Gene expression and DNA methylation changes in response to hypoxia in toxicant-adapted Atlantic killifish (Fundulus heteroclitus)

Abstract

Coastal fish populations are threatened by multiple anthropogenic impacts, including the accumulation of industrial contaminants and the increasing frequency of hypoxia. Some populations of the Atlantic killifish (Fundulus heteroclitus), like those in New Bedford Harbor (NBH), Massachusetts, USA, have evolved a resistance to dioxin-like polychlorinated biphenyls (PCBs) that may influence their ability to cope with secondary stressors. To address this question, we compared hepatic gene expression and DNA methylation patterns in response to mild or severe hypoxia in killifish from NBH and Scorton Creek (SC), a reference population from a relatively pristine environment. We hypothesized that NBH fish would show altered responses to hypoxia due to trade-offs linked to toxicant resistance. Our results revealed substantial differences between populations. SC fish demonstrated dose-dependent changes in gene expression in response to hypoxia, while NBH fish exhibited a muted transcriptional response to severe hypoxia. Interestingly, NBH fish showed significant DNA methylation changes in response to hypoxia, while SC fish did not exhibit notable epigenetic alterations. These findings suggest that toxicant-adapted killifish may face trade-offs in their molecular response to environmental stress, potentially impacting their ability to survive severe hypoxia in coastal habitats. Further research is needed to elucidate the functional implications of these epigenetic modifications and their role in adaptive stress responses.

Keywords: Cost of tolerance; DNA methylation; Evolved resistance; Hypoxia; Mummichog; RNAseq.

Fig. 1.

Experimental overview. (A) Map of Southeastern Massachusetts showing the collection sites of sensitive (SC) and resistant (NBH) Atlantic killifish. (B) Relative expression of cyp1a in SC and NBH fish embryos in response to PCB126 (50 nM) exposure. *** represents statistically significant difference from the control group. ** p.value = 0.0001 and * p.value = 0.01. (C) Illustration of the experimental setup. Mild and severe hypoxia exposures were conducted by pumping oxygen containing either 5% or 10% air saturation into the chambers, respectively. Control group was maintained outside under ambient conditions.

Fig. 2.

Differentially expressed genes in response to hypoxia in NBH and SC fish. Venn diagrams showing unique and common genes in response to mild and severe hypoxia in (A) SC fish and (B) NBH fish.

Fig. 3.

Gene Ontology (Molecular Function) terms enriched among differentially expressed genes (DEGs) in mild hypoxia (A) and severe hypoxia (B) treatment groups in SC fish. Only top 10 terms enriched among up- and downregulated genes are shown. Entire list of GO biological process and molecular function terms are provided in the supplementary information. The numbers in the parenthesis represent the number of DEGs represented in each GO term. Detailed description of filtering of GO terms to remove redundancy is described in Materials and Methods. GO terms enriched among upregulated DEGs are in blue and those from downregulated genes are in red.

Fig. 4.

Gene Ontology (Molecular Function) terms enriched among differentially expressed genes (DEGs) in mild hypoxia (A) and severe hypoxia (B) treatment groups in NBH fish. Only top 10 terms enriched among up- and down-regulated genes are shown. Entire list of GO biological process and molecular function terms are provided in the supplementary information. The numbers in the parenthesis represent the number of DEGs represented in each GO term. Detailed description of filtering of GO terms to remove redundancy is described in Materials and Methods. GO terms enriched among upregulated DEGs are in blue and those from downregulated genes are in red.

Fig. 5.

Reaction norm plots showing gene expression patterns in response to two levels of hypoxia in NBH and SC fish. Mean expression (Log counts per million (cpm)) of all the differential expressed genes in response to mild and severe hypoxia were plotted for up- (A) and downregulated (B) genes in NBH and SC fish.

Fig. 6.

Comparison of hypoxia treatment groups between populations. (A) Venn diagram of differentially expressed genes (DEGs) in NBH fish in comparison to SC among control and hypoxia treatment groups. Up- and downregulated genes are in red and downregulated genes are in blue color. (B) Gene Ontology (molecular function) terms enriched among DEGs in NBH control group in comparison to SC control group and (C) Gene Ontology (molecular function) terms enriched among DEGs between NBH and SC hypoxia treatment groups. Due to small number of DEGs in mild hypoxia group, we pooled both mild and severe hypoxia groups for GO analysis. The complete list of GO biological process and molecular function terms are provided in the supplementary information. The numbers in the parenthesis represent the number of DEGs represented in each GO term. Detailed description of filtering of GO terms to remove redundancy is described in Materials and Methods. GO terms enriched among upregulated DEGs are in blue and those from downregulated genes are in red.

Fig. 7.

DNA methylation landscape in F. heteroclitus. (A) CpG DNA methylation density plots showing proportion of CpG methylation in different population and treatment groups. Inset shows the density plots of CpG sites with methylation levels below 25%. (B) Percent of methylated (>0% methylation; dark grey) and unmethylated (0% methylated; light grey) CpGs in various population and treatment groups. The average number of methylated and unmethylated CpGs are shown.

Fig. 8.

Volcano plot showing differentially methylated regions (DMRs) in response to (A) 10% (mild) and (B) 5% (severe) hypoxia exposure in NBH fish. Mean methylation difference (x-axis) between severe hypoxia and control group is plotted against q-value (y-axis). Each green and red spot represents a statistically significant hypo- and hypermethylated region, respectively.