Nutritional impact of a novel iron-containing protein meal on gastrointestinal tract functioning in larval zebrafish Danio rerio: characterisation of microbial communities and mRNAseq gene expression analysis

Zebrafish has been explored as nutritional fish model with the purpose to assess the impact of two dietary inclusion levels of a novel iron-containing protein meal (IPM) of animal origin on the gastrointestinal tract (GIT) of the developing zebrafish. The development of the microbial community has been assessed over the first 21 days post egg fertilisation (dpf) through 16S rRNA gene-based microbial composition profiling by quantitative PCR (qPCR) and pyrosequencing using a Genome Sequencer FLX. The molecular regulation of physiological processes by differentially expressed genes in the GIT is demonstrated at 21 dpf by whole transcriptome sequencing (mRNAseq) using an Illumina HiSeq2000 focusing on genes that are functionally involved in iron uptake and homeostasis. Differential diet dependent phylogenetic diversity occurred. Larvae fed at high inclusion level of IPM differed from those fed at low level in early presence of Bacteroidetes, then an increase of Firmicutes and other phyla at the expense of the Actinobacteria. Finally with Firmicutes and Actinobacteria still present, Proteobacteria dominated. The abundance of Firmicutes in the larvae fed at high inclusion level of IPM at 14 and 21 dpf was much higher than those fed at low inclusion level which probably relates to their iron oxidizing capacity that may coincide with higher pH in the GIT. mRNAseq revealed that 328 genes were differentially expressed: expression of 214 genes was up-regulated and 114 genes down-regulated in larvae fed at high vs. low inclusion levels of IPM. Dominant gene groups representing ribosome components and activity and transport were up-regulated in the GIT of these larvae. 27 genes were identified as involved in iron homeostasis but were non-differentially expressed at a fold change 0.27 – 1.54. Functional classification of genes revealed that the GIT of larvae fed at higher IPM level are more active in transmembrane ion transport and protein synthesis. The marked differences in microbial constitution over time and gene expression reveal major impact of inclusion level of the novel IPM. These differences suggest that larvae fed at high inclusion level spend more energy on nutrient uptake and their innate immune response may be activated. The specific impact of iron level on the host-microbial symbiosis is indicated by the abundance of Firmicutes but not reflected by changes in expression of specific marker genes involved in iron homeostasis. Zebrafish can be applied as vertebrate and fish model to determine the nutritional impact on the microbial constitution and the GIT response