This post details experimental design thoughts and brainstorming for the WSG and USDA project efforts.

In my previous post I detailed the background information on this project. Check that out first.

Experimental design ideas

Steven and Eric and I talked about potential experimental design ideas. Here are the main takeaway ideas.

We all agree that running a latent acute stress would be interesting and worth doing. Note that individuals exposed to the first acute stress were all sampled and not available for secondary exposure. This would produce the following treatment groups for each lifestage:

Conditioning treatment - latent acute treatment

Control-Control
Control-Treated
Treated-Control
Treated-Treated

Hypotheses

This would provide interesting information on a few things.

1. First, it would provide information on whether the conditioning exposure produces differential stress tolerance in a latent acute stress. The hypothesis is that pre-conditioning elevates stress tolerance.
2. Second, it would provide information on lifestage-specific conditioning capacity. Which lifestages can be influenced by conditioning and how does conditioning affect performance in a latent stress between stages? The hypothesis would be that earlier developmental stages would be more receptive to conditioning effects as a product of developmental plasticity that does not exist in adults.
3. Third, we could compare the results of the first acute stress to the latent acute stress. This would be interesting to determine whether conditioning provides immediate or latent effects for stress response. The hypothesis is that conditioning does not improve (and may actually reduce) immediate stress tolerance, but provides positive latent effects on stress tolerance.

Possible measurements

We will need to think about these hypotheses and what we would like to measure. Here are some of the ideas we discussed:

• RNA analyses: 1) qPCR as Eric is planning to do to look at expression of target genes. These target genes may include stress tolerance, heat shock proteins, membrane integrity, energy metabolism, metabolic stress, oxidative response, and related genes. 2) 3’ or TagSeq of a subsample to look at transcriptome-scale shifts in functional roles of expressed genes. We only have RNA data available from the acute stress, so this is our only comparison available to assess hypothesis in point #3 above.

• Growth: We have group means for growth in all of the treatments, but we do not have growth for all individuals. This could provide interesting information on energetic costs or benefits of conditioning. For example, if the oysters that underwent conditioning are smaller, there may be an energetic cost of this exposure. This could also be different by lifestage, and would help us evaluate hypothesis #2 above .

• Metabolomics/lipidomics: It would be really neat to do metabolomics and lipidomics approaches on a subset of samples (n=5 per treatment x lifestage group; N=16 groups; approx. 80 samples). RNA provides us with the molecular, immediate response of the organism. Looking at metabolites would tell us about metabolic phenotype and the metabolic pathways used to produce energy to deal with stress. Lipidomics would tell us about the composition of lipid energy stores in the animals and how they are using those lipids to survive. These would be relevant in examining all three hypotheses above. For example, perhaps the younger lifestages will use more lipids in their metabolism because they have greater proportional energy demands than adults due to fast developmental growth. Further, we would be able so see how well the animals are able to handle the metabolic demands of stress and how that is impacted by conditioning history - maybe those that have been conditioned are less metabolically stressed and use less lipids than those that were not conditioned, for example.

• Aerobic scope and metabolic rates: We would like to take respiration measurements of animals after they go through the latent acute stress. This would give us an organism-level reading on the energy demands required to handle the acute stress. This would allow us to assess all three hypotheses above. If the animals have higher metabolic rates, that could indicate metabolic stress. On the other hand, if the animals reduce their metabolic rates, this could be a product of cellular energy savings and metabolic depression as a strategy to survive, or could be due to a collapse in cellular energy systems. We could try to assess this with oxygen evolution approaches or with rapid assay approaches (Steven had some ideas on this).

• Stress assays: We need to identify stress assays that would be 1) feasible in an aquaculture setting and 2) provide accurate indicators of stress. This would address the three hypotheses above. Examples could include antioxidant capacity, ATPase activity, mitochondrial activity, central metabolism enzyme activity. I am going to spend some time reading papers to see what our best options are.

• Epigenetics: It would be super cool to do epigenetic work (i.e., methylation) on the oysters. This would allow us to understand, if there are differential effects of conditioning, if epigenetic modifications are a mechanism. This is expensive, however, so we will need to think about this approach perhaps on a subset of samples. This would also help us address all three hypotheses!

Possible sampling

If we were able to proceed with all of these sampling protocols, we would need to collect the following from each sampled individual:

• 2 RNA samples (gill and mantle) in RNA/DNA later or shield
• 1 DNA sample in RNA/DNA later or shield
• 2 metabolomics samples (gill and mantle) snap frozen in liquid nitrogen
• 2 lipidomic samples (gill and mantle) snap frozen in liquid nitrogen
• 2-3 tissue samples (gill and mantle) for stress assays

These samples would be in addition to anything that is done on live organisms.

Supplies

Some of the supplies we would need to do all of these things.

• Heaters, tanks, containers, Apex equipment and anything else needed to manipulate temperature treatments
• Temperature loggers
• Mechanical stress for adults (salad spinners) - if we do this
• Tubes, labels, pipettes, scalpels, razer blades, knives, clippers, etc. for sampling
• RNA/DNA later or shield
• Liquid nitrogen in a dewar
• Ethanol, bleach, and DI water for sterilizing eqiupment
• Plastic or bench paper for sampling and dissecting
• Assays or equipment for live metabolic rate measurements

Preparing for summer outplanting

After this lifestage carry over experiment, we will transition to the next phase of the experiment.

