## Title Differential DNA methylation in *Mytilus trossulus* mantle tissue from contrasting PAH exposure sites in Puget Sound ## Keywords *Mytilus trossulus*, DNA methylation, PAH, epigenetics\ Maybe add: WGBS, biomarker, bivalve ## Abstract Legacy PAH contamination in nearshore marine environments presents persistent monitoring challenges because of the temporal mismatch between the monitoring program and short windows of enzymatic activity measured by traditional biomarkers. In bivalves, DNA methylation occurs in a mosaic pattern preferentially associated with constitutively expressed genes, creating a molecular landscape in which differentially methylated loci may reflect cellular responses to environmental stressors rather than direct regulation of canonical biotransformation pathways. Despite the appeal of methylation-based biomarkers for contaminant monitoring, locus-level characterization of field exposed bivalve populations remains limited, particularly for *Mytilus trossulus* in Pacific Northwest waters. We applied whole genome bisulfite sequencing to mantle tissue from *M. trossulus* collected at high- and low- Σ16 PAH sites in Puget Sound (n = 3 sites per group, n = 4 individuals per site) as part of the 2021--2022 WDFW Nearshore Monitoring deployment. Differential methylation analysis identified 12 differentially methylated loci (DML) across 12 annotated genes, comprising five hyper-methylated and seven hypo-methylated loci in high-PAH mussels relative to low-PAH mussels. No DMLs mapped to canonical xenobiotic biotransformation genes. Affected loci span genes involved in membrane dynamics, vesicle trafficking, cell signaling, stress-activated apoptosis, DNA repair, and cell adhesion, suggesting a coordinated cellular response rather than direct epigenetic regulation of detoxification machinery. These candidate loci provide a foundation for targeted, cost-effective methylation biomarkers deployable at the spatial scale of regional nearshore monitoring programs. ## Introduction Legacy contaminants remain a persistent challenge in nearshore ecosystem monitoring because they are chemically stable, hydrophobic, and bioaccumulate in sediments and organism tissues (Dourdin et al., 2024; U.S. Environmental Protection Agency). Many of these compounds are also classified as xenobiotics as they are foreign to biological systems and can require physiological processing, sequestration, transformation, or elimination after exposure. In nearshore environments, legacy xenobiotics such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), pesticides, and other persistent organic pollutants can remain biologically relevant long after their initial release, creating chronic exposure conditions for sessile filter-feeding organisms. The Σ16 PAHs were designated as priority pollutants under the U.S. Clean Water Act, and they are among the first chemical groups recognized as probable human carcinogens by the U.S. Environmental Protection Agency. They are derived from the incomplete combustion of fossil fuels and biomass as well as natural processes; unlike PCBs and organochlorine pesticides such as dichlorodiphenyltrichloroethane (DDT), PAHs are still actively produced as combustion byproducts. Long-term monitoring programs such as Washington Department of Fish and Wildlife's Nearshore Monitoring Program, have generated extensive Σ16 PAH concentration data across the Puget Sound region, but comparable site-resolved data on foundational organism impact remain sparse. Traditional biomarkers provide valuable evidence of organismal condition, but field-collected mussels integrate multiple exposure histories, environmental conditions, and contaminant mixtures that can obscure the molecular pathways underlying observed physiological responses. Most enzyme-activity biomarkers are responsive only within a narrow temporal window of induction, which is difficult to align with the sampling logistics of regional programs. Epigenetic mechanisms, particularly DNA methylation, offer