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Additional file 1: Figure S1. Rarefaction curves based on observed amplicon sequence variants (ASVs) for the different sample types. The ASV table was rarefied based on minimum sequence size (10,332) in the sample for normalization of the sequence for computation of two of the beta-diversity indices (Jaccard distance and unweighted UniFrac distance). CD: control diet; IM: full-fat BSF (black soldier fly) larvae meal diet; DFIM: defatted BSF larvae meal diet; DCIM: de-chitinized BSF larvae meal diet; IO: BSF larvae oil diet; EX: BSF larvae exoskeleton diet. Figure S2. Principal component analysis (PCA) on standardized amplicon sequence variants (ASVs). Score plots for PC1 and PC2 (a) and PC1 and PC3 (b), mean scores (dark) with 95% confidence intervals for PC1 (c), PC2 (d), and PC3 (e), and percentage of variance explained by PCs (f). PC: principal component, CD: control diet; IM: full-fat BSF (black soldier fly) larvae meal diet; DFIM: defatted BSF larvae meal diet; DCIM: de-chitinized BSF larvae meal diet; IO: BSF larvae oil diet; EX: BSF larvae exoskeleton diet. Figure S3. Principal component analysis (PCA) on metabolic reaction abundances (z-scores). Score plots for PC1 and PC2 (a) and PC1 and PC3 (b), mean scores (dark) with 95% confidence intervals for PC1 (c), PC2 (d), and PC3 (e), and percentage of variance explained by PCs (f). PC: principal component, CD: control diet; IM: full-fat BSF (black soldier fly) larvae meal diet; DFIM: defatted BSF larvae meal diet; DCIM: de-chitinized BSF larvae meal diet; IO: BSF larvae oil diet; EX: BSF larvae exoskeleton diet. Figure S4. Expected and observed taxonomic profiles of the mock microbial community standard. Mock_1, Mock_2: observed taxonomic profiles of the mock. Mock_Exp: expected taxonomic profile of the mock. Figure S5. Most abundant taxa in feed samples. Top 10 most abundant taxa at phylum (comprised 100% of abundance) (a) and top 15 most abundant taxa at genus or lowest taxonomy level (comprised 61���75% of abundance) (b) in feed samples. The plots display mean abundance of each taxon within the same diet. CD: control diet; IM: full-fat BSF (black soldier fly) larvae meal diet; DFIM: defatted BSF larvae meal diet; DCIM: de-chitinized BSF larvae meal diet; IO: BSF larvae oil diet; EX: BSF larvae exoskeleton diet. Figure S6. Linear discriminant analysis (LDA) effect size (LEfSe) results on gut microbiota of fish fed CD and DFIM diets. Circular cladogram reporting LEfSe results presents the identified amplicon sequence variants (ASVs) distributed according to phylogenetic characteristics around the circle (a). The dots closest to the center represent ASVs at the phylum level, whereas the outer circle of dots represent ASVs at the genus level. The color of the dots and sectors indicate the dietary group in which the respective ASVs are most abundant. The color explanation is given in the upper left corner. Yellow color indicates ASVs that showed similar abundance in all dietary groups. The colored sectors give information on phylum, class (full name in outermost circles, given only for phylum or class showing significant difference between groups), order, family, and genus (indicated by letter). The explanation is given below the cladogram. Indicator taxa with LDA scores of 4 or greater in the microbial communities (b). p: phylum; c: class; o: order; f: family; g: genus; CD: control diet; DFIM: defatted black soldier fly larvae meal diet. Figure S7. Linear discriminant analysis (LDA) effect size (LEfSe) results on gut microbiota of fish fed CD and DCIM diets. Circular cladogram reporting LEfSe results presents the identified amplicon sequence variants (ASVs) distributed according to phylogenetic characteristics around the circle (a). The dots closest to the center represent ASVs at the phylum level, whereas the outer circle of dots represent ASVs at the genus level. The color of the dots and sectors indicate the dietary group in which the respective ASVs are most abundant. The color explanation is given in the upper left corner. Yellow color indicates ASVs that showed similar abundance in all dietary groups. The colored sectors give information on phylum, class (full name in outermost circles, given only for phylum or class showing significant difference between groups), order, family, and genus (indicated by letter). The explanation is given below the cladogram. Indicator taxa with LDA scores of 4 or greater in the microbial communities (b). p: phylum; c: class; o: order; f: