described by Ptak et al. [26] was applied. The amino acid profile was determined using an AAA-400 automatic amino acid analyser (Ingos Ltd., Prague, Czech Republic) and ninhydrin for postcolumn derivatization. Before carrying out profile analyses, hydrolysis in 6 N HCl for 24 h at 110°C was applied according to the AOAC procedure (994.12) [25]. The chemical composition and amino acid profile of the ingredients are presented in Table 3, while the results for experimental diets can be found in Table 1.

2.3. Animal Handling and Nutritional Trial

At the Experimental Station of Feed Production Technology and Aquaculture in Muchocin, a total of 375 Atlantic salmon (S. salar) presmolts were housed. These fish were randomly divided into five experimental groups, and the trial was conducted in three replications (tanks) per diet, each containing 25 fish, within tanks with a water capacity of 260 L. The average initial fish weight was 201.3 g. The water supply originated from a natural watercourse and was filtered through gravel-filled filters before being distributed into two equalizing tanks, each with a capacity of 5 m³. Aeration was provided using Hiblow HP200 aerators from Hiblow, Japan, and water was distributed to the tanks via a network of pipes. Water parameters, including temperature (ranging from 14.2 to 16.3°C) and oxygen concentration (ranging from 7.8 to 8.4 mg/L), were monitored daily throughout the 60-day experiment. The photoperiod was maintained at L16:D8. Feeding was automated using belt feeders (FIAP Fishtechnik GmbH, Germany), with a discharge time of 12 h. The feed ratio was based on a feeding chart designed for Atlantic salmon, considering the average body weight and the water temperature [27]. Fish mortality and health conditions were monitored daily.

2.4. Sampling

At the conclusion of the experiment, five fish per treatment were randomly chosen for sampling. Animals were euthanized using an overdose of MS-222 [28] and decapitated for dissection. The contents of both the proximal and distal sections of the GIT were carefully extracted, placed in cryotubes containing 99% ethanol and promptly stored at –80°C. These samples were preserved for subsequent analysis of microbial populations using next-generation sequencing (NGS).

2.5. Bacterial DNA Extraction and 16S rRNA Sequencing

DNA extraction was conducted using a commercial kit (Sherlock AX, A&A Biotechnology, Poland) following the manufacturer’s instructions. The samples underwent mechanical lysis on FastPrep-24 Zirconia beads (A&A Biotechnology, Poland) and were subsequently subjected to enzymatic lysis to eliminate bacteria. To confirm the presence of bacterial DNA in the samples, real-time PCR was performed using an Mx3000P thermocycler (Stratagene, USA) with SYBR Green as the fluorochrome. Universal reaction primers (1055F 5′-ATGGCTGTCGTCAGCT-3′ and 1392R 5′-ACGGGCGGTGTGTAC-3′) were employed for the amplification of 16S rDNA. The temperature conditions of the reaction were 95°C, 3 min; 95°C, 15 s; 58°C, 30 s; 72°C, 30 s; and Tm 65°C → 95°C.

DNA quantification was carried out using a NanoDrop spectrophotometer and standardized to a concentration of 5 ng/µL. The study of microbial diversity involved sequencing the amplified V3–V4 region of the 16S rRNA gene using specific primer pairs. The PCR procedure comprised an initial denaturation step at 95°C for 3 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 5 min and a holding step at 4°C. The expected size of the PCR products after amplification was approximately 550 base pairs, as confirmed by Bioanalyzer analysis. Subsequently, the PCR products were purified using AMPure XP beads.

For library preparation, the purified PCR products were combined with sequencing adapters and dual indices using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) as per the 16S Metagenomic Sequencing Library Preparation protocol (Illumina, San Diego, CA, USA). PCR with Nextera XT Index Primers was performed with initial denaturation at 95°C for 3 min, followed by eight cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 5 min and a holding step at 4°C. The resulting libraries were cleaned once more using AMPure XP beads and validated for the expected size on a Bioanalyzer trace, which was approximately 630 base pairs for the final library. Finally, the libraries were quantified using a fluorometric quantification method with dsDNA binding dyes, and individual concentrations were determined in nanomolar (nM) units based on the size of the DNA amplicons, as analysed by an Agilent Technologies 2100 Bioanalyzer.

For sequencing, each individual library was diluted to a concentration of 4 nM, denatured using 10 mM Tris buffer (pH 8.5) and supplemented with 20% (v/v) PhiX as an internal control. Aliquots containing 5 µL of diluted DNA were combined for pooling during library preparation for MiSeq analysis (Illumina, San Diego, CA, USA). Each sample underwent sequencing to generate more than 100,000 reads.