a developing but increasingly tractable approach for identifying molecular responses that may bridge xenobiotic exposure and subsequent physiological change. Epigenetics refers to modifications that influence gene expression without altering the underlying DNA sequence, and operates through four broad mechanisms: DNA methylation, histone modification, chromatin remodeling, and non-coding RNA activity. DNA methylation, the addition of a methyl group to the 5-carbon position of cytosine residues, is the most well characterized of these. In bivalves, methylation occurs almost exclusively at CpG sites and is established and maintained by a conserved set of DNA methyltransferases (DNMT1, DNMT3) with active demethylation mediated by TET enzymes (Männer et al., 2021; Gerdol et al., 2020). Unlike vertebrate genomes where methylation is typically distributed across gene bodies, promoters, and intergenic regions, and is broadly associated with transcriptional silencing, bivalve and other molluscan genomes display a mosaic methylation pattern in which methylated and unmethylated stretches alternate along the chromosome (Männer et al., 2021; Roberts and Gavery, 2012). In bivalves, regions of elevated methylation are preferentially associated with the gene bodies of constitutively expressed housekeeping genes, while genes subject to inducible or tissue-specific regulation tend to occupy hypo-methylated genomic regions. Gene-body methylation in invertebrates has been proposed to support transcriptional stability through mechanisms including reduced spurious transcription and modulated alternative splicing, rather than the strong promoter-driven silencing characteristic of vertebrates (Männer et al., 2021; Rivière, 2014; Roberts and Gavery, 2012). Because methylation in bivalves is enriched within gene bodies, identifying both the genomic location and the functional annotation of differentially methylated loci is essential for interpreting candidate methylation marks as biomarkers of environmental exposure. Here, we leveraged Σ16 PAH contaminant concentration data from the 2021--2022 WDFW Nearshore Monitoring deployment and applied whole-genome bisulfite sequencing to compare DNA methylation patterns in the mantle tissue of *M. trossulus* collected from high- and low-PAH sites in Puget Sound. We aimed to identify loci that were differentially methylated between exposure groups, annotate their genomic context and associated genes, and evaluate whether the affected genes pointed toward canonical xenobiotic biotransformation pathways. ## Methods **Figure 1**. Sampling Sites. All sampling sites ranked by Σ16 PAH concentrations (ng/g) from the 2021-22 WDFW Nearshore Monitoring deployment. Sites indicated in pink have the lowest concentrations amongst the group whereas sites indicated in purple, the highest. The reference site is indicated in grey. **Study design and sample selection**\ A subset of mussels from the 2021--2022 WDFW deployment was selected for DNA methylation analysis based on site-level PAH exposure profiles. Sites were ranked by the sum of 16 polycyclic aromatic hydrocarbons (Σ16 PAHs), the priority pollutant set identified under the U.S. Clean Water Act. Mussels from sites representing the upper (high PAH; n = 3 sites) and lower (low PAH; n = 3 sites) tails of the Σ16 PAH distribution were selected for sequencing (n= 24 samples). From each site four individual mussels were selected for whole genome bisulfite sequencing. Two additional high-PAH sites in Commencement Bay were excluded prior to extraction due to tissue degradation. **Table 1**. Sampling sites by treatment group and Σ16 PAH concentrations. | Sites | Σ16 PAH Group | Concentration Range (ng/g) | |------------------------|------------------------|:-----------------------| | Elliot Bay Myrtle Edwards Seattle Aquarium Pier 59 Smith Cove (Terminal 91) | High | 3304.96 - 12078.42 | | Hood Canal Holly Aiston Preserve Broad Spit | Low | 97.4 - 127.26 | **Library preparation and sequencing**\ DNA from mantle