Biomarkers of whale shark health: a metabolomic approach

Abstract

In a search for biomarkers of health in whale sharks and as exploration of metabolomics as a modern tool for understanding animal physiology, the metabolite composition of serum in six whale sharks (Rhincodon typus) from an aquarium collection was explored using (1)H nuclear magnetic resonance (NMR) spectroscopy and direct analysis in real time (DART) mass spectrometry (MS). Principal components analysis (PCA) of spectral data showed that individual animals could be resolved based on the metabolite composition of their serum and that two unhealthy individuals could be discriminated from the remaining healthy animals. The major difference between healthy and unhealthy individuals was the concentration of homarine, here reported for the first time in an elasmobranch, which was present at substantially lower concentrations in unhealthy whale sharks, suggesting that this metabolite may be a useful biomarker of health status in this species. The function(s) of homarine in sharks remain uncertain but it likely plays a significant role as an osmolyte. The presence of trimethylamine oxide (TMAO), another well-known protective osmolyte of elasmobranchs, at 0.1-0.3 mol L(-1) was also confirmed using both NMR and MS. Twenty-three additional potential biomarkers were identified based on significant differences in the frequency of their occurrence between samples from healthy and unhealthy animals, as detected by DART MS. Overall, NMR and MS provided complementary data that showed that metabolomics is a useful approach for biomarker prospecting in poorly studied species like elasmobranchs.

Figure 1. Typical ¹H NMR spectrum of whale shark serum:

(a) Full spectrum: the signal at 3.27 ppm corresponds to trimethylamine-N-oxide (TMAO). Two other major peaks are due to an internal reference (TMSP; used for referencing the chemical shift scale) and residual protons in the solvent. (b) Spectrum (a) after pre-processing (see Methodology). The grey bars depict spectral regions which were excluded from PCA.

Figure 2. PCA scores plots from analysis of (a) NMR and (b) MS metabolomics datasets of only those whale shark serum samples that were analysed by both methods.

: unhealthy individual 1 : unhealthy individual 2 : healthy individual 3 (n = 2) : healthy individual 4 (n = 3) : healthy individual 5 (n = 5).

Figure 3. PCA of ¹H NMR spectra of extracted whale shark serum (42 samples, showing PC1 scores plotted against time of sampling for the unhealthy animals.

: unhealthy individual 1 : unhealthy individual 2 : average for healthy individual 3 (n = 2) : average for healthy individual 4 (n = 3) : average for healthy individual 5 (n = 5).

Figure 4. Differences in concentration of homarine (A) and trimethylamine-oxide (TMAO) (B) in serum samples from two unhealthy (animals 1–2) and three healthy (3–5) whale sharks.

Figure 5. Loading plot for PC1 as a means to identify NMR spectroscopic features corresponding to relevant metabolites within the serum of whale sharks.