Insights on the Organ-Dependent, Molecular Sexual Dimorphism in the Zebra Mussel, Dreissena polymorpha, Revealed by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Metabolomics

Abstract

The zebra mussel, Dreissena polymorpha, is extensively used as a sentinel species for biosurveys of environmental contaminants in freshwater ecosystems and for ecotoxicological studies. However, its metabolome remains poorly understood, particularly in light of the potential molecular sexual dimorphism between its different tissues. From an ecotoxicological point of view, inter-sex and inter-organ differences in the metabolome suggest variability in responsiveness, which can influence the analysis and interpretation of data, particularly in the case where males and females would be analyzed indifferently. This study aimed to assess the extent to which the molecular fingerprints of functionally diverse tissues like the digestive glands, gonads, gills, and mantle of D. polymorpha can reveal tissue-specific molecular sexual dimorphism. We employed a non-targeted metabolomic approach using liquid chromatography high-resolution mass spectrometry and revealed a significant sexual molecular dimorphism in the gonads, and to a lesser extent in the digestive glands, of D. polymorpha. Our results highlight the critical need to consider inter-sex differences in the metabolome of D. polymorpha to avoid confounding factors, particularly when investigating environmental effects on molecular regulation in the gonads, and to a lesser extent in the digestive glands.

Keywords: ecotoxicology; gender and organ specificity; metabolome; zebra mussel.

Figure 1

Heatmap with hierarchical classification of the whole set of metabolites (n = 2634) determined by mass spectrometry in the digestive glands (red), gills (green), gonads (dark blue), and mantle (light blue) of male (n = 12) and female (n = 12) D. polymorpha.

Figure 2

Principal component analysis (A1,B1,C1,D1) and volcano plots (A2,B2,C2,D2) of the metabolite data from gonads, digestive glands, gills, and mantles of male (n = 12, blue) and female (n = 12, red) D. polymorpha. The variance of the PCA is given on the axis of PC-1 and -2. In the volcano plots, the fold changes in intensity of individual metabolites between males and females are plotted on the x-axis (fold change ≥ 2) with the significance of the differences between males and females (p < 0.1) being determined by t-test (y-axis). The statistical significance for down-regulated metabolites is indicated in blue and for up-regulated metabolites in red for females compared to males (the statistically unvarying metabolites are shown in pale grey, p < 0.1).

Figure 3

Molecular networks generated from 579 MS/MS spectra obtained from the gonads, digestive glands, gills, and mantle of D. polymorpha males (n = 12) and females (n = 12), using the GNPS and t-SNE tools. The diagram displays connected metabolites sharing a structural similarity based on the similarity of their respective fragmentation patterns. Annotated compounds are indicated in the dashed-line circles. Relative concentrations of annotated compounds are indicated in (A) each tissue (in green for gills, in red in digestive glands, in blue in gonads and in turquoise in mantles), and (B) in each sex (in blue in males and in red in females).

Figure 4

Heatmap with hierarchical classification of 198 putatively annotated metabolites measured by MS/MS in the digestive glands (red), gills (green), gonads (dark blue), and mantle (light blue) of male (n = 12) and female (n = 12) D. polymorpha.

Figure 5

List of most discriminated metabolites among the 198 annotated molecules between males and females according to their molecular family and to the different tissues. The metabolites with concentrations higher in females are shown in red, while those higher in males are shown in blue. The darker the color the greater the fold change. Statistically significant results (ANOVA, p < 0.01) are indicated with a star.

Figure 6

The top 25 most discriminated metabolites among the 198 annotated molecules between D. polymorpha males and females. The metabolites expressed principally in males are shown in green and in females in red. Abundances (mean area of the MS peak ± SD, n = 12) of the top four metabolites in the digestive glands, gills, gonads, and mantle are depicted in box plots (green for males and red for females).