tissue was extracted using Zymo's Quick-DNA Miniprep kit, followed by an additional ethanol cleanup step to improve purity based on initial NanoDrop assessments. DNA yield was quantified using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific) on a Qubit fluorometer, and integrity was confirmed by 1% agarose gel electrophoresis. Whole genome bisulfite sequencing libraries (paired end 150bp) were prepared by Psomagen (Rockville, MD) and sequenced on an Illumina NovaSeq platform. Raw reads were assessed with FastQC v0.12.1 and aggregated reports were generated with MultiQC v1.12. Adapter sequences and low-quality bases were trimmed with fastp v1.3.2 using a 20-bp hard trim from both the 5ʹ and 3ʹ ends of each read, a parameterization consistent with bisulfite-sequencing best practices for invertebrate WGBS (Bogan et al., 2023; Trigg et al., 2022). Trimmed reads were re-assessed with FastQC and MultiQC to confirm improvements in per-base quality and per-sequence quality scores. **Alignment and methylation calling**\ A bisulfite-converted version of the *Mytilus trossulus* reference genome (NCBI assembly GCF_036588685.1) was prepared with Bismark v0.25.1. Trimmed reads were aligned to the converted reference using Bismark in conjunction with Bowtie2 v2.5.5 under established bisulfite parameters (Venkataraman et al., 2020). Aligned BAM files were deduplicated using the Bismark deduplication module and coordinate-sorted with SAMtools v1.23.1. Mapping efficiency, duplication rate, and global CpG methylation percentages were monitored at each step (supplement) to confirm consistency with reported values for marine-invertebrate WGBS (Trigg et al., 2022; Venkataraman et al., 2020). **Filtering, differential methylation, and annotation**\ CpG sites with a minimum read depth of five across all 24 samples were retained for downstream analysis using the methylKit package (Akalin et al., 2012) in R (version 4.4.2). Differential methylation in mussel mantle tissue from high- and low-PAH sites was examined at the per-CpG level using logistic regression. P-values were adjusted for multiple comparisons. Differentially methylated loci (DMLs) were defined by a q-value \< 0.01 and an absolute methylation difference of at least 55%. DMLs were intersected with the NCBI annotation of the *M. trossulus* reference using BEDTools v2.30.0 (Quinlan and Hall, 2010) to identify overlapping annotated gene features and to recover associated gene IDs. Annotated genes were assigned to functional categories based on NCBI gene descriptions, Gene Ontology, and a manual literature review. ## Results Sequencing produced 2.23 billion paired-end read pairs across 24 libraries (mean: 92.9 million read pairs per library; range: 86.2--108.1 million). Raw reads were 151 bp in length with low GC content consistent with bisulfite-converted libraries (mean: 19.0%; range: 17--22%). Mean per-base Phred quality across libraries was 38.9, and an average of 98.3% of reads per library exceeded Q30. Trimmed reads were aligned to the *M. trossulus* reference genome with a mean mapping efficiency of 47.1%. After deduplication, duplicate alignments averaged 11.2% (range: 7.6--15.5%), yielding a mean of 38.5 million deduplicated alignments per sample. Global CpG methylation averaged 12% (range: 10.3 - 13%), consistent with published values for invertebrate WGBS (Trigg et al., 2022; Venkataraman et al., 2020). After applying a minimum 5x per-sample coverage filter, X CpG sites were retained from X total sites, and X sites were submitted to differential methylation analysis in methylKit. Differential methylation analysis identified 12 DMLs meeting the significance and effect-size thresholds described in Methods. These 12 DMLs mapped to 12 unique annotated genes. Five DMLs were hyper-methylated in mussels at high-PAH locations and seven DMLs were hypo-methylated; no locus was bidirectional, and each affected gene contained a single DML. **Table 2**. DML Summary. | Functional Group | | Gene ID | q-value | Methylation