Introduction
The aquaculture industry is one of the fastest growing industries of its sector, mostly due to the incremental demand of fish production. However, this generates resource management issues mainly in the use of fish meal (FM), which becomes both ecologically and economically unsustainable. To respond to this issue, novel dietary ingredients are being studied to formulate aquafeeds with suitable dietary profiles for each species. With a high protein and lipid contents, a balanced amino acid profile and a recent authorization for the incorporation in aquafeeds in the European Union, insect meal (IM) positions itself as a strong candidate for FM substitution. Introduction of novel ingredients, however, can impact over the fish immune status and/or growth performance. One of the known effects of introducing novel dietary ingredients is the potential to generate gut microbial communities shifts which are recognized to impact on fish growth, metabolism, and immune status (Ringø et al. 2015; Ringø et al. 2018; Serra et al. 2021). In fact, prebiotic effects have been attributed to IM, with these being commonly associated with the increase of dietary chitin, a structural polysaccharide present in the insects exoskeleton (Perry et al. 2020) However, information about the potential gut microbiota shifts occurring as a consequence of dietary IM inclusion are scarce (Perry et al. 2020). Thus, this study aims to assess the effect of three different IM, namely, Hermetia illucens larvae meal (HM), Tenebrio molitor larvae meal (TM), and H. illucens exuviae meal (HEM) on European seabass gut microbiota modulation.
Materials and methods
To access gut microbiota modulation due to dietary IM, five isoproteic (45%) and isolipidic (18%) diets were used: a control diet (CTR); a diet similar to the CTR with 5% commercial chitin (CHIT5), and three diets with 25% inclusion of HM25, TM25 and HEM2, respectively. The experimental diets were randomly assigned to 5 groups of 15 fish with an initial mean body weight of 53.7 ± 2.67 g. The trial was conducted in a recirculating aquaculture system equipped with 100 L water capacity tanks, thermo-regulated to 22.7 ± 0.3 °C. Fish were fed by hand twice a day, 6 days per week, until apparent visual satiation for 8 weeks. The allochthonous (digesta) and autochthonous (mucosa) communities were aseptically collected, had their DNA extracted and were subjected to a 2-step PCR barcoding of the V4 region of the 16S rRNA followed by Illumina Miseq sequencing (paired-end mode; 2× 300 bp). Amplicon pools were extracted from the raw sequencing data using the FASTQ workflow in BaseSpace (Illumina), demultiplexing was performed with the python package demultiplex (Laros JFJ, github.com/jfjlaros/demultiplex) and barcodes, linkers and primers were trimmed off using BBDuk (BBTools, sourceforge.net/projects/bbmap). DADA2 R package version 1.14.1 (https://www.r-project.org/, R 3.6.2) was used for demultiplexing amplicon sequencing variants (ASVs). Taxonomy assignment was based on SILVA taxonomy database (release 138). Amplicon sequence libraries were analysed using the vegan (v2.4.3) and phyloseq (v1.30.0) packages of the software R. DESeq2 (v1.26.0) implemented in phyloseq was used to determine statistical significant differences in ASV abundances between diets and/or sites. Finally, quantitative PCR of the conserved chiA gene was used to assess if the observed microbial shifts were accompanied by a genomic shift towards chitinase-encoding genes.
Results
Overall, the European sea bass gut microbiota was dominated by the phylum Proteobacteria, followed by Firmicutes, Actinobacteria and Bacteroidota. A beta-diversity analysis of the allochthonous and autochthonous communities revealed a stronger impact of the sampled site (digesta versus mucosa) when compared with the impact of the tested diets on community composition, with digesta communities being more diverse than their mucosal counterparts. Nevertheless, an overall impact of diet on the composition of the European sea bass gut microbiota (p=0.004, r2=0.244, PERMANOVA) could also be observed. While the mucosa-associated microorganisms were more resilient to dietary changes, digesta-associated communities were significantly altered upon HM-based diets leading to an increase in several families belonging to the Firmicutes and Actinobacteria phyla, with HEM25 presenting a stronger influence. Although genomic potential for chitinolytic activity is recognized for bacteria in both phyla, our results showed that only HEM25 was able to promote a shift towards chitinase-encoding taxa, mainly due to the increase of the Paenibacillaceae family, which was not found to significantly increase in HM25. When screening our samples for the presence of chitinase A gene (chiA) using primers targeting a conserved region of ChiA, we detected chiA in the digesta of fish in all diets, but fish fed the HEM25 diet had significantly higher copy numbers of chiA compared to fish fed any of the other diets (p=0.0077, ANOVA). These results show an increased chiA-driven chitonolytic potential of the European sea bass digesta microbiota fed a HEM25 diet.
In conclusion, our results showed a metabolic shift in fish gut microbiota towards chitin-degrading microorganisms due to dietary IM inclusion.
Acknowledgements
This research was supported by national funds through FCT - Foundation for Science and Technology within the scope of UIDB/04423/2020 and UIDP/04423/2020. P.E. and C.R.S. had scientific employment contracts supported by national funds through FCT. The researcher F.R. was supported by a FCT grant, Portugal (SFRH/BD/138375/2018).
References
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