Application of Passive Sampling to Characterise the Fish Exometabolome

Abstract

The endogenous metabolites excreted by organisms into their surrounding environment, termed the exometabolome, are important for many processes including chemical communication. In fish biology, such metabolites are also known to be informative markers of physiological status. While metabolomics is increasingly used to investigate the endogenous biochemistry of organisms, no non-targeted studies of the metabolic complexity of fish exometabolomes have been reported to date. In environmental chemistry, Chemcatcher^® (Portsmouth, UK) passive samplers have been developed to sample for micro-pollutants in water. Given the importance of the fish exometabolome, we sought to evaluate the capability of Chemcatcher^® samplers to capture a broad spectrum of endogenous metabolites excreted by fish and to measure these using non-targeted direct infusion mass spectrometry metabolomics. The capabilities of C18 and styrene divinylbenzene reversed-phase sulfonated (SDB-RPS) Empore™ disks for capturing non-polar and polar metabolites, respectively, were compared. Furthermore, we investigated real, complex metabolite mixtures excreted from two model fish species, rainbow trout (Oncorhynchus mykiss) and three-spined stickleback (Gasterosteus aculeatus). In total, 344 biological samples and 28 QC samples were analysed, revealing 646 and 215 m/z peaks from trout and stickleback, respectively. The measured exometabolomes were principally affected by the type of Empore™ (Hemel Hempstead, UK) disk and also by the sampling time. Many peaks were putatively annotated, including several bile acids (e.g., chenodeoxycholate, taurocholate, glycocholate, glycolithocholate, glycochenodeoxycholate, glycodeoxycholate). Collectively these observations show the ability of Chemcatcher^® passive samplers to capture endogenous metabolites excreted from fish.

Keywords: DIMS; FT-ICR; bile acid; environmental metabolomics; exogenous; fish; metabolic footprinting.

Figure 1

Principal components analysis (PCA) scores plots from analysis of the fish-derived metabolites that were extracted using Chemcatcher^® passive samplers and measured via direct infusion Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry metabolomics. (a) Analysis of both the quality control (QC) and biological samples, highlighting the high technical reproducibility of the metabolomics measurements of the 28 QC samples. Key: QC (red) and biological samples (black); (b) Biological samples only, labelled so as to highlight any differences in the exometabolomes that were excreted by rainbow trout and three-spined stickleback that were captured onto C18 (non-polar) and styrene divinylbenzene reversed-phase sulfonated (SDB-RPS) (polar) Empore™ disks. Key: C18 disk (blue), SDB-RPS disk (red), stickleback (square) and trout (triangle); (c) Biological samples only, labelled to highlight any differences in the exometabolomes excreted across the 4-week investigation. Multivariate analysis of variance (MANOVA) of PCA scores for three-factor model, p < 1.0 × 10⁻³ for type of disk, fish species and sampling time; two-way interaction of disk x time was significant (p < 1.0 × 10⁻³), interaction of disk x species was significant (p = 3.7 × 10⁻⁵), and interaction of species x time was not significant (p = 6.4 × 10⁻¹). Key: metabolites captured in week 1 (red), week 2 (orange), week 3 (green) and week 4 (blue), for stickleback (square) and trout (triangle).

Figure 2

Venn diagrams highlighting the complementarity of C18 and SDB-RPS Empore™ disks in a Chemcatcher^® passive sampler, by showing the numbers of putatively annotated metabolites captured from the water by each type of disk for rainbow trout and three-spined stickleback. Relatively few metabolites are captured by both receiving phases.

Figure 3

Venn diagrams highlighting the larger number of exometabolic peaks arising from rainbow trout versus three-spined stickleback, by showing the numbers of putatively annotated metabolites excreted from each fish species using C18 and SDB-RPS Empore™ disks in a Chemcatcher^® passive sampler. The majority of peaks measured from stickleback are also observed in the trout datasets.

Figure 4

Multiple correspondence analysis of the metabolites captured on the SDB-RPS receiving phase in the trout tanks and measured via mass spectrometry metabolomics. Metabolite data were represented in an indicator matrix (presence/absence). MANOVA of scores, p < 1.0 × 10⁻³, and all pairwise comparisons between four time points, p ≤ 1.6 × 10⁻³). Key: metabolites captured in week 1 (red), week 2 (orange), week 3 (green) and week 4 (blue).