Many December goals were comlepeted, but some work is still incomplete in filling in the gaps from the first round data, specifically finishing alkalinity sample analysis and incorporating this data to determine in situ pH. I will also continue to work on creating the transcriptome library, and begin the second round of the clam OA experiment and any remaining prep beforehand.

This is a running daily post of current actions in January

Most October goals were comlepeted except for writing on the manuscript and any RNA extraction from sperm. In place of a completed manuscript for the clam OA project, the current goal is to create a detailed report of methods and results so far completed, to be used in part for the manuscript but also for reference when needing to provide details about the project status.

Staging Manila and Littleneck clams based on histology requires a good rubric to compare to samples and establish consistant assessment. In order to establish this rubric for our samples, here are collected a number images illustrating different stages.

Histology Staging

All September goals were comlepeted except the full development of a plan for examining histology slides

All clams not used for dry tissue weight were used in ATPase activity analysis. The data can be found here

ATPase activity is a measurement of the amount of NADH which is transformed to NAD+ over a specific period of time while Na+/K+ ATPase is inhibited by ouabain in one set of samples and uninhibited in another set. In clam gill tissue, ATPase activity is a reflection of the overall metabolic activity in the tissue, given it’s central role in mitochondrial function. We hope to use ATPase activity assays to understand whether OA has an effect on the energetics of clam gills.

ATPase

A preliminary experiment was conducted to determine the natural uptake of fluorescent dextran dye (Dextran, Fluorescein, 3000 MW, Anionic from ThermoFisher) in C. gigas eggs. Eggs were collected and held overnight in a refrigerator before the experiment. They were then placed in a solution containing dye for 20 minutes, washed several times in filtered seawater, and imaged. Below are light microscope images and fluorescent images using a FITC filter.

Data collected from regular assessment of water pH using a spectrometer must be calibrated each time a new batch of dye is used. m-Cresol Purple dye alters the pH of a solution slightly, and since batches of dye may have slightly different concentrations, the effect of dye concentration on pH must be assessed. This is done by dosing samples twice with the same volume of dye (referred to as a double shot) and comparing the change between the first and second addition. After acquiring a sufficient number of double shot comparisons, approximately 20, data is filtered to eliminate spectrometer readings which do not meet two requirements. Firstly, readings variance in measurements at 730 nm should not exceed 0.01

Experiments with the goal of understanding the effects of ocean acidification rely on accurate determination of pH in seawater. There are a number of ways of measuring pH, but one of the most accurate is the use of a spectrometer and a pH sensetive dye. In our experiment, pH was determined initially using a durefet sensor, which is an type of ion sensitive field effect transistor (ISFET). These probes were connected to relays which injected CO ₂ when sensed pH was determined to be above the setpoint. Nonetheless, probes can vary in accuracy, and so regular spectrometery was needed to determine the actual pH in treatments and adjust relay setpoints as necessary.

As previously described, approximately 320 clams of two species were collected for an experiment to understand the impact of ocean acidification (OA) conditions on clams in the Puget Sound. One species, Leukoma staminea, is natively distributed in the intertidal zone of the Eastern Pacific Ocean from Alaska to Northern Mexico. It has traditionally been an important wild harvested seafood source in the Puget Sound, and anecdotal accounts indicate that populations may be declining. The other species, Ruditapes philippinarum, is native to the Western Pacific and is raised globally in aquaculture production. R. philippinarum is naturalized in the Puget Sound, and is also raised in hatcheries and dispersed in order to boost the quantity of clams harvested from beaches.

This post describes the beginning of a project assessing the comparative impacts of Ocean Acidification on two species of clams found locally in the Puget Sound