DNA methylation changes in response to ocean acidification at the time of larval metamorphosis in the edible oyster, Crassostrea hongkongensis

https://doi.org/10.1016/j.marenvres.2020.105214Get rights and content

Highlights

  • Low pH stress resulted in hyper- and hypo-methylated genes in the pediveliger larvae of the Hong Kong oyster.

  • Differentially methylated loci were concentrated in the exon region within the gene bodies.

  • High capability of oyster larvae to acclimate to low pH stress within single generation despite poor choice of attachment.

  • Differential methylation is associated to higher metamorphosis rate and poor larval substratum selection under low pH stress.

Abstract

Unprecedented rate of increased CO2 level in the ocean and the subsequent changes in carbonate system including decreased pH, known as ocean acidification (OA), is predicted to disrupt not only the calcification process but also several other physiological and developmental processes in a variety of marine organisms, including edible oysters. Nonetheless, not all species are vulnerable to those OA threats, e.g. some species may be able to cope with OA stress using environmentally induced modifications on gene and protein expressions. For example, external environmental stressors including OA can influence the addition and removal of methyl groups through epigenetic modification (e.g. DNA methylation) process to turn gene expression “on or off” as part of a rapid adaptive mechanism to cope with OA. In this study, we tested the above hypothesis through testing the effect of OA, using decreased pH 7.4 as proxy, on DNA methylation pattern of an endemic and a commercially important estuary oyster species, Crassostrea hongkongensis at the time of larval habitat selection and metamorphosis. Larval growth rate did not differ between control pH 8.1 and treatment pH 7.4. The metamorphosis rate of the pediveliger larvae was higher at pH 7.4 than those in control pH 8.1, however over one-third of the larvae raised at pH 7.4 failed to attach on optimal substrate as defined by biofilm presence. During larval development, a total of 130 genes were differentially methylated across the two treatments. The differential methylation in the larval genes may have partially accounted for the higher metamorphosis success rate under decreased pH 7.4 but with poor substratum selection ability. Differentially methylated loci were concentrated in the exon regions and appear to be associated with cytoskeletal and signal transduction, oxidative stress, metabolic processes, and larval metamorphosis, which implies the high potential of C. hongkongensis larvae to acclimate and adapt through non-genetic ways to OA threats within a single generation.

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).

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