Exploration of the gut-brain axis in adult zebrafish exposed to Perfluorononanoic Acid (PFNA): correlating the intestinal microbiome with neural gene expression.

and reproductive success in zebrafish. Evaluating how neurotoxicants affect bidirectional communication between the brain and gut microbiome has also become an intriguing new area of research in the field of toxicology. Here we attempt to infer the biological processes and molecular pathways that may underpin the neurotoxic endpoints and downstream effects of PFNA exposure in adult zebrafish using analysis of differential transcript abundance across a dose gradient. Additionally, we investigate modulation of the gut-brain axis in adult zebrafish exposed to Perfluorononanoic acid (PFNA) through correlation of intestinal microbiome abundance and neural gene counts. Total RNA from brain and DNA from intestinal tissues was collected from 48 adult zebrafish across an exposure gradient of PFNA. RNAseq and 16S taxonomic profiling data were processed using nf-core rnaseq and ampliseq pipelines built with Nextflow, respectively. Relevant features from metataxonomic and transcriptomic datasets were filtered by applying differential abundance analysis utilizing multivariable associations with linear models (MaAsLin2). Hierarchical All-against-All association (HALLA) was used to find any significant relationships between gene expression and relative abundance of gut microbes. We report evidence of significantly differentiated genes across all concentrations, with consistent downregulation of WNT1 and CHRNB4. However, no apparent dose-related patterns in gene ontology were observed save for changes in transcripts related to sensory processing and transcriptional activity in most concentrations. Integrative Pathway Analysis (IPA) showed significantly decreased abundance of transcripts related to downstream inhibition for various pathways across multiple concentrations, including Autism Signaling Pathway and Molecular Mechanisms of Cancer (0.3nM PFNA), Pulmonary Fibrosis (3nM PFNA), Potassium Channels (30nM), and GNRH, Hepatic Fibrosis and RAR Activation (300nM PFNA). All differentially abundant phyla from Maaslin2 showed a positive correlation across the PFNA dosing gradient. Analysis with HALLA showed a positive correlation between Desulfobacterota and multiple genes associated with properties in the nervous system, including sphingolipid biosynthesis regulator serinc1 and ornithine decarboxylase inhibitor oaz2b. Additionally, Myxococcota, Chloroflexi, Desulfobacterota, MBNT15, Crenarchaeota, and Patescibacteria where all negatively correlated with gxp3, which plays a protective role against oxidative stress. We conclude that PFNA exposure may cause potential adverse effects on synaptic plasticity, sensory processes, regeneration capability and hormonal regulation. Although further study is we required, the data suggest that gut-brain axis modulation plays an important role in the toxicity profile of PFNA and subsequent adverse outcomes in adult zebrafish.