2.6. Metagenomic Analyses

Microbiome sequences were characterized based on the V3 and V4 amplicons and analysed using a database of 16S rRNA data. To amplify and prepare the library, specific sequences 341F and 785R were utilized. The PCR reaction conditions were carried out according to the specifications provided by the manufacturer for the Q5 Hot Start High-Fidelity 2X Master Mix. Sequencing was performed on the MiSeq platform using paired-end (PE) technology at 2 × 250 nucleotides with an Illumina v2 kit. The initial data analysis was conducted on the MiSeq instrument using MiSeq Reporter (MSR) v2.6 software. This analysis involved two main stages: automatic demultiplexing of samples and the generation of fastq files containing raw reads. Subsequently, the sequencing output provided a classification of reads at various taxonomic levels including kingdom, phylum, class, order, family, genus and species. Quality analysis of the sequences was performed through stringent quality control and filtration steps to obtain high-quality sequences. Valid sequences were selected from samples based on the barcode present at both ends of the sequence and were corrected for orientation using the primer sequences. All valid and filtered sequences were then clustered into operational taxonomic units (OTUs) based on a 97% similarity threshold in the 16S rRNA gene sequence identity. These OTUs were further analysed using basic local alignment search tool (BLAST) against the Greengenes database to determine their phylogenetic classification. The results were classified at multiple taxonomic levels including phylum, family and genus. Relative abundance profiles of the proximal and distal microbiota were established based on the abundance of OTUs belonging to different taxonomic groups.

2.7. Statistical Analysis

R studio software v1.4.14 was used to analyse the data obtained in this study. To determine the normality of the data distribution and equality of variances, the Shapiro–Wilk and Bartlett’s tests were used. In the case of a normal distribution, one-way ANOVA was used. When there were significant differences between treatments, further analysis was performed by correcting for Duncan’s post hoc test. For nonnormally distributed data, the Kruskal‒Wallis test was used. When there were significant differences between treatments, further analysis was performed by correcting Dunn’s post hoc test. The data are presented as the means ± standard error of the mean (SEM). The statistical significance level was set at p  < 0.05. The packages used in the statistical analyses were Agricolae, Dplyr, FSA, Ggplot2, Rcmdr, Rcompanion, UpSetR and Vegan.

3.1. GIT Microbiota Diversity

During the experiment, no mortality or health issues in fish were observed. The NGS analyses were conducted on a total of 25 samples, resulting in the generation of 2,455,779 raw sequence reads. Bacterial abundance was observed across all experimental groups, ranging from 99.92% to 100.00%. Additionally, traces of archaea were detected in the samples, comprising up to 0.02% of the total microbial composition. No significant differences (p  > 0.05) were observed in the alpha diversity indices for the proximal and distal intestine microbial compositions of the groups, irrespective of the intestinal section analysed (Figures 1 and 2).

3.2. Microbial Population Intersections Among the Treatments

The majority of the identified OTUs are unique to specific experimental diets, with minimal overlap observed between groups. This pattern holds true for both the proximal and distal sections of the intestine. In the proximal intestine, the CON group exhibited the highest number of distinct phyla (24), while the group supplemented with 20% BSFM had 15 characteristic phyla. Similarly, in the distal intestine, the CON group displayed the highest number of specific phyla (25). Increasing the inclusion of BSFM in the diet led to a reduction in the presence of specific phyla. Only one phylum was shared among all experimental groups in the proximal region, while two phyla were shared in the distal region. Additionally, eight phyla in the proximal section and 10 phyla in the distal section were shared between the CON group and other experimental groups (Figure 3). Similar trends were observed at the family level (Figure 4), with the CON group exhibiting the largest number of specific families in both the proximal (59) and distal (51) sections. Increasing the proportion of BSFM in the diet correlated with an increase in microbiota diversity in both sections of the intestine, with a comparable effect size. Regarding genus-level analysis (Figure 5), the CON group displayed the highest number of specific microbiota in both the proximal (77) and distal (64) sections. There were 54 genera recorded for the proximal section and 55 genera for the distal section shared between the two experimental groups. In contrast, the number of genera unique to each group was lower, with 26 genera in the proximal section and 30 genera in the distal section.

3.3. Relative Abundances of GIT Microbial Populations

The relative abundances at three levels (phylum, family and genus) in the proximal and distal sections are presented in Figures 6–8. For the proximal region at the phylum level (Table 4), an increase in the amount of Actinobacteria was recorded in BSFM5, BSFM10 and BSFM20 (p = 0.001). Increasing insect meal inclusion led to a decrease in the Fusobacteria population (p = 0.041). At the family level, Corynebacteriaceae increased in abundance in response to the application of BSFM5, BSFM10 and BSFM20 (p  < 0.001). BSFM10 showed an increase in the abundance of Staphylococcaceae. However, higher levels of Enterococcaceae were noted only in BSFM5 and BSFM10 (p = 0.042). Similar results were recorded at the genus level, where increases in Enterococcus were observed only in BSFM5 and BSFM10 (p = 0.042). In the case of Lactobacillaceae and Lactobacillus, no difference compared to CON was observed in BSFM5 and BSFM10 groups. Statistical analysis revealed no significant effect of the applied insect diets on the distal area microbiome (Table 5). Despite the lack of significant differences, Bacilliaceae family abundance increased from 20.08% in CON to 68.36% in BSFM20 treatment. while at the genus level, Bacillus increased from 7.78% in CON to 45% in BSFM20 in proximal intestine. Similarly in the distal part of the GIT at family level, numerical changes were observed in Bacillaceae which increased from 2.36% in CON to 30.06 and 27.13 in BSFM15 and BSFM20 groups, respectively. Bacillus population increased from 1.51% to 18.74 in BSFM 20. All groups with the use of BSFM resulted in Aeromonadaceae populations bellow 2%, while in CON it was 27.04%.