Differential DNA methylation in Pacific oyster reproductive tissue in response to ocean acidification

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Venkataraman, Y. R., White, S. J., & Roberts, S. B. Differential DNA methylation in Pacific oyster reproductive tissue in response to ocean acidification. BMC Genomics, 23(1), (2022): 556, https://doi.org/10.1186/s12864-022-08781-5.Background There is a need to investigate mechanisms of phenotypic plasticity in marine invertebrates as negative effects of climate change, like ocean acidification, are experienced by coastal ecosystems. Environmentally-induced changes to the methylome may regulate gene expression, but methylome responses can be species- and tissue-specific. Tissue-specificity has implications for gonad tissue, as gonad-specific methylation patterns may be inherited by offspring. We used the Pacific oyster (Crassostrea gigas) — a model for understanding pH impacts on bivalve molecular physiology due to its genomic resources and importance in global aquaculture— to assess how low pH could impact the gonad methylome. Oysters were exposed to either low pH (7.31 ± 0.02) or ambient pH (7.82 ± 0.02) conditions for 7 weeks. Whole genome bisulfite sequencing was used to identify methylated regions in female oyster gonad samples. C- > T single nucleotide polymorphisms were identified and removed to ensure accurate methylation characterization. Results Analysis of gonad methylomes revealed a total of 1284 differentially methylated loci (DML) found primarily in genes, with several genes containing multiple DML. Gene ontologies for genes containing DML were involved in development and stress response, suggesting methylation may promote gonad growth homeostasis in low pH conditions. Additionally, several of these genes were associated with cytoskeletal structure regulation, metabolism, and protein ubiquitination — commonly-observed responses to ocean acidification. Comparison of these DML with other Crassostrea spp. exposed to ocean acidification demonstrates that similar pathways, but not identical genes, are impacted by methylation. Conclusions Our work suggests DNA methylation may have a regulatory role in gonad and larval development, which would shape adult and offspring responses to low pH stress. Combined with existing molluscan methylome research, our work further supports the need for tissue- and species-specific studies to understand the potential regulatory role of DNA methylation.This work was funded by National Science Foundation award 1634167 to SBR. The Hall Conservation Genetics Research Fund (YRV) supported sequencing for this project

Characterizing host-pathogen interactions between Zostera marina and Labyrinthula zosterae

Introduction Seagrass meadows serve as an integral component of coastal ecosystems but are declining rapidly due to numerous anthropogenic stressors including climate change. Eelgrass wasting disease, caused by opportunistic Labyrinthula spp., is an increasing concern with rising seawater temperature. To better understand the host-pathogen interaction, we paired whole organism physiological assays with dual transcriptomic analysis of the infected host and parasite. Methods Eelgrass (Zostera marina) shoots were placed in one of two temperature treatments, 11° C or 18° C, acclimated for 10 days, and exposed to a waterborne inoculation containing infectious Labyrinthula zosterae (Lz) or sterile seawater. At two- and five-days post-exposure, pathogen load, visible disease signs, whole leaf phenolic content, and both host- and pathogen- transcriptomes were characterized. Results Two days after exposure, more than 90% of plants had visible lesions and Lz DNA was detectable in 100% percent of sampled plants in the Lz exposed treatment. Concentrations of total phenolic compounds were lower after 5 days of combined exposure to warmer temperatures and Lz, but were unaffected in other treatments. Concentrations of condensed tannins were not affected by Lz or temperature, and did not change over time. Analysis of the eelgrass transcriptome revealed 540 differentially expressed genes in response to Lz exposure, but not temperature. Lz-exposed plants had gene expression patterns consistent with increased defense responses through altered regulation of phytohormone biosynthesis, stress response, and immune function pathways. Analysis of the pathogen transcriptome revealed up-regulation of genes potentially involved in breakdown of host defense, chemotaxis, phagocytosis, and metabolism. Discussion The lack of a significant temperature signal was unexpected but suggests a more pronounced physiological response to Lz infection as compared to temperature. Pre-acclimation of eelgrass plants to the temperature treatments may have contributed to the limited physiological responses to temperature. Collectively, these data characterize a widespread physiological response to pathogen attack and demonstrate the value of paired transcriptomics to understand infections in a host-pathogen system

