Experiment planning update!

I’m back from a lovely vacation and am ready to really crank through experiment prep! Here’s a running list of some updates.

This year

• THE SUPER GLUE LABELS WORKED. Three weeks after I labelled the crabs with waterproof paper and either a nail polish or super glue coating, all the nail polish labels fell off. However, nine of the ten super glue labels stayed! Obviously there are a few caveats, namely that the crabs had way more space in the water table than they would in a tank. But these buddies like to climb on top of eachother, so I think this labelling system is what I should use moving forward. I will modify it by using different color/shape tags for treatment (or tank if possible) so it’s easy to identify crabs in case the label peels off. It also seems like the super glue has pitting on it from the crabs climbing on top of eachother, so part of label maintenance may involve re-upping a super glue layer.
• Ordered my experimental materials after discussing which items I need to purchase vs. which ones the lab will purchase.
• Lauren has a biochemistry background and is interested in bringing that to the crab project. Using crabs that are already going to be sampled for metabolomics, Carolyn and I thought she could do a specific biochemical assay that would complement the assays I’m already thinking of. Here are the options:
  • Citrate synthase assay: initial enzyme of TCA cycle, exclusive marker of the mitochondrial matrix. overall metabolic activity of the sample being tested, so you can examine metabolic changes in response to various conditions or diseases. also an indicator of mitochondrial density. Olivia in the Roberts Lab has used this for oysters, so I posted this discussion to get her insight and protocol. I’m leaning towards this assay because a lot of troubleshooting has already been done and Olivia is confident that her protocol should be usable by an undergraduate student. I’ve also seen papers with Carcinus spp. that have used this assay, so there are crab-specific protocols to pull from too.
  • HIF-1a assay + qPCR: A HIF-1a transcription factor assay can tell you about HIF-1a binding activity. I think this would also be interesting because HIF-1a is one of the genes in the putative inversion, so there would potentially be a genotype effect in addition to a treatment effect. I’ve seen different colormetric assays for HIF-1a transcription factor activity.
  • Have also seen glucose (energy status), lactate dehydrogenase (measures anaerobic activity), ATP, dependent isocitrate dehydrogenase (NADP+), and superoxide dismutase assays for measuring ROS response or other baseline metabolism responses in crustaceans.
  • After talking to Carolyn, we decided that a citrate synthase assay is the way to go. If Lauren has time, she can also try a HIF-1a assay!
• Thinking about the amount of work I have to do each sampling day, I don’t think it’s feasible for me to also collect heart rate information. However, I think heart rate would be an interesting variable for Julia to explore. She’s also interested in reversible acclimation, so I think it could be interesting for her to take crabs acclimated at different temperatures and expose them to a heat shock (30ºC) for a short period of time, then return them to their acclimation temperatures.
  • It would also be awesome if Lauren wanted to hop in on the same project and sample crabs for a citrate synthase assay! This way Julia and Lauren can work together on their own experimental maintenance, but they would have different, independent datasets.

Last year

• Contacted the Rutgers metabolomics facility to get a sequencing quote. They’re swamped currently and said they’d contact me in three months. This is fine since I don’t anticipate having time for labwork until after the summer. I know Shelly used the UC Davis Metabolomics core for her Dungeness crab project, so I contacted them to get a rough quote and determine if they would be able to process my samples without me having to do the extractions. Shelly did both metabolomics and lipidomics because they were not sure what may be more influenced by pH and O₂ in crabs, and there was a deal with the core. I will also look into this with the facility. If I can tell which one is more influenced with the 2022 East Coast crabs, I can make a decision on what to sequence with the 2023 West Coast crabs. For Shelly’s experiment, the metabolome was more influenced by stress in crabs.

Going forward

1. Order 2023 experiment materials
2. Create running to-do lists and expectation documents for Julia and Lauren
3. Finalize demographic data analysis
4. Test formatting and analysis for one set of respirometry data
5. Respirometry analysis
6. Test chiller and heater in ESL
7. Update methods
8. Update results