family; g: genus; CD: control diet; DCIM: de-chitinized black soldier fly larvae meal diet. Figure S8. Linear discriminant analysis (LDA) effect size (LEfSe) results on gut microbiota of fish fed CD and EX diets. Circular cladogram reporting LEfSe results presents the identified amplicon sequence variants (ASVs) distributed according to phylogenetic characteristics around the circle (a). The identified ASVs are distributed according to phylogenetic characteristics around the circle. The dots closest to the center represent ASVs at the phylum level, whereas the outer circle of dots represent ASVs at the genus level. The color of the dots and sectors indicate the dietary group in which the respective ASVs are most abundant. The color explanation is given in the upper left corner. Yellow color indicates ASVs that showed similar abundance in all dietary groups. The colored sectors give information on class (full name in outermost circles, given only for phylum or class showing significant difference between groups), order, family, and genus (indicated by letter). The explanation is given below the cladogram. Indicator taxa with LDA scores of 4 or greater in the microbial communities (b). c: class; o: order; f: family; g: genus; CD: control diet; EX: black soldier fly larvae exoskeleton diet. Figure S9. Linear discriminant analysis (LDA) effect size (LEfSe) results on gut microbiota of fish fed CD and IO diets. Circular cladogram reporting LEfSe results presents the identified amplicon sequence variants (ASVs) distributed according to phylogenetic characteristics around the circle (a). The dots closest to the center represent ASVs at the phylum level, whereas the outer circle of dots represent ASVs at the genus level. The color of the dots and sectors indicate the dietary group in which the respective ASVs are most abundant. The color explanation is given in the upper left corner. Yellow color indicates ASVs that showed similar abundance in all dietary groups. The colored sectors give information on phylum, class (full name in outermost circles, given only for phylum or class showing significant difference between groups), order, family, and genus (indicated by letter). The explanation is given below the cladogram. Indicator taxa with LDA scores of 4 or greater in the microbial communities (b). p: phylum; c: class; o: order; f: family; g: genus; CD: control diet; IO: black soldier fly larvae oil diet. Figure S10. Venn���s diagram showing the shared and unique core ASVs in digesta samples belong to insect-based groups (a), insect meal groups (b), insect lipid containing groups (c) and insect chitin containing groups (d). The core ASVs were computed using a prevalence threshold of 80%. CD: control diet; IM: full-fat BSF (black soldier fly) larvae meal diet; DFIM: defatted BSF larvae meal diet; DCIM: de-chitinized BSF larvae meal diet; IO: BSF larvae oil diet; EX: BSF larvae exoskeleton diet. Figure S11. The beta-diversity (based on Jaccard, unweighted UniFrac, and Aitchison distance matrices) of gut microbiota in fish fed experimental diets. PCo: principal coordinate; CD: control diet; IM: full-fat BSF (black soldier fly) larvae meal diet; DFIM: defatted BSF larvae meal diet; DCIM: de-chitinized BSF larvae meal diet; IO: BSF larvae oil diet; EX: BSF larvae exoskeleton diet. Figure S12. The boxplots for homogeneity of multivariate dispersions in gut microbiota of fish fed experimental diets. CD: control diet; IM: Full-fat BSF (black soldier fly) larvae meal diet; DFIM: Defatted BSF larvae meal diet; DCIM: De-chitinized BSF larvae meal diet; IO: BSF larvae oil diet; EX: BSF larvae exoskeleton diet. Figure S13. Number of ASVs mapped to genome-scale metabolic models. Number of samples matched to models at different taxonomic levels (a) and the number of models mapped to each sample by taxonomic level (b). Figure S14. Results from t-tests comparing reaction abundances between pairs of diets. The t-statistic for each reaction is shown along with the mean across all reactions with 95% confidence interval for all significantly enriched subsystems. IO and CD (a), EX and IO (b), DCIM and CD (c), IM and CD (d), IO and DFIM (e), IO and DCIM (f), DFIM and IM (g), EX and CD (h), EX and DCIM (i), IO and IM (j), DCIM and IM (k), DFIM and CD (l), EX and DFIM (m), EX and IM (n) and DCIM and DFIM (o) groups. CD: control diet; IM: full-fat BSF (black soldier fly) larvae meal diet; DFIM: defatted BSF larvae meal diet; DCIM: de-chitinized BSF larvae meal diet; IO: BSF larvae oil diet; EX: BSF larvae exoskeleton diet.