Difference | Methylation Status | |------------|:-----------|:----------:|------------|------------|------------| | Membrane dynamics and vesicle trafficking | LOC134725187 | | 5.77E-14 | 56.45 | Hypermethylated | | | LOC134690221 | | 1.16E-11 | 56.32 | Hypermethylated | | | LOC134714536 | | 2.92E-13 | 59 | Hypermethylated | | | LOC134695637 | | 4.17E-14 | -57.86 | Hypomethylated | | Cell signaling and regulation | LOC134707074 | | 1.10E-13 | 58.27 | Hypermethylated | | | LOC134720671 | | 7.20E-13 | -55.24 | Hypomethylated | | Stress response, apoptosis, and nucleic acid metabolism | LOC134711256 | | 8.98E-18 | -60.69 | Hypomethylated | | | LOC134685297 | | 1.06E-12 | -56.07 | Hypomethylated | | | LOC134687110 | | 6.02E-14 | -55.83 | Hypomethylated | | | LOC134687290 | | 5.83E-13 | -56.50 | Hypomethylated | | Uncharacterized | LOC134712818 | | 1.32E-13 | 55.88 | Hypermethylated | | | LOC134724475 | | 2.52E-16 | -60.82 | Hypomethylated | The 12 DMLs and their associated genes, methylation direction, and functional annotations are summarized in Table 2. The annotated genes span three broad functional categories: membrane dynamics and vesicle trafficking, cell signaling and regulation, and stress response, apoptosis, and nucleic acid metabolism. Two DMLs mapped to uncharacterized proteins. No DMLs were annotated to canonical xenobiotic biotransformation genes such as cytochrome P450 enzymes or glutathione-associated detoxification pathways ## Discussion This study identifies 12 differentially methylated loci in *Mytilus trossulus* mantle tissue from contrasting PAH exposure environments, mapping to 12 unique annotated genes. These loci are not located in canonical xenobiotic biotransformation genes, instead, they map to genes involved in membrane dynamics and vesicle trafficking, cell signaling and regulation, and stress response, apoptosis, and nucleic acid metabolism. This pattern suggests that mantle tissue methylation may reflect cellular maintenance and signaling processes that accompany contaminant response, rather than direct regulation of the biotransformation pathway itself. In the invertebrate mosaic methylation framework, housekeeping and constitutively active genes, including the core biotransformation machinery, are typically associated with stable, maintained methylation rather than dynamic methylation changes (Männer et al., 2021). The absence of CYP450 DMLs is therefore consistent with stable methylation in those loci, and with methylation not being the primary regulatory layer for those genes in this tissue and exposure context. Hyper-methylated loci in high-PAH mussels were associated with genes involved in membrane-associated signaling and intracellular vesicle transport, processes that are both energetically costly and central to cellular homeostasis. GRAM domain-containing proteins are highly conserved lipid sensors involved in sterol movement and membrane trafficking; in bivalves, PAH exposure is associated with reduced cell membrane stability, decreased phagocytic and adhesion capacity, and lysosomal destabilization (Mansour et al., 2017; Ciacci et al., 2011). Coatomer subunit proteins mediate intracellular vesicle coat assembly; their expression has been linked to an integrated stress response operating as an energy-conserving on-demand mechanism in mussels under thermal and environmental stress (Gleason et al., 2023). Membrane protein BRI3-like, a membrane-associated protein implicated in immune defense, was similarly hyper-methylated (Panteleev et al., 2023; Ćetković et al., 2018). Hypermethylation of housekeeping-associated gene bodies is consistent with stable or maintained transcription in the invertebrate mosaic methylation framework (Männer et al., 2021) and may indicate preserved rather than suppressed function at these loci. Hypo-methylated loci in high-PAH mussels corresponded to genes associated with membrane permeability, cellular repair, and stress-activated apoptosis. Otoferlin-like proteins are Ca2+-sensor transmembrane