Adult low pH exposure influences larval abundance in Pacific oysters (Crassostrea gigas)

As negative effects of ocean acidification are experienced by coastal ecosystems, there is a growing trend to investigate the effect ocean acidification has on multiple generations. Parental exposure to ocean acidification has been shown to induce larval carryover effects, but whether or not an acute exposure to a stressor as an adult can influence the larval generation long after the stress has been removed has yet to be tested. To assess how a temporary exposure to experimental ocean acidification affects the ecologically and commercially relevant Pacific oyster (Crassostrea gigas), adult oysters were exposed to either low pH (7.31 ± 0.02) or ambient pH (7.82 ± 0.02) conditions for seven weeks. Oysters were then held for eight weeks in ambient conditions, and subsequently reproductively conditioned for four weeks at ambient pH. After conditioning, oysters were strip-spawned to create four families based on maternal and paternal ocean acidification exposure. The number of D-hinge larvae were counted eighteen hours post fertilization. A sex-specific broodstock response was observed, where female exposure to low pH conditions resulted in fewer D-hinge larvae. This study demonstrates that the effects of ocean acidification can last beyond the time from when the environmental perturbation is experienced. Broadening the understanding of environmental memory will be valuable when considering an organism’s ability to persist in the face of environmental change

Seawater carbonate chemistry and larval response to parental low pH exposure in the pacific oyster Crassostrea gigas

As negative effects of ocean acidification are experienced by coastal ecosystems, there is a growing trend to investigate the effect ocean acidification has on multiple generations. Parental exposure to ocean acidification has been shown to induce larval carryover effects, but whether acute exposure to a stressor as an adult can influence the larval generation long after the stress has been removed has yet to be tested. To assess how a temporary exposure to experimental ocean acidification affects the ecologically and commercially relevant Pacific oyster Crassostrea gigas, adult oysters were exposed to either low pH (7.31 +- 0.02) or ambient pH (7.82 +- 0.02) conditions for 7 wk. Oysters were then held for 8 wk in ambient conditions, and subsequently reproductively conditioned for 4 wk at ambient pH. After conditioning, the oysters were strip-spawned to create four families based on maternal and paternal ocean acidification exposure. The number of D-hinge larvae was counted 18 h postfertilization. A sex-specific brood stock response was observed, where female exposure to low pH conditions resulted in fewer D-hinge larvae. This study demonstrates that the effects of ocean acidification can last beyond the time from when the environmental perturbation is experienced. Broadening the understanding of environmental memory will be valuable when considering organismal ability to persist in the face of environmental change

Invertebrate methylomes provide insight into mechanisms of environmental tolerance and reveal methodological biases

There is a growing focus on the role of DNA methylation in the ability of marine invertebrates to rapidly respond to changing environmental factors and anthropogenic impacts. However, genome-wide DNA methylation studies in nonmodel organisms are currently hampered by a limited understanding of methodological biases. Here, we compare three methods for quantifying DNA methylation at single base-pair resolution—whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and methyl-CpG binding domain bisulfite sequencing (MBDBS)—using multiple individuals from two reef-building coral species with contrasting environmental sensitivity. All methods reveal substantially greater methylation in Montipora capitata (11.4%) than the more sensitive Pocillopora acuta (2.9%). The majority of CpG methylation in both species occurs in gene bodies and flanking regions. In both species, MBDBS has the greatest capacity for detecting CpGs in coding regions at our sequencing depth, but MBDBS may be influenced by intrasample methylation heterogeneity. RRBS yields robust information for specific loci albeit without enrichment of any particular genome feature and with significantly reduced genome coverage. Relative genome size strongly influences the number and location of CpGs detected by each method when sequencing depth is limited, illuminating nuances in cross-species comparisons. As genome-wide methylation differences, supported by data across bisulfite sequencing methods, may contribute to environmental sensitivity phenotypes in critical marine invertebrate taxa, these data provide a genomic resource for investigating the functional role of DNA methylation in environmental tolerance