Exposure to plastic debris alters expression of biomineralization, immune, and stress-related genes in the eastern oyster (Crassostrea virginica)

Abstract

The degradation of marine plastic debris poses a threat to organisms by fragmenting into micro- and nano-scale pieces and releasing a complex chemical leachate into the water. Numerous studies have investigated harms from plastic pollution such as microplastic ingestion and exposure to single chemicals. However, few studies have examined the holistic threat of plastic exposure and the synergistic impacts of chemical mixtures. The objective of this study was to measure changes in gene expression of gill and gonadal tissue of the eastern oyster (Crassostrea virginica) in response to plastic debris exposure during their first year, using RNA-seq to explore multiple types of physiological responses. Shell and polyethylene terephthalate plastic were used as substrate for the metamorphosis of larval oysters in a settlement tank. Substrate pieces were then transferred to metal cages and outplanted in pairs - shell cage and plastic cage - onto restoration reefs in the St. Mary's River, Maryland, USA. After 10 months of growth, the oysters were collected, gill and gonadal tissue removed, and sex identified. The tissues of six oysters from each sex and substrate type were then analyzed in RNA-seq. Both gill and gonadal tissue samples had altered expression of immune and stress-response genes in response to plastic exposure. Genes upregulated in response to plastic were enriched for gene ontology functions of proteolysis and fibrinolysis. Downregulated genes were involved in shell biomineralization and growth. One male oyster exposed to plastic had "feminized" gene expression patterns despite developing mature sperm, suggesting plastic leachate can alter gene expression and shift protandric individuals to develop as females. Plastic pollution may therefore reduce shell growth, initiate immune and stress responses, alter sex differentiation, and impact reproductive output of eastern oysters through changes in transcription.

Fig 1. Venn diagrams of differentially expressed genes by model factor in each tissue.

Venn diagrams indicating the number of reference genes differentially expressed in response to model factors sex, substrate, and sex-by-substrate for (A) gill tissue, (B) gonad tissue, and (C) gonad tissue after the removal of the P8 male on plastic sample.

Fig 2. Multidimensional scaling plots of samples for each tissue.

Multidimensional scaling plots indicating the clustering of female and male samples on shell and plastic substrate based on patterns of read counts in all expressed genes for (A) gill tissue, (B) gonad tissue, and (C) gonad tissue after the removal of the P8 male on plastic sample.

Fig 3. Volcano plots of genes expressed in gill tissue by model factor.

Volcano plot log₂ fold change in expression and false discovery rate of reference genes (n = 41,899) expressed in gill tissue in response to model factors (A) sex, (B) substrate, and (C) sex-by-substrate with differentially expressed genes indicated in red and a subset of genes labeled with gene annotation.

Fig 4. Volcano plots of genes expressed in gonad tissue by model factor.

Volcano plot log₂ fold change in expression and false discovery rate of reference genes expressed in gonad tissue (n = 35,736) in response to model factors (A) sex, (B) substrate, and (C) sex-by-substrate and in gonad tissue after the removal of male on plastic sample P8 (n = 37,676) in response to model factors: (D) sex, (E) substrate, and (F) sex-by-substrate with differentially expressed genes indicated in red and a subset of genes labeled with gene annotation.

Fig 5. Enriched gene ontology terms of differentially expressed genes in gonadal tissue by sex.

Enriched gene ontology terms for genes differentially expressed in gonadal tissues indicating the number of genes and the false discovery rate of each term for (A) males, (B) males after the removal of male on plastic P8, and (C) females after the removal of male on plastic P8.

Fig 6. Enriched gene ontology terms of differentially expressed genes in gonadal tissue by plastic.

Enriched gene ontology terms for genes differentially expressed in gonadal tissues indicating the number of genes and the false discovery rate of each term for (A) oysters on plastic and (B) oysters on plastic after the removal of male on plastic P8.

Fig 7. Model results for twenty-four genes differentially expressed in both gill and gonadal tissue.

Model results for each of three factors (Sex, Substrate, and Sex-by-substrate) for twenty-four genes differentially expressed in both gill and gonadal tissue. For genes with differential expression for a model factor, the log fold change is provided. The sex factor indicates male expression relative to female ( + male/- female). The substrate factor indicates shell expression relative to plastic ( + shell/- plastic). The sex-by-substrate (SExSU) factor indicates male on shell expression relative to all other groups. Heat map colors are red for positive log-fold changes and blue for negative log-fold changes. Non-significant factors for a gene are indicated as 0 log-fold change.