Environmental "memory" through dynamic DNA methylation provides acclimatization of geoduck clams to ocean acidification

Abstract
We tested the sensitivity of early life stages and the potential for geoduck clams to display acclimatization to ocean acidification through a series of  repeat exposure experiments.  First, we exposed larval geoducks to ambient (~8.0) and low pH (~7.4) for 10 days and found that larval mortality is decreased and shell size increased in low pH conditions. Second, we exposed juvenile geoduck to ambient (~8.0), low (~7.4) and lower (~7.0) pH for 23 days, placed them in ambient common garden for several months, then re-exposed them to ambient (~8.0) pH and low pH (~7.4) for another 23 days. In geoduck juveniles there was a size benefit of preconditioning to low pH. Juvenile growth initially declined at pH ~7.4 and 7.0 in the first exposure, but when replaced in the ambient conditions, the initial exposure to low pH resulted in compensatory growth, such that the juveniles grew larger. Growth in the pre-exposed juveniles was also more resistant to low pH in the second exposure. The role of DNA methylation as a mechanism of environmental memory was tested using reduced representation bisulfite sequencing. This suggests that acclimatization to OA can result in benefits to geoduck growth, with exposure memory that is potentially linked to epigenetic mechanisms such as DNA methylation.
Introduction
Materials and Methods
Geoduck juveniles were exposed to a three component experiment (Fig. 1) designed to test for: 1) the effects of acute exposure to ocean acidification, 2) the potential for latent effects due to initial exposure, and 3) the potential for initial exposure to provide preconditioning and acclimatization to a secondary exposure. The experiments were conducted at the Kenneth K. Chew Center for Shellfish Research and Restoration in Manchester, WA. Juvenile geoduck clams were received at ~3month of age (Taylor Hatchery Quilcene, WA) on 16 March 2016.  Animals were placed in 5L replicate tanks per treatment (35 x 21 x 12cm, lxwxh).   The geoducks were allowed to bury themselves as desired in ~4cm of graded sand of the same source and grade from which the clams were collected at the hatchery.

Ocean Acidification Control System and Seawater Chemistry Analysis
The experiments were conducted using a flow-through, pH-stat system, with 1µm filtered seawater. The pH in the header tanks was continuously monitored with DuraFET pH probes (Honeywell, Morristown NJ, USA) that fed data back into a solenoid controlled injection system. The pH set points were achieved by the injection of ambient air or pure \(CO_2\) into water cycling lines of the header tanks, which continually cycled water from the bottom to the top of each header for even mixing and equilibration of \(CO_2\), as well as pumping it to the treatment tanks.

Seawater chemistry was assessed in each tank following best practices standards (Riebesell 2010). Seawater pH, salinity and temperature were measured in each tank using a handheld probes. pH was measured in mV (resolution = 0.01, DG115SC glass probe, Mettler Toledo, Ohio, USA) and calculated for the in situ temperature against a linear regression of a tris standard (Batch 2/14/16 salinity 27.5) as a function of temperature. Temperature was measured simultaneously, with a traceable digital thermometer (Accuracy: ±0.05°C, resolution: 0.001°, temperature Range: –50 to 150°C, VWR, USA), and salinity with a traceable digital portable conductivity meter (Accuracy: 0.3%, Temperature Range: –30.0 to 130.0°C, VWR, USA). Simultaneously with the probe measurements, 120 ml water samples were collected and stored in sealed borosilicate glass bottles and poisoned with 50µl of HgCl for total alkalinity analysis. Total alkalinity was processed at the X and measured using an open cell gran titration (Dickson SOP3, Dickson 2007). Carbonate chemistry parameters were calculated using the seacarb package in R (