Exploration of Response Mechanisms in the Gills of Pacific Oyster (Crassostrea gigas) to Cadmium Exposure through Integrative Metabolomic and Transcriptomic Analyses

Abstract

Marine mollusks, including oysters, are highly tolerant to high levels of cadmium (Cd), but the molecular mechanisms underlying their molecular response to acute Cd exposure remain unclear. In this study, the Pacific oyster Crassostrea gigas was used as a biological model, exposed to acute Cd stress for 96 h. Transcriptomic analyses of their gills were performed, and metabolomic analyses further validated these results. In our study, a total of 111 differentially expressed metabolites (DEMs) and 2108 differentially expressed genes (DEGs) were identified under acute Cd exposure. Further analyses revealed alterations in key genes and metabolic pathways associated with heavy metal stress response. Cd exposure triggered physiological and metabolic responses in oysters, including enhanced oxidative stress and disturbances in energy metabolism, and these changes revealed the biological response of oysters to acute Cd stress. Moreover, oysters could effectively enhance the tolerance and detoxification ability to acute Cd exposure through activating ABC transporters, enhancing glutathione metabolism and sulfur relay system in gill cells, and regulating energy metabolism. This study reveals the molecular mechanism of acute Cd stress in oysters and explores the molecular mechanism of high tolerance to Cd in oysters by using combined metabolomics and transcriptome analysis.

Keywords: Crassostrea gigas; cadmium stress; energy metabolism; metabolomic; transcriptomic.

Figure 1

Biochemical alterations of the acute Cd exposure C. gigas. (a) Increased intracellular Cd level in Cd-exposed oyster individuals. The bar chart depicts mean levels and standard deviation (SD) values, with n = 3. Significance differences were determined by t-test (p < 0.05), denoted by asterisks (*). (b) The survival rate of oysters after 96 h of acute Cd exposure. (c–f) After Cd exposure, oxidative stress markers such as SOD (c), CAT (d), MDA (e), and GPx (f) were measured. Data were presented as the mean ± SD (n = 3). Distinguishing letters were assigned to indicate significant differences (p < 0.05). Star plots depict the impact of Cd exposure on gill samples.

Figure 2

Analysis of oyster transcriptome following a 96-hour exposure to Cd. (a) A volcano plot of DEGs is graphically represented with upregulated genes in red and downregulated genes in blue. (b) Hierarchical clustering based on the DEGs, where red signifies upregulation and blue signifies downregulation. (c) Enrichment of DEGs in GO terms categorized into cellular components, biological processes, and molecular functions. (d) KEGG pathway enrichment analysis of DEGs, with colors indicating p-value significance and bubble size reflecting the count of enriched genes.

Figure 3

Analysis of oyster metabolomes following a 96-hour exposure to Cd. (a) OPLS-DA score plot of the metabolomic data. (b) PLS-DA sorting test plot of the metabolomic data. (c) Hierarchical clustering was performed using 111 DEMs, with red indicating upregulation and blue representing downregulation, respectively. (d) Sample comparisons for the matchstick diagram. The top 20 metabolites of up and down are displayed in the matchstick diagram. The x-axis of the matchstick diagram represents log₂ (Fold Change) values, the y-axis represents metabolites, and the size of the points corresponds to VIP values. The metabolites that are upregulated and downregulated are represented by the red and blue points, respectively.

Figure 4

Pathway analysis of the DEMs. (a) Top 20 KEGG pathway results. p-values are represented by colors, while pathway impact is indicated by the size of the bubbles. (b) KEGG regulatory network diagram. Red circles represent individual metabolic pathways, yellow circles depict enzyme information related to specific substances, green circles indicate background substances for a metabolic pathway, purple circles represent information on molecular modules of a certain substance category, blue circles represent chemical interactions involving a specific substance, and green squares denote differentially expressed substances identified in this comparison.

Figure 5

Correlation heatmap analysis of DEGs and DEMs. The DEMs are depicted on the y-axis, while the x-axis displays the DEGs. A correlation coefficient less than 0 is indicative of a negative correlation, and a coefficient greater than 0 indicates a positive correlation. Negative correlations are symbolized by the color blue, while positive correlations are represented by the color red.

Figure 6

Pathway enrichment analysis of DEGs and DEMs. (a) Bubble plot of KEGG enrichment for DEGs and DEMs. The x-axis represents the ratio of the number of enriched differential metabolites or genes annotated to metabolites or genes in the pathway to the total number in that pathway (ratio). The y-axis represents KEGG pathways jointly enriched in the metabolome and transcriptome. The count indicates the number of enriched metabolites or genes in the pathway. The colors represent p-values, with brighter colors indicating smaller p-values and more significant pathway enrichment. (b) iPath pathway map of shared enriched pathways. Colored boxes represent enriched pathways, nodes depict various biochemical molecules, lines represent biochemical reactions, and blue lines within the pathways indicate pathways jointly enriched with DEGs and DEMs.

Figure 7

Main biological pathway responses to acute Cd exposure in oysters. The blue boxes represent four core Cd exposure response pathways. The orange boxes represent DEMs associated with the main pathways. The green boxes represent downregulated DEGs related to the main pathways. The red elliptical frames represent upregulated DEGs associated with the main pathways.