Our goal is to conduct thermal conditioning on seed and larvae and outplant those individuals to our commercial partners for summer grow out and assessment of survival and growth.

Our primary goal is getting seed and larvae conditioned and grown to outplant in the field.

We are also talking with Neil Thompson next week regarding source animals for this project.

Here are the numbers that we committed to outplanting in the field for each project.

Scroll down to the bottom of this section for a grand summary of numbers - there is lots of math in this section as I think through this.

Ariana made this online worksheet to calculate all the things!

USDA project

• [Objective 1: Larval conditioning] Broodstock (5 families) will be strip spawned and larvae from each family challenged with environmental conditions over a 4 month period. Field deployments of spat will take place at Discovery Bayy and Totten Inlet (Puget Sound) and Bay Center and Goose Point (Willapa Bay) with Taylor Shellfish Co. and Nisbet Oyster Co.

• [Objective 2: Broodstock conditioning] Expose broodstock (5 families) before spawning to stress exposures from Objective 1, undergo strip spawning and rear larvae. Spat from these treated broodstock will be deployed at the same size as described above.

In both objectives, performance and growth and metabolic response will be measured before deployment and 6 months after deployment.

For each objective, the proposed estimate for sample size is 200 oysters from each family x treatment group at each deployment site. 30 per treatment group will be required for lab assays.

If we do a simple conditioned vs non-conditioned approach (2 treatments), that would generate 10 family x treatment combinations. If we want to outplant 200 per family x treatment x site, that would be 2,000 outplanted individuals at each site. If we choose 4 sites, that is a total of 8,000 individuals (800 per group total). This would be 200 oysters per family per treatment per site.

Larvae numbers

If we are able to start with larvae, we will need to back calculate accounting for larval survival, settlement success, and post-settlement survival bottlenecks.

For example, if only 10% of larvae make it to deployment as spat, we would need to start with 80,000 larvae to get 8,000 at the end.

If only 5% of larvae make it to deployment, we would need to start with 160,000 to get 8,000 at the end.

This estimate may be different for survival depending on treatments. The more stressful the treatment, the higher mortality we expect. This is a minimum estimate.

Seed numbers

If we start with seed, we would assume a higher survival rate through to deployment.

For example, if 80% of seed make it to deployment as spat, we would need to start with 10,000 to get 8,000 per group at the end.

If only 50% of seed make it to deployment, we would need to start with 16,000 to get 8,000 per group at the end.

WSG project

• [Objective 2: Resistant seed conditioning] OsHV-1 resilient osyter seed from USDA undergo sublethal exopsrue to simulated heat waves in the hatchery. Spat are outplanted and assessed for summer surval in Puget Sound and Willapa Bay (hatcheries as described above). Seed from 10 families. In Year 1, spat undergo stress priming using control, acute and repeated exposure to heat and dessication and combined heat and dessication (5 treatments total). Proportion will be outplanted for field evaluation. Taylor Shellfish Co and Westcott Bay Shellfish Co (Puget Sound) committed to outplanting 30 bags of oysters at 3 sites and Nisbett Bay agreed to 30 bags outplanted at 1 site. This adds to 4 total sites with 30 bags each = 120 bags. If we have 5 treatments, we could do 6 bags per treatment per site. The number of oysters is not specified.

Seed numbers

If we want to outplant a total of 120 bags of oysters (30 bags per each of 4 sites), we need to know about how many oysters we want in each bag. If we base this off of the 200 oyster estimate at each site from above, that would be approximately 33 oysters per bag. Lets increase this to 100 oysters per bag for now for estimating. This would be 100 oysters * 120 bags = 12,000 oysters total (3,000 per site). If we outplant 3,000 per site, that would be a total of 600 oysters per treatment per site (or 60 oysters per family per treatment per site). Lot’s of math happening here!

If we start with seed, we would assume a higher survival rate through to deployment.

For example, if 80% of seed make it to deployment as spat, we would need to start with 15,000 seed total to get 12,000 at the end per site or 48,000 total.

If only 50% of seed make it to deployment, we would need to start with 24,000 seed to get 12,000 at the end per site or 48,000 total.

Adding up all the numbers

In summary, we want to have the following final numbers to go out in the field:

USDA = 200 oysters per family per treatment per site x 5 families x 2 treatments x 4 sites = 8,000 outplanted spat total

WSG = 60 oysters per family per treatment per site x 10 families x 5 treatments x 4 sites = 12,000 outplanted spat total

If we source the USDA grant from seed this year and use the same source of seed as the WSG project, that would be 20,000 total outplanted spat. We need to think about mortality estimates in order to start the rearing process and consider what we would take for sampling.

Note that these numbers also need to take into account any sampling. We will want to have an n=30 for sampling for each assay. Assays could include RNA/DNA, metabolomics, tissue assays, and respirometry.

Ariana made this online worksheet to calculate all the things!

Equipment preparations

We will need to rework out current tank system. Here are some pictures:

We are going to be running larval and seed experiments at the same time, so we ideally would have a tank system with a shelf of HUDL larval systems and water baths or shelves for spat pipe systems (as shown in the picture).

We will primarily focus on temperature treatments for this year, so we will need capacity to manipulate temperature. We could also see what other space is available at Point Whitney (e.g., large conical tanks).

I’ll work on this more over the coming weeks!