DNA methylation changes in response to ocean acidification at the time of larval metamorphosis in the edible oyster, Crassostrea hongkongensis
Introduction
Molecular mechanisms such as natural selection and genetic drift have long been known to explain the evolutionary trajectory of organisms (Kronholm et al., 2017; Darwin and Wallace, 1858). In light of rapid adaptation, epigenetic processes have gained attention for their capability to decipher stress plasticity resulting from the production of epigenetic variants via environmental induction (Vogt, 2017; Waddington, 1953; Lamarck, 1802). Studies have shown that genetic variation only explains partly the adaptive strategy (Gavery et al., 2018; Kronholm et al., 2017). While adaptive evolution is accomplished by genetic changes through mutation and selection, the process is expected to take thousands of years for many species. On the other hand, epigenetic mechanisms can provide an alternative source of phenotypic change for rapid adjustments to environmental fluctuations (Liu et al., 2018). DNA methylation, being the most-widely studied epigenetic modification has been proposed to be an important mechanism of gene regulation in response to external environmental stresses (Thiebaut et al., 2019). Stress exposure sets a series of functionally relevant modifications in the genome without changing the underlying original sequence of DNA (Eirin-Lopez and Putnam, 2019), affecting gene activity through adding or removing a methyl group to the DNA cytosine (Suzuki and Bird, 2008; Song et al., 2005). DNA methylation is therefore a pivotal mechanism to understand stress plasticity in the face of changing climate that includes the rising carbon dioxide (CO2) level in marine habitats (Fox et al., 2019).
The unprecedented increase rate of CO2 level in the ocean is one of the prominent environmental stressors for many calcifying organisms in marine ecosystem. The phenomenon of nearly one-third of CO2 emission of anthropogenic sources soaked into the ocean resulting in a shift in seawater carbonate chemistry towards a reduced pH environment is termed as ocean acidification (OA) (Caldeira and Wickett, 2003). As compared to open oceans, the estuarine and coastal regions experienced more land and anthropogenic disturbance and thus are highly sensitive to OA (Bauer et al., 2013). Response of calcifying marine invertebrates to OA, including edible oyster species, appears to be highly species-specific. For instance, early-stage larvae of Eastern oysters (Crassostrea virginica) showed no significant differences in shell growth between control (pH7.9/pCO2 = 803 μatm) and low pH treatment (pH7.5) (Boulais et al., 2017; Gobler and Talmage, 2014). In contrast, Suminoe oysters (Crassostrea ariakensis) and Sydney Rock oysters (Saccostrea glomerata) showed hindered larval shell growth with a decrease in 0.3 pH difference from control condition (Miller et al., 2009; Watson et al., 2009). Larvae of Pacific oyster (Crassostrea gigas) from the Yellow Sea had reduced metabolic and clearance rates but displayed higher shell growth rate after settlement under pH7.4 compared to pH8.1 (Ginger et al., 2013). Larvae of the Hong Kong oyster (Crassostrea hongkongensis), which is an estuarine species, showed no significant delay in calcification or shell growth rates in response to low pH 7.4 when compared to control pH 8.1 (Dineshram et al., 2013). Although few recent studies have shown the ability of many terrestrial organisms such as mammals (Rolland et al., 2018) and plants (Zlotorynski, 2019; Vannier et al., 2015) to rapidly adapt to external stresses through epigenetic mechanisms, existence of similar mechanisms is scarce in marine invertebrates, especially Mollusca. Therefore, exploration of DNA methylation changes and associated epigenetic modifications as a key biological process linked to stress and phenotypic plasticity could help to explain why some oyster species are resilient to OA.
DNA methylation can be triggered by the environmental stresses and is present in calcifying marine organisms (Hoffmann, 2017) such as corals (Liew et al., 2018; Torda et al., 2017) and shellfishes including mussels (Ardura et al., 2018; Chin, 2018) and oysters (Venkataraman et al., 2020; Gavery and Roberts, 2010). The availability of an annotated genome from C. gigas (Zhang et al., 2012) allows an opportunity of inter-species mapping and a comprehensive analysis of gene interactions and pathways of a closely related oyster species. Recent studies have described DNA methylation patterns in the C. gigas genome, with methylation level varying significantly across different functional categories of genes (Gavery and Roberts, 2010). These pioneering DNA methylation studies in oysters have focused on developmental stages such as gametes and early larvae (Wang et al., 2014; Olson and Roberts, 2014; Rivière et al., 2017), spats (Rivière et al., 2017; Zhang et al., 2018), as well as adult tissues such as gill (Gavery and Roberts, 2014; Rivière, 2014) and mantle (Wei et al., 2018; Song et al., 2017). In particular, DNA methylation patterns correspond to different functional gene transcriptions that control reproductive (2–8 cells) and post-metamorphosis (spats) stages (Rivière et al., 2017). DNA methylation on the exon level was found to be associated with mRNA expression in marine invertebrates (Flores et al., 2012) including C. gigas mantle tissue, suggesting that alternative splicing could be affected during transcription (Song et al., 2017). In C. gigas male gamete tissue, 15% of 7.6 million CpG sites was methylated in intragenic regions, where methylation and expression were found positively correlated in genes bodies and promoter regions (Olson and Roberts, 2014).
