Hermetia illucens in diets for zebrafish (Danio rerio): A study of bacterial diversity by using PCR-DGGE and metagenomic sequencing

Abstract

In the present research, bacterial diversity was studied during a 6-month feeding trial utilizing zebrafish (Danio rerio) fed Hermetia illucens reared on different substrates with an emphasis on fish gut bacterial diversity. A polyphasic approach based on viable counting, PCR-DGGE and metagenomic 16S rRNA gene amplicon target sequencing was applied. Two different H. illucens groups were reared on coffee by-products (C) or a mixture of vegetables (S). Viable counts showed a wide variability based on substrate. PCR-DGGE and Illumina sequencing allowed the major and minor bacterial taxa to be detected. Both samples of larvae and their frass reared on the S substrate showed the highest richness and evenness of bacterial communities, whereas zebrafish (ZHC) fed H. illucens reared on substrate C and zebrafish (ZHS) fed H. illucens reared on substrate S had the lowest bacterial richness and evenness. A stimulating effect of bioactive compounds from coffee by-products on the occurrence of Lactobacillaceae and Leuconostoccaceae in H. illucens reared on substrate C has been hypothesized. Zebrafish gut samples originating from the two feeding trials showed complex microbial patterns in which Actinobacteria and Alteromonadales were always detected, irrespective of the diet used. Enterobacteriaceae in fish guts were more abundant in ZHS than in ZHC, thus suggesting an influence of the bioactive compounds (chlorogenic and caffeic acids) in the substrate on Enterobacteriaceae in fish guts. ZHC showed a higher abundance of Clostridia than did ZHS, which was likely explained by stimulating activity on the bacteria in this class by the bioactive compounds contained in H. illucens reared on substrate C. An influence of the microbiota of H. illucens or insect-derived bioactive compounds on the gut microbiota of zebrafish has been suggested. The presence of bacteria consistently associated with zebrafish guts has been found irrespective of the diet, thus attesting to the likely stability of the core fish microbiota.

Fig 1. Bacterial DGGE profiles of the DNA extracted directly from the analyzed samples and amplified with primers 338fGC and 518r.

The bands indicated by the letters were excised, reamplified and subjected to sequencing. The identities of the bands are given in Table 2. C, coffee by-product; HC, H. illucens reared on coffee by-product; HCF, frass of H. illucens reared on coffee by-product; S, vegetable substrate; HS, H. illucens reared on vegetable substrate; HSF, frass of H. illucens reared on vegetable substrate; ZHC, gut content of zebrafish fed HC; ZHS, gut content of zebrafish fed HS.

Fig 2. Relative abundances (%) of bacterial community in the analyzed samples identified by analysis of V3-V4 region of the 16S rRNA gene through MiSeq Illumina.

C, coffee by-product; HC, H. illucens reared on coffee by-product; HCF, frass of H. illucens reared on coffee by-product; S, vegetable substrate; HS, H. illucens reared on vegetable substrate; HSF, frass of H. illucens reared on vegetable substrate; ZHC, gut content of zebrafish fed HC; ZHS, gut content of zebrafish fed HS.