Additional file 2: Table S1. Chemical composition (%, as is) of meals and fractions of black soldier fly (BSF) larvae. Table S2. Fatty acid composition (% of total fatty acids) of the lipid fraction of black soldier fly larvae. Table S4. Pair-wise comparison of alpha diversity indices of gut microbiota in fish fed experimental diets containing meals and fractions of black soldier fly (BSF) larvae ��� adjusted p values. Table S5. PERMANOVA analysis for beta-diversity of gut microbiota in fish fed experimental diets containing meals and fractions of black soldier fly (BSF) larvae ��� adjusted p value. Table S6. Test of homogeneity of multivariate dispersions among dietary groups.

Additional file 2. Relative abundance of bacterial genera in the sequenced mock community standards along with their expected abundance.

Additional file 4. Differentially abundant ASVs between the yeast and control diets (ileum, caecum). A ileum, d 7 PW, B ileum, d 14 PW, C ileum, d 14 PW (family level), D caecum, d 7 PW (phylum level), E caecum, d 14 PW (phylum level). All taxonomic entities appeared as differentially abundant at FDR = 0.05.

Additional file 5. Differentially abundant ASVs between the yeast and control diets (caecum). A, caecum, d 7 PW, B caecum, d 14 PW, C caecum, d 7 PW (family level), D caecum, d 14 PW (family level). All taxonomic entities appeared as differentially abundant at FDR = 0.05.

Additional file 6. Differentially abundant ASVs between the yeast and control diets (colon). A colon, d 7 PW, B colon, d 14 PW, C colon, d 7 PW (family level), D colon, d 14 PW (family level). All taxonomic entities appeared as differentially abundant at FDR = 0.05.

Additional file 3. Alpha bacterial diversity as measured by a) Observed species and b) Shannon diversity index. The average values between the control and yeast group are represented as mean values with standard deviation (SD) along with medians with inter-quartile ranges (IQR). Comparison pairs that correspond to p-values less than 0.05 (MWW test) are given in bold.

Additional file 8. Association of liver index with individual bacterial groups, and colonic butyrate concentration. Panel A: Principal component analysis performed on the relative abundance of Spirochaetaceae, Faecalibacterium and the liver index of pigs measured at days 7 and 14 PW (n = 32 but 2 dots are not shown). The dots are coloured by the diet (control pink; yeast dark cyan). The vectors represent the degree of correlation between the bacterial groups data and the liver index data. Panel B: Correlation plot of colonic butyrate concentration (measured in μM per gram of intestinal contents) against liver index measured in the control group pigs at days 7 and 14 PW (n = 16). The dots are coloured by the diet (control pink). Pearson’s rho is reported above the regression line. Panel C: Correlation plot of colonic butyrate concentration (μM/gram of intestinal contents) against liver index measured in the yeast group pigs at days 7 and 14 PW (n = 16). The dots are coloured by the diet (yeast dark cyan). Pearson’s rho is reported above the regression line.

Additional file 7. Association of colonic butyrate concentration with individual bacterial groups. Panel A: Correlation plot of colonic butyrate concentration (measured in μM per gram of intestinal contents) against F. prausnitzii relative abundance measured at days 7 and 14 PW (n = 32). The dots are coloured by the diet (control pink; yeast dark cyan). Pearson’s rho is reported above the regression line. Panel B: Principal component analysis performed on the relative abundance of Spirochaetaceae, Faecalibacterium, Dialister and molarities of butyrate in the colon of pigs measured at days 7 and 14 PW (n = 32 but 3 dots are not shown). The dots are coloured by the diet (control pink; yeast dark cyan). The vectors represent the degree of correlation between the bacterial groups data and the butyrate concentration data.

Additional file 1. Comparison of bacterial CFUs on the selective agar plates. The values are the group medians (IQR) of logCFUs per gram of lumen contents. The bold font indicates statistically significant level (p

Apparent total-tract digestibility (ATTD) of nutrients could be an alternative measure of feed efficiency (FE) when breeding for robust animals that are fed fiber-rich diets. Apparent total-tract digestibility of nutrients requires measuring individual feed intake of a large number of animals which is expensive and complex. Alternatively, ATTD of nutrients and feces chemical composition can be predicted using fecal near-infrared reflectance spectroscopy (FNIRS). The objective of this study was to assess if the feces chemical composition and ATTD of nutrients can be predicted using FNIRS that originate from various pig-experimental datasets. Fecal samples together with detailed information on the feces chemical composition and ATTD of nutrients were obtained from four different pig experiments. Feces near-infrared spectroscopy was analyzed from fecal samples of a complete dataset. The model was calibrated using the FNIRS and reference samples of feces chemical composition and ATTD of nutrients. The robustness and predictability of the model were evaluated by the r(2) and the closeness between SE of calibration (SEC) and SE of cross-validation (SECV). Prediction of the feces chemical components and ATTD of nutrients were successful as SEC and SECV were equivalent. Calibration model was developed to estimate the ATTD of nutrients and fecal chemical composition from the FNIRS and worked well for OM (r(2) = 0.94; SEC = 48.5; SECV = 56.6), CP (r(2) = 0.89; SEC = 18.1; SECV = 18.8), GE (r(2) = 0.92; SEC = 1.2; SECV = 1.4), NDF (r(2) = 0.94; SEC = 55; SECV = 60.2), OM digestibility (r(2) = 0.94; SEC = 5.5; SECV = 6.7), GE digestibility (r(2) = 0.88; SEC = 2.3; SECV = 2.6), and fat digestibility (r(2) = 0.79; SEC = 6, SECV = 6.8). However, the SE of prediction was slightly higher than what has been reported in another study. The prediction of feces chemical composition for fat (r(2) = 0.69; SEC = 11.7, SECV = 12.3), CP digestibility (r(2) = 0.63; SEC = 2.3; SECV = 2.7), and NDF digestibility (r(2) = 0.64, SEC = 7.7, SECV = 8.8) was moderate. We conclude that the FNIRS accurately predicts the chemical composition of feces and ATTD of nutrients for OM, CP, and GE. The approach of FNIRS is a cost-effective method for measuring digestibility and FE in a large-scale pig-breeding programs.