Not only does DNA methylation mediate rapid adaptive response to environmental stressors, but organisms could also use this mechanism to quickly respond to environmental cues and metamorphose in optimal habitats. Metamorphosis involves transformation with regards to physiological and morphological body changes prepared for a species such as the invertebrates to transit from planktonic to benthic adult phases (Holstein and Laudet, 2014). Consequently, modification in regulating gene expression is expected. A previous study has shown that sea lamprey (Petromyzon marinus) exhibited differentially methylation pattern in genes related to morphogenesis, water balance and osmotic homeostasis, in response to environmental stressors (Covelo-Soto et al., 2015). Significant differences in brain methylation pattern was found in European eels (Anguilla L.) linking to morphological changes (Trautner et al., 2017). In oysters, the start of metamorphosis is signaled by development of a foot and a pigmented eyespot on a larval shell, of which the foot is required to attach on a substrate in response to microbial biofilm cues (Tamburri et al., 1992) to indicate that pediveliger larvae are competent for attachment. To date, data attributing DNA methylation to metamorphosis in oysters are limited. Wang et al. (2014) reported marked difference in the expression level of DNA methylation related gene, DNMT3 of C. gigas pediveliger in comparison to other early developmental stages. Indeed, differentially methylated regions, especially the higher proportion of methylation in exons than introns were found highly regulated in C. gigas spats during post-metamorphosis (Rivière et al., 2017). Investigating into the potential role of DNA methylation in oyster metamorphosis is therefore crucial especially in the face of unprecedented OA rates.
Although information on the effect of OA on DNA methylation in calcifying marine invertebrates is limited, a recent study reported an impact of high pCO2 (~2550 μatm) on the gonad tissue of C. virginica, where a majority of differentially methylated loci were in exons and the rest in intron (Venkataraman et al., 2020). Similarly, Putnam et al. (2016) found two-fold increase in DNA methylation level and discrimination capacity in metabolic profiles of a scleractinian coral species, P. damicornis at low pH (7.6–7.35) as compared to ambient (7.9–7.65) condition, corresponding to a reduced calcification rate. The role of DNA methylation as rapid adaptive response in oyster larvae under OA remains unknown despite the fact that such information could improve the understanding of sustained adaptation strategies in marine bivalves, especially at its critical developmental stages. Here, we seek to fulfill the knowledge gap by answering 1) how oyster larvae changes their DNA methylation pattern in response to OA, and, 2) how larval metamorphosis and growth rates are related to OA-induced differential methylation pattern. This study represents the first controlled experiment to determine the extent of changes in DNA methylation in response to OA at the time of larval metamorphosis in one of the commercially important estuary edible oyster species, C. hongkongensis.