proteins involved in vesicle-mediated transport and membrane fusion events (Pangrsic et al., 2012); hypo-methylation at this locus is consistent with increased transcriptional accessibility for membrane repair or barrier maintenance processes. Tumor protein p53-inducible protein 11-like, classified as an anti-apoptotic factor in *M. galloprovincialis* under xenobiotic exposure (Mezzelani et al., 2016), showed reduced methylation, a pattern concordant with upregulation of stress response and apoptosis gene networks reported in mussels following xenobiotic or NSAID exposure (Mezzelani et al., 2016). Receptor-type tyrosine-protein phosphatase N2-like regulates cell signaling, metabolic, and immune processes; disruption of this signaling axis has been documented in oysters under polybrominated diphenyl ethers (PBDEs) exposure, including imbalances in apoptosis and cell proliferation (Yin et al., 2022). Roundabout homolog 1-like, involved in cell adhesion and guidance signaling, was also hypomethylated; under metal and contaminant stress, reduction in cell adhesion and ATP turnover has been observed in bivalve hemocytes (Qiu et al., 2020; Ivanina et al., 2016). The cancer-related nucleoside-triphosphatase homolog, which functions in nucleic acid metabolism, was hypo-methylated, as were two additional stress-response loci: an alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-like, which mediates direct repair of alkylation damage in DNA and RNA, and a mitogen-activated protein kinase kinase kinase 5-like (ASK1/MAP3K5), the canonical stress-activated kinase that initiates apoptotic signaling under oxidative and xenobiotic stress (Ćetković et al., 2018). Together, the affected gene categories suggest a coordinated cellular economy rather than a single pathway response. Hyper-methylation of membrane trafficking and vesicle transport gene bodies may reflect maintained constitutive activity in these energetically demanding processes, while hypo-methylation of membrane-repair, apoptotic, and cell-adhesion loci may increase transcriptional flexibility for damage-response and cell-clearance functions. This pattern is consistent with a cellular strategy in which mussels at high-PAH sites conserve stable transport processes while mobilizing barrier maintenance and damaged-cell removal, functions that are complementary to, rather than duplicative of, the canonical biotransformation pathways operating primarily in the digestive gland. The BRI3-like (hyper-methylated), NTPase homolog, alkB homolog 3-like, and ASK1-like kinase (all hypo-methylated) signals further suggest that immune defense, DNA repair, and stress-activated apoptosis are engaged under exposure, consistent with roles as dual-function defense and damage-response proteins (Panteleev et al., 2023; Ćetković et al., 2018). Compared with prior molluscan methylation studies examining xenobiotic exposure, this work extends the field-based, locus-level perspective to *M. trossulus* under environmentally realistic PAH conditions. Global DNA hypo-methylation has been reported in response to acute benzo$$a$$pyrene exposure in blood clam *Tegillarca granosa* (Guo et al., 2021), microplastic exposure in *Mytilus galloprovincialis* (Ortiz-Moriano et al., 2024), and mercury contamination at field-deployed *Crassostrea virginica* sites (Rowe et al., 2020). In contrast, locus-specific promoter hyper-methylation of the endocrine gene hsd17b has been documented in scallop *Chlamys farreri* under benzo$$a$$pyrene stress, where it co-occurred with reduced hsd17b expression (Tang et al., 2020). Genome-wide WGBS studies of Pacific oyster *Crassostrea gigas* under herbicide exposure identified DMRs enriched in coding sequences with generally weak coupling between methylation change and steady-state transcription (Rondon et al., 2017). The present study is consistent with the broader pattern that bivalve methylation responses to xenobiotics are not tightly constrained to canonical detoxification genes, and that the magnitude and direction