Inclusion of up to 20% Black Soldier Fly larvae meal in broiler chicken diet has a minor effect on caecal microbiota

Abstract

Background: The Black Soldier Fly larvae (BSFL) are a source of nutrients and bioactive compounds in broiler diets. Some components of the BSFL may serve as a prebiotic or may impact the intestinal microbiota of the broilers by other modes of action, which in turn can affect the health and performance of broilers. Here, we investigate the impact of up to 20% BSFL in broiler diets on the diversity and composition of the broiler's microbiota.

Methods: Four hundred broilers were fed five iso-nutritious experimental diets with increasing levels of BSFL meal reaching 0%, 5%, 10%, 15%, 20% in the finisher diets. Eight caecal content samples coming from each of the eight replicates per treatment were collected at two time points (day 21 and day 42) for DNA extraction and sequencing of the V3-V4 regions using Illumina MiSeq 2 × 300 bp pair-end sequencing with 341f and 805r primers. Analysis of variance and Spearman's correlation were performed, while QIIME2, DADA2, and Calypso were used for data analysis.

Results: When broilers were 21 days of age, the abundance of two groups of sequence variants representing Enterococcus and unclassified Christensenellaceae was significantly lower (p-value = 0.048 and p-value = 0.025, respectively) in the 20% BSFL group compared to the 0% BSFL group. There was no relevant alteration in the microbiota diversity at that stage. On day 42, the Spearman correlation analysis demonstrated that the sequence variants representing the genus Coprococcus showed a negative relationship with the BSFL inclusion levels (p-value = 0.043). The sequence variants representing the genus Roseburia and Dehalobacterium demonstrated a positive relationship with the BSFL dietary inclusion (p-value = 0.0069 and p-value = 0.0034, respectively). There was a reduction in the dissimilarity index (ANOSIM) caused by the 20% BSFL dietary inclusion.

Conclusion: The addition of up to 20% BSFL in broiler diets did not affect the overall caeca microbiota diversity or composition at day 21. On day 42, there was a reduction in the beta diversity caused by the 20% BSFL dietary inclusion. The abundance of the bacterial group Roseburia was increased by the BSFL dietary inclusion, and it may be beneficial to broiler immunity and performance.

Keywords: Broiler chickens; Hermetia illucens; Insects; Microbiota; Poultry.

Figure 1. Stacked bar plots showing general microbiota profile at phylum and genus level.

(A) Phylum level taxa at day 21. (B) Phylum level taxa at day 42. (C) Top 20 genera at day 21. (D) Top 20 genera at day 42. T1 (20%), T2 (15%), T3 (10%), T4 (5%), and T5 (0%).

Figure 2. (A) Chao1 diversity of 21 days old broilers fed 0%, 5%, 10% 15% or 20% Black Soldier Fly larvae (BSFL). (B) Chao1 diversity of 42 days old broilers fed 0 (red), 5 (green), 10 (blue), 15 (grey) or 20% (yellow) BSFL.

Box plots representing the variation of the Chao1 diversity (alpha diversity) index.

Figure 3. (A) Shannon index at genus level of 21 days old broilers fed 0%, 5%, 10%, 15%, or 20% BSFL. (B) Shannon index at amplicon sequence variant (ASV) level of 42 days old broilers fed 0%, 5%, 10%, 15%, or 20% BSFL.

Shannon Index in broilers caeca microbiota of different groups was compared through ANOVA analysis.

Figure 4. (A) ANOSIM Bray-Curtis (genus level) in the caeca of 21 days old broilers fed 0 (red), 5 (green), 10 (blue), 15 (grey), or 20% (yellow) Black Soldier Fly larvae (BSFL).

(B) ANOSIM Bray-Curtis at in the caeca of 42 days old broilers fed 0%, 5%, 10%, 15%, or 20% BSFL. Analysis of Similarities (ANOSIM Bray-Curtis) in broiler caeca community structure.

Figure 5. The microbial composition of 21 (A) and 42 (B) days old broilers ordinated by principal component analysis (PCA) of broilers fed 0%, 5%, 10%, 15%, or 20% BSFL.

The red circle represents the control group, green square represents the 5% BSFL group, the blue rhombi represent the 10% BSFL group, the gray triangle represent the BSFL group, and the yellow upside-down triangle represents the 20% BSFL group in the PCA analysis.

Figure 6. (A) Abundance of Enterococcus in the caeca of 21 days old broilers fed 0% (red), 5% (green), 10% (blue), 15% (grey), or 20% (yellow) BSFL. (B) Abundance of unclassified Christensenellaceae in the caeca of 21 days old broilers fed 0%, 5%, 10%, 15%, or 20% BSFL.

Abundance plot of bacterial population in 21 days old broilers caeca microbiota of different groups was compared through analysis of variance (ANOVA).

Figure 7. Relative abundance of microbiome at genus level in the caeca of 21 (A) and 42 (B) days old broilers.

On top of Figs. 6A and 6B (x-axis) there are red, green, blue, grey, and yellow representing the BSFL percentage groups (0% (red), 5% (green), 10% (blue), 15% (grey), and 20% (yellow)). Different colours in the squares at the legend boxes represent the bacterial groups found in the broilers caeca samples analysed. On the y-axis, the abundance of the bacterial groups found is represented from 0 to 40.

Figure 8. Spearman correlation was performed to evaluate the relative abundance of Coprococcus (A), Roseburia (B), and Dehalobacterium (C).

All three graphs are representing the bacterial abundance increases at genus level. The positive correlation of the Black Soldier Fly larvae (BSFL) with the Roseburia abundance in the caeca microbiome of the 42 days old broiler chickens is most likely linked with a broilers performance improvement.