Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

These data water quality readings for oysters exposed to ocean acidification at multiple timepoints over an 80 day period. The experiment was conducted in the summer of 2017 at the Northeastern University's Marine Science Center with a flow-through seawater system that draws water from Broad Sound in Nahant, Massachusetts (42.416884, −70.907564) using oysters collected from Plum Island Sound, Massachusetts, USA (Site 1, 42.751636, −70.837023; Site 2, 42.725186, −70.855022; Site 3, 42.681764, −70.813498) in late April.

Calcification Rate: Net calcification rate was calculated for oysters surviving to either 50 or 80 days (n = 35) by buoyantly weighing oysters prior to exposure (BW1) and on day 33 or 34 of the exposure (BW2) following the methods of Ries et al. (2009). Buoyant weight was measured in a 27.65 liter tank (48 cm long, 24 cm wide and 24 cm deep) filled with seawater from the flow-through system and was maintained at treatment temperature by an Aqua Euro USA Model MC-1/4HP aquarium chiller. Buoyant weight was measured by completely submerging the oyster on a flat platform suspended from a bottom-loading scale (Cole Parmer Symmetry S-PT 413E, precision = 0.001 g). Care was taken to ensure no bubbles were trapped inside oyster shells. Oysters were weighed three times in the weighing basket, removing the oyster from the basket between each measurement. If replicate measurements varied by more than 0.01 g, oysters were re-weighed. A standard of known weight was weighed every 20 oysters to ensure that no drift was occurring in the scale. To establish a relationship between buoyant weight and dry weight for the purpose of estimating net calcification rate, shells of oysters sampled for tissue within four days of a buoyant weight measurement were soaked in 10% ethanol to remove salts, dried, and weighed. The dry weight was then regressed against the buoyant weight measurement to establish an empirical dry-buoyant weight relationship:

DryWgtBWi(mg)  = 1.87*BWi −2.74

This empirical relationship was then used to calculate dry shell weight at each buoyant weight time point via linear regression. Calculated dry weights were then used to calculate daily calcification rate:

calcification rate (%) = (DryWgtBW2 −DryWgtBW1 )/n * 100/DryWgtBW1

where dry weight (DryWgtBWi) was calculated for each individual using the buoyant weight pre-exposure (BW1) and 33-34 days into the exposure (BW2) and n was the number of days between the two measurements. Lastly, the average daily change in dry weight was divided by initial dry weight to standardize calcification rate for allometric effects, and multiplied by 100 to convert that fraction into a percent.

Extrapallial Fluid: Oyster pHEPF was measured by removing each oyster from its tank, inserting a 5 mL syringe with a flexible 18-gauge polypropylene tip through the luer-lock port into the oyster's extrapallial cavity, and extracting ~0.5–2 mL of extrapallial fluid. Care was taken to avoid puncturing the mantle tissue and inadvertently sampling either the hemolymph or stomach fluid. The pHEPF was measured immediately after extraction with an Orion 91'10DJWP Double Junction micro-pH probe calibrated with pH 7.01 and 10.01 NBS buffers (for slope) and Dickson seawater Certified Reference Material (for intercept).

Link to data processing: https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse

NSF Award Abstract:
Marine ecosystems worldwide are threatened by ocean acidification, a process caused by the unprecedented rate at which carbon dioxide is increasing in the atmosphere. Since ocean change is predicted to be rapid, extreme, and widespread, marine species may face an "adapt-or-die" scenario. However, modifications to the DNA sequence may be induced in response to a stress like ocean acidification and then inherited. Such "epigenetic" modifications may hold the key to population viability under global climate change, but they have been understudied. The aim of this research is to characterize the role of DNA methylation, a heritable epigenetic system, in the response of Eastern oysters (Crassostrea virginica) to ocean acidification. The intellectual merit lies in the integrative approach, which will characterize the role of DNA methylation in the intergenerational response of oysters to ocean acidification. These interdisciplinary data, spanning from molecular to organismal levels, will provide insight into mechanisms that underlie the capacity of marine invertebrates to respond to ocean acidification and lay the foundation for future transgenerational studies. Ocean acidification currently threatens marine species worldwide and has already caused significant losses in aquaculture, especially in Crassostrea species. This research has broader impacts for breeding, aquaculture, and the economy. Under the investigators' "Epigenetics to Ocean" (E2O) training program, the investigators will build STEM talent in bioinformatics and biogeochemistry, expose girls in low-income school districts to careers in genomics, and advance the field through open science and reproducibility.

This research will specifically test if intermittent exposure to low pH induces a methylation response with downstream beneficial effects for biomineralization. These methylation states could be inherited and confer a fitness advantage to larvae that possess them. Phase 1 of the project will use an exposure experiment to determine the degree to which DNA methylation is altered and regulates the response to OA. Data from this experiment will be used to test the hypotheses that (i) DNA methylation, induced in the tissue of shell formation (i.e., mantle tissue), is correlated with changes in transcription and regulation of pallial fluid pH (calcifying fluid pH, measured by microelectrode), and (ii) that methylation changes induced in the mantle tissue are also induced in the germline --indicating that such changes are potentially heritable. Phase 2 of the project will use a pair-mated cross experiment to test the hypothesis that parental exposure to OA alters larval traits (calcification rate, shell structure, and polymorph mineralogy). Larvae will be generated from parents exposed to OA or control seawater, and then raised under control or OA conditions. Results will be used to (i) characterize inheritance of induced methylation states, (ii) estimate the variance in larval traits explained by genotype, non-genetic maternal/paternal effects, adult OA exposure, larval OA exposure, and parental methylome, and (iii) test the hypothesis that adult exposure alters the heritability (a quantity that predicts evolutionary response) of larval traits. Since the effects of epigenetic phenomena on estimates of heritability are highly debated, the results would advance understanding of this important issue. Because the investigators could discover that DNA methylation is a mechanism for heritable plastic responses to OA, knowledge of this mechanism would significantly improve and potentially transform predictive models for how organisms respond to global change.