of methylation change are context-dependent, varying by contaminant type, tissue, life stage, and exposure duration Biomarkers identified in this study provide a set of candidate loci that can be tested as molecular indicators in future contaminant monitoring work. DNA methylation has been repeatedly proposed as a more stable indicator of environmental exposure than short-window enzyme biomarkers (Lu et al., 2025; Suarez-Ulloa et al., 2015), and the candidate loci reported here are amenable to targeted bisulfite sequencing or methylation-sensitive PCR assays that could be deployed at the spatial scale of the Nearshore Monitoring Program without requiring sequencing of every individual. ## References Akalin, A., Kormaksson, M., Li, S., Garrett-Bakelman, F. E., Figueroa, M. E., Melnick, A., & Mason, C. E. (2012). methylKit: A comprehensive R package for the analysis of genome-wide DNA methylation profiles. *Genome Biology*, *13*(10), R87. \ Bogan, S. N., Johns, J., Griffiths, J. S., Davenport, D., Smith, S. J., Schaal, S. M., Downey‐Wall, A., Lou, R. N., Lotterhos, K., Guidry, M. E., Rivera, H. E., McGirr, J. A., Puritz, J. B., Roberts, S. B., & Silliman, K. (2023). A dynamic web resource for robust and reproducible genomics in nonmodel species: Marineomics.io. *Methods in Ecology and Evolution*, *14*(11), 2709--2716. \ Ćetković, H., Halasz, M., & Herak Bosnar, M. (2018). Sponges: A Reservoir of Genes Implicated in Human Cancer. *Marine Drugs*, *16*(1), 20. \ Ciacci, C., Barmo, C., Fabbri, R., Canonico, B., Gallo, G., & Canesi, L. (2011). Immunomodulation in Mytilus galloprovincialis by non-toxic doses of hexavalent Chromium. *Fish & Shellfish Immunology*, *31*(6), 1026--1033. \ Dourdin, T. S., Berthelin, C., Guyomard, K., Morin, A., Morandi, N., Elie, N., Villain-Naud, N., Rivière, G., & Sussarellu, R. (2024). The Pacific oyster reproduction is affected by early-life exposure to environmental pesticide mixture: A multigenerational study. *Science of The Total Environment*, *937*, 173569. \ Gerdol, M., Moreira, R., Cruz, F., Gómez-Garrido, J., Vlasova, A., Rosani, U., Venier, P., Naranjo-Ortiz, M. A., Murgarella, M., Greco, S., Balseiro, P., Corvelo, A., Frias, L., Gut, M., Gabaldón, T., Pallavicini, A., Canchaya, C., Novoa, B., Alioto, T. S., ... Figueras, A. (2020). Massive gene presence-absence variation shapes an open pan-genome in the Mediterranean mussel. *Genome Biology*, *21*(1), 275. \ Gleason, L. U., Fekete, F. J., Tanner, R. L., & Dowd, W. W. (2023). Multi-omics reveals largely distinct transcript- and protein-level responses to the environment in an intertidal mussel. *Journal of Experimental Biology*, *226*(22), jeb245962. \ Guo, B., Feng, D., Xu, Z., Qi, P., & Yan, X. (2021). Acute benzo$$a$$pyrene exposure induced oxidative stress, neurotoxicity and epigenetic change in blood clam Tegillarca granosa. *Scientific Reports*, *11*(1), 18744. \ Ivanina, A. V., Hawkins, C., & Sokolova, I. M. (2014). Immunomodulation by the interactive effects of cadmium and hypercapnia in marine bivalves Crassostrea virginica and Mercenaria mercenaria. *Fish & Shellfish Immunology*, *37*(2), 299--312. \ Long, E. R., Macdonald, D. D., Smith, S. L., & Calder, F. D. (1995). Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments. *Environmental Management*, *19*(1), 81--97. \ Lu, G., Wang, J., Luo, M., & Wang, C. (2025). Epigenetic mechanisms and applications in aquaculture and fisheries, with perspectives on technology and analysis. *Aquaculture and Fisheries*, S2468550X2500139X. \ Männer, L., Schell, T., Provataris, P., Haase, M., & Greve, C. (2021). Inference of DNA methylation patterns in molluscs. *Philosophical Transactions of the Royal Society B*, *376*(1825), 20200166. \ Mansour, C., Guardiola, F. A., Esteban, M. Á., & Mosbahi, D. S. (2017). Combination of polycyclic aromatic hydrocarbons and temperature exposure: In vitro effects on immune response of European clam (Ruditapes decussatus). *Fish & Shellfish Immunology*, *67*, 110--118. \ Meador, J. P., Collier, T. K., & Stein, J. A. (2001). *Use of tissue and sediment based threshold concentrations of polychlorinated biphenyls (PCBs) to protect juvenile salmonids listed under the Endangered Species Act*. NOAA Technical Memorandum NMFS. \ Mezzelani, M., Gorbi, S., Fattorini, D., d'Errico, G., Benedetti, M., Milan, M., Bargelloni, L., & Regoli, F. (2016). Transcriptional and cellular effects of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in experimentally exposed mussels, Mytilus galloprovincialis. *Aquatic Toxicology*, *180*, 306--319. \ Ortiz-Moriano, M. P., Masiá, P., Acle, S., Ardura, A., Garcia-Vazquez, E., & Machado-Schiaffino, G. (2024). Changes in global methylation patterns of Mytilus galloprovincialis exposed to microplastics. *Aquatic Toxicology*, *276*, 107115. \ Pangršič, T., Reisinger, E., & Moser, T. (2012). Otoferlin: A multi-C2 domain protein essential for hearing. *Trends in Neurosciences*, *35*(11), 671--680. \ Panteleev, P. V., Safronova, V. N., Duan, S., Komlev, A. S., Bolosov, I. A., Kruglikov, R. N., Kombarova, T. I., Korobova, O. V., Pereskokova, E. S., Borzilov, A. I., Dyachenko, I. A., Shamova, O. V., Huang, Y., Shi, Q., & Ovchinnikova, T. V. (2023). Novel BRICHOS-Related Antimicrobial Peptides from the Marine Worm Heteromastus filiformis: Transcriptome Mining, Synthesis, Biological Activities, and Therapeutic Potential. *Marine Drugs*, *21*(12), 639. \ Qiu, L., Chen, H., Zhou, Z., Zhang, H., Liu, R., Yi, Q., Yang, C., Gao, L., & Wang, L. (2020). Transcriptomic profile of oyster Crassostrea gigas hemocyte after short-term cadmium exposure and bacteria stimulation. *Fish & Shellfish Immunology*, *98*, 138--146. \ Quinlan, A. R., & Hall, I. M. (2010). BEDTools: A flexible suite of utilities for comparing genomic features. *Bioinformatics*, *26*(6), 841--842. \ Rivière, G. (2014). Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates. *Frontiers in Physiology*, *5*. \ Roberts, S. B., & Gavery, M. R. (2012). Is There a Relationship between DNA Methylation and Phenotypic Plasticity in Invertebrates? *Frontiers in Physiology*, *2*. \ Rondon, R., Grunau, C., Fallet, M., Charlemagne, N., Sussarellu, R., Chaparro, C., Montagnani, C., Mitta, G., Bachère, E., Akcha, F., & Cosseau, C. (2017). Effects of a parental exposure to diuron on Pacific oyster spat methylome. *Environmental Epigenetics*, *3*(1). \ Rowe, G. T., Fernando, H., Elferink, C., Ansari, G. A. S., Sullivan, J., Heathman, T., Quigg, A., Petronella Croisant, S., Wade, T. L., & Santschi, P. H. (2020). Polycyclic aromatic hydrocarbons (PAHs) cycling and fates in Galveston Bay, Texas, USA. *PLOS ONE*, *15*(12), e0243734. \ Suarez Ulloa, M. V. (2017). *Transcriptomic and Epigenetic Responses to Environmental Stress in Marine Bivalves with a Focus on Harmful Algal Blooms* $$Doctor of Philosophy    Biology, Florida International University$$. \ Tang, Y., Rong, J., Guan, X., Zha, S., Shi, W., Han, Y., Du, X., Wu, F., Huang, W., & Liu, G. (2020). Immunotoxicity of microplastics and two persistent organic pollutants alone or in combination to a bivalve species. *Environmental Pollution*, *258*, 113845. \ Trigg, S. A., Venkataraman, Y. R., Gavery, M. R., Roberts, S. B., Bhattacharya, D., Downey‐Wall, A., Eirin‐Lopez, J. M., Johnson, K. M., Lotterhos, K. E., Puritz, J. B., & Putnam, H. M. (2022). Invertebrate methylomes provide insight into mechanisms of environmental tolerance and reveal methodological biases. *Molecular Ecology Resources*, *22*(4), 1247--1261. \ Venkataraman, Y. R., Downey-Wall, A. M., Ries, J., Westfield, I., White, S. J., Roberts, S. B., & Lotterhos, K. E. (2020). General DNA Methylation Patterns and Environmentally-Induced Differential Methylation in the Eastern Oyster (Crassostrea virginica). *Frontiers in Marine Science*, *7*, 225. \ Yin, C., Sun, Z., Ji, C., Li, F., & Wu, H. (2022). Toxicological effects of tris(1,3-dichloro-2-propyl) phosphate in oyster Crassostrea gigas using proteomic and phosphoproteomic analyses. *Journal of Hazardous Materials*, *434*, 128824.