Green Crab Experiment Part 7

Treatment Days 3 and 4

Day 3

Today I intended to do time-to-right and respirometry for the batch of crabs I meant to follow throughout the experiment.

  • Mikayla and I did time-to-right (TTR) for half of the crabs in T2 and set up crabs for a one-hour acclimation in the respirometry chamber
  • Finished TTR for half of T5 crabs
  • Issue with respirometer! Oxygen leaked into the chambers and we were unable to get reliable data. We used plastic lids to seal of the chambers, but plastic is permeable to oxygen. We used deoxygenated water to test our sealing system and confirmed that oxygen was leaking into the chambers. We decided to scrap respirometry for today and to get TTR measurements for every crab in each tank, then measure oxygen consumption for our selected crabs tomorrow.
  • Finished TTR for all crabs in T2 and T5
  • One mortality in T6 (crab 58)
  • Went to start TTR for the remaining tanks when we saw that the crabs had climbed all over eachother and scratched off any paint pen labelling! Since we could no longer track individual crabs, doing TTR and then trying to pair it with a respirometry measurement would be difficult. I decided to just scrap sampling today and get ready for tomorrow.
  • Got glass lids and confirmed that oxygen did not leak in or out of the chambers using those lids.
  • Replaced all aquarium filters
  • Checked ammonia concentrations in all tanks
  • T1: 0.25
  • T2: 1.0
  • T3: 8.0
  • T4: 4.0
  • T5: 0.50
  • T6: 8.0
  • Performed a water change and added 1/2 tsp conditioner to each tank
  • Likely feeding crabs too much as there is lots of food debris on the bottom. Cleaned tansk to starve crabs properly before respirometry tomorrow
  • Obtained HOBO data. T6 temperature dropped around 7:30 a.m. Back probe reading at 21ºC, front probe reading at 36ºC.
    • T6 temperature too low because the front probe was not accurately reading temperature! I confirmed this by dipping the front probe in the water bath and noting that the temperature in the water bath was read too high.
    • Replaced the front probe and confirmed that the temperature was reading correctly.
    • T3: Front probe was out of water, shoved back in
    • Both treatments took 1/2 day to go from ambient to the target temperature!
  • Obtained an extra tank, heater, and aerator. Set up an acclimation tank for respirometry in the cold room

Day 4

Today we ACTUALLY got data! Just took an extra day of tweaking things to make things run smoothly.

  • Protocol for today
    • Performed TTR for seven randomly selected crabs in a tank. Sexed, gauged color, measured, and weighed each sampled crab until we had three that had weights between 20-30 g for respirometry
    • Acclimated three selected crabs for one hour
    • Measured oxygen consumption. The goal was to measure consumption until the chambers reached 75-80% oxygen.
    • Removed crabs from the respirometry chambers. Resealed the chamber and measured background respiration for 20 minutes. Empty chambers and wipe down with 70% ethanol.
    • Use refractometer to get salinity measurement for acclimation tank
    • Sampled crabs immediately after respirometry. Flash froze heart tissue in dry ice, then moved the cooler with dry ice and samples to the freezer in ESL. Moved them to the -80ºC in the lab whenever I had the time (usually after two tanks).
    • Notes
    • Usually set up acclimation for one tank while performing TTR for the next tank
    • T5: One crab respired for an hour and barely got down to 82% I stopped it at the hour mark, forgot to wipe chambers with 70% EtOH before starting respiration trial
    • T2 samples in T1 tubes, T5 samples in T2 tubes, T1 samples in T5 tubes
    • T3: Recalibrated probe 1 before running trials so it starts at 100% oxygen saturation. Probe 2 starting at 96% saturation
    • T3 and T6: The warm crabs respired QUICKLY! They reached 70% so quickly that by the time I removed them from their chambers they was between 30-60% oxygen in each chamber.
    • T4: 1 molt/dead crab. Probe 1 starting at 80%, unable to calibrate probe 3
  • Salinity
    • T1/T4: 34
    • T2/T5: 37
    • T3/T6: 34
  • Checked ammonia concentrations in all tanks
    • T1: 0.50
    • T2: 0.25
    • T3: 8.0
    • T4: 1.0
    • T5: 0.25
    • T6: 8.0
    • Added conditioner to T3 and T6
  • Performed water change for T3 and T6 and added 1 tsp conditioner (miscommunication, should have been 1/2 tsp)
  • Fed crabs 1/4 tsp in cold and ambient tanks, 1/2 tsp in warm tanks. Ambient crabs were fed before respirometry (miscommunication)
  • When I left the lab around midnight I noticed that T6 temperature was too low. I was too braindead to figure out how to fix it so I just left it for tomorrow.

Going forward

  1. Continue with experiment
  2. Update protocols
  3. Organize project repository and datasheets
Written on July 8, 2022