Mussel mass mortality in the Clinch River, USA: metabolomics detects affected pathways and biomarkers of stress

Abstract

Biologists monitoring freshwater mussel (order Unionida) populations rely on behavioral, often subjective, signs to identify moribund ("sick") or stressed mussels, such as gaping valves and slow response to probing, and they lack clinical indicators to support a diagnosis. As part of a multi-year study to investigate causes of reoccurring mortality of pheasantshell (Ortmanniana pectorosa; synonym Actinonaias pectorosa) in the Clinch River, Virginia and Tennessee, USA, we analyzed the hemolymph metabolome of a subset of mussels from the 2018 sampling period. Mussels at the mortality sites were diagnosed in the field as affected (case) or unaffected (control) based on behavioral and physical signs. Hemolymph was collected in the field by non-lethal methods from the anterior adductor muscle for analysis. We used ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectroscopy to detect targeted and untargeted metabolites in hemolymph and compared metabolomic profiles by field assessment of clinical status. Targeted biomarker analysis found 13 metabolites associated with field assessments of clinical status. Of these, increased gamma-linolenic acid and N-methyl-l-alanine were most indicative of case mussels, while adenine and inosine were the best indicators of control mussels. Five pathways in the targeted analysis differed by clinical status; two of these, purine metabolism and glycerophospholipid metabolism, were also indicated in the untargeted analysis. In the untargeted nalysis, 22 metabolic pathways were associated with clinical status. Many of the impacted pathways in the case group were catabolic processes, such as degradation of amino acids and fatty acids. Hierarchical clustering analysis matched clinical status in 72% (18 of 25) of mussels, with control mussels more frequently (5 of 16) not matching clinical status. Our study demonstrated that metabolomic analysis of hemolymph is suitable for assessing mussel condition and complements field-based indicators of health.

Keywords: Unionida; hemolymph; metabolites; mussel die-off; pheasantshell.

Figure 1

Sampling locations on the Clinch River, Tennessee and Virginia, USA (adapted from Richard et al., 2020, original image published under a Creative Commons Attribution 4.0 International License).

Figure 2

Four targeted metabolites identified as potential biomarkers based on AUC. GLA (A) and N-methyl-l-alanine (B) abundances were relatively greater in case vs. control mussels. Adenine (C) and inosine (D) abundances were slightly greater in control versus case mussels.

Figure 3

Volcano plots of both targeted and untargeted metabolites in hemolymph of pheasantshell (O. pectorosa) diagnosed as case or control. All metabolites were used in the volcano plot. Points in the upper left quadrant are metabolites with lower abundance in control mussels. Points in the upper right quadrant are metabolites with higher abundance in case mussels Metabolite differences between case and control mussels were deemed significant when FC (>1.0) and the t test (P < 0.1).

Figure 4

Heat map of 25 significant metabolites and hierarchical cluster of case and control pheasantshell (O. pectorosa) mussels by metabolite concentration. The horizontal axis is the unique mussel identification T1 = September, T2 = October sample. The vertical axis is the mass and mode of detection (p = positive, n = negative).