Section snippets
Study animal
Adult Hong Kong oyster, the C. hongkongensis (>2 years old) were collected from an oyster aquaculture farm in Guangxi area, China. They were cleaned, scrubbed, and subsequently acclimated for 20 days in the oyster commercial hatchery “Zhanjiang Haixiang Aquatic” located near the estuary of Zhanjiang, China. Adults were fed adtum libido with a mix of Isochrysis sp. and Chetoceros sp. and conditioned at a salinity of 16 ppt to match conditions at the collection site. Seawater supply was filtered
pH perturbation and carbonate system
The pH values throughout maintaining the larval cultures were significantly different between the control and low pH tanks at pH 8.12 ± 0.04 and pH 7.40 ± 0.04, respectively (ANOVA, pH, F1,166 = 17,681, p < 0.01). However, no significant differences were found in temperature, salinity or total alkalinity (ANOVA, temperature, F1, 166 = 0.986, p > 0.05; salinity, F1,86 = 2.205, p > 0.05; total alkalinity, F1,86 = 0.21, p > 0.05). The daily pH measurements and carbonate system in different pH
Discussion
To understand the process of rapid adaptive responses to climate change-associated environmental stressors better, it is important to learn whether external environmental pressures created by ocean acidification (OA) is able to alter methylation pattern of DNA, i.e. the mediator of gene expression that ultimately expressed in functional molecule level such as proteins in an organism (Cavalli and Heard, 2019; Pinel et al., 2019; Hoffmann, 2017; Suarez-Ulloa et al., 2015). Our intragenerational
Conclusion
We provided the first study of changes in DNA methylation profile of oyster larvae in response to ocean acidification (OA) using one of the commercially important edible oyster species as model. Through Methyl-RAD method, low pH stress associated with OA has been shown to impact the CpG methylation state in larval DNA after three weeks of exposure, resulting a total of 130 hyper- and hypo-methylated genes. CpG methylated sites were found to concentrate in the exon region within the gene bodies,
CRediT authorship contribution statement
Yong-Kian Lim: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Khan Cheung: Formal analysis, Investigation, Writing - review & editing. Xin Dang: Formal analysis, Investigation, Writing - review & editing. Steven B. Roberts: Formal analysis, Writing - review & editing. Xiaotong Wang: Formal analysis, Writing - review & editing. Vengatesen Thiyagarajan: Conceptualization, Methodology, Formal analysis, Resources,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was funded by two HKSAR’s RGC grants (Nos 17304619 and 17303517). This work was also supported by funding from State Key Laboratory for Marine Pollution (AR170078).
References (98)
- et al.
Effect of sea-water acidification on fertilization and larval development of the oyster Crassostrea gigas
Journal of Experimental Biology and Ecology
(2013) - et al.
Does DNA methylation regulate metamorphosis? The case of the sea lamphrey (Petromyzon marinus) as an example
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
(2015) - et al.
A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media
Deep-Sea Res. Part A
(1987) - et al.
Protein degradation and the stress response
Semin. Cell Dev. Biol.
(2012) - et al.
Life-history evolution: at the origins of metamorphosis
Curr. Biol.
(2014) - et al.
Calcium carbonate unit realignment under acidification: a potential compensatory mechanism in an edible estuarine oyster
Mar. Pollut. Bull.
(2019) - et al.
Dissociation constants of carbonic acid in seawater as a function of salinity and temperature
Mar. Chem.
(2006) - et al.
Environmental epigenetics: a promising venue for developing next-generation pollution biomonitoring tools in marine invertebrates
Mar. Pollut. Bull.
(2015) - et al.
Larval growth response of the Portuguese oyster (Crassostrea angulata) to multiple climate change stressors
Aquaculture
(2012) - et al.
Metamorphosis and transition between developmental stages in European eel (Anguilla Anguilla, L) involve epigenetic changes in DNA methylation patterns
Comp. Biochem. Physiol. Genom. Proteonomics
(2017)
Consistent inverse correlation between DNA methylation of the first intron and gene expression across tissues and species
Epigenet. Chromatin
Differential expression analysis for sequence count data
Genome Biol.
Stress related epigenetic changes may explain opportunistic success in biological invasions in antipode mussels
Sci. Rep.
The Ca2+/Mn2+ pumps in the golgi apparatus
Biochim. Biophys. Acta Mol. Cell Res.
The changing carbon cycle of the coastal ocean
Nature
CpG-rich islands and the function of DNA methylation
Nature
Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI
Genome Res.
Oyster reproduction is compromised by acidification experienced seasonally in coastal regions
Sci. Rep.
The role of epigenetics in aquatic toxicology
Environ. Toxicol. Chem.
Oceanography: anthropogenic carbon and ocean pH
Nature
Advances in epigenetics link genetics to the environment and disease
Nature
Characterizing the Role of DNA Methylation Patterns in the California Mussel, Mytilus californianus. Thesis (Doctorate of Philosophy)
A program for annotating and predicting the effects of single nucleotide polymorphism SnpEff: SNPs in the genome of Drasophila melanogaster strain w1118; iso-2; iso-3
Fly
On the tendency of species to form varieties; and on the perpetuation of varieties and species by natural means of selection
Zoology Journal of the Linnean Society
Proteomic Analysis of Oyster Larvae Reveals Molecular Mechanism of Ocean Acidification and Multiple Stressor Effects (Doctorate of Philosophy)
Elevated CO2 alters larval proteome and its phosphorylation status in the commercial oyster, Crassostrea hongkongensis
Mar. Biol.
Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification
Proteomics
Marine environmental epigenetics
Annual Review of Marine Science
Environment Hong Kong
Genome-wide association between DNA methylation and alternative splicing in an invertebrate
BMC Genom.
Beyond buying time: the role of plasticity in phenotypic adaptation to rapid environmental change
Phil. Trans. Biol. Sci.
Induction of invertebrate larval settlement; different bacteria, different mechanisms?
Sci. Rep.
DNA methylation patterns provide insight into epigenetic regulation in the pacific oyster (Crassostrea gigas)
BMC Genom.
A context dependent role for DNA methylation in bivalves
Briefings in Functional Genomics
Epigenetic considerations in aquaculture
PeerJ
Characterization of genetic and epigenetic variation in sperm and red blood cells from adult hatchery and natural-origin steelhead, Oncorhynchus mykiss
G3 (Bethesda)
Larval and post-larval stages of pacific oysters (Crassostrea gigas) are resistant to elevated CO2
PloS One
Physiological response and resilience of early life-stage eastern oysters (Crassostrea virginica) to past, present and future ocean acidification
Conservation Physiology
Effect of cyprid age on the settlement balanus amphitrite Darwin in presense to natural biofilms
Biofouling
Minor impacts od reduced pH on bacterial biofilms on settlement tiles along natural pH gradients at two CO2 seeps in Papua New Guinea
ICES (Int. Counc. Explor. Sea) J. Mar. Sci.
Epigenetic in marine metazoans
Frontiers in Marine Science
Bacterial biofilm communities and coal larvae settlement at different levels of anthropogenic impact in the Spermonde Archipelago, Indonesia
Frontiers in Marine Sciences
Epigenetic and genetic contributions to adaptation in Chlamydomas
Mol. Biol. Evol.
Effects of increased seawater CO2 on early development of the oyster Crassostrea gigas
Aquat. Biol.
Recherches sur l'organisation des corps vivans
Caspase-7: a protease involved in apoptosis and inflammation
Int. J. Biochem. Cell Biol.
Fast gapped-read alignment with Bowtie 2
Nat. Methods
Dynamic changes in the human methylome during differentiation
Genome Res.
Regulation of actin cytoskeleton dynamics in cells
Mol. Cell.
Cited by (25)
Starvation-induced changes in sex ratio involve alterations in sex-related gene expression and methylation in Pacific oyster Crassostrea gigas
2023, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular BiologyEpigenetic analytical approaches in ecotoxicological aquatic research
2023, Environmental PollutionReview of warming and acidification effects to the ecotoxicity of pharmaceuticals on aquatic organisms in the era of climate change
2023, Science of the Total EnvironmentOcean acidification drives gut microbiome changes linked to species-specific immune defence
2023, Aquatic ToxicologyWild oyster population resistance to ocean acidification adversely affected by bacterial infection
2023, Environmental PollutionCitation Excerpt :The carbonate chemistry of pH 7.3-driven OA treatment was more extreme than the projected open ocean surface in 2100 (IPCC et al., 2022) but in line with the predicted conditions of estuarine and coastal areas (Calosi et al., 2017). In addition, pH 7.3–7.4 has been commonly used in previous studies to represent the OA conditions for C. hongkongensis (Chandra Rajan et al., 2021; Lim et al., 2021a; Lim et al., 2021b) because of its resistance to moderate acidifying treatment (i.e., pH 7.6) (Leung et al., 2022; Meng et al., 2019). The seawater was utterly changed every two days and 50 mL seawater was preserved with HgCl2 every week for total alkalinity (TA) measurement.
Microplastics can aggravate the impact of ocean acidification on the health of mussels: Insights from physiological performance, immunity and byssus properties
2022, Environmental PollutionCitation Excerpt :For instance, energy deficiency caused by microplastics can lead to abnormal gametogenesis and gonadal resorption in oysters (Gardon et al., 2018), thereby reducing reproductive output. In contrast, ocean acidification is a relatively slow process due to the gradual rate of change in seawater pH over time, where marine organisms may be able to accommodate its impacts through adaptive responses (Ellis et al., 2015; Castillo et al., 2017; Leung et al., 2017b, 2020b; Lim et al., 2021; Nordio et al., 2021). Nevertheless, the impact of microplastics is subject to their size, shape and polymer type (De Sá et al., 2018).