Experiment is UNDERWAY

Brian and the lovely team at WDFW sent me ~200 crabs from Ocean Shores Airport in Gray’s Harbor. My team (Sarah, Julia, Lauren, and McCaela) helped me distribute crabs across tanks! With each crab shipment, we:

• sorted crabs into blue/green males, blue/green females, and yellow crabs
• distributed blue/green females as evenly as possible
• adjusted the distribution with yellow females
• added blue/green males to tanks
• evened out everything we could using the remaining yellow males

While trying to evenly distribute sex and integument colors across tanks, we also tried to spread out really big or really small crabs.

Labelling protocol

In between crab shipments on 6/22/23 and 6/24/23, we labelled crabs in T1-T6. Here’s the rough protocol:

Materials:

• 95% ethanol
• Flame kit
• Dissection tools (scissors, tweezers)
• Tubes labelled with crab ID and filled with 1 mL ethanol
• Tube rack
• Paper towels
• Labels for crabs (our case: Rite-in-the-Rain paper with color-coded number labels)
• Super glue
• Calipers
• Weigh boats
• 100 g scale
• Data sheets

Method:

1. Start flame and sterilize dissection equipment
2. Place all crabs from a tank in a cooler with a little bit of water
3. Randomly select a crab
4. Cut of the last two leg joints on the right third leg of the crab. Place the leg segment in the labelled tube with ethanol. Cover the leg segment with additional ethanol as needed. Place tube in a tube rack.
5. Dry crab using paper towels.
6. Measure carapce width and length with calipers to the nearest mm.
7. Tare the scale with two weigh boats. Place the crab in one weigh boat and cover with another (looks like a takeout container). Weigh the crab and record weight.
8. Super glue the appropriate label to the crab’s carapace. Cover the label in a “shell” of super glue. Dry the crab for ~5 minutes. It helps to blow on the super glue to accelerate the drying process.
9. Place labelled crab back into the cooler with other crabs. Repeat for remaining crabs.
10. Place crab back in experimental tank. Feed.

Experiment checks

6/22/23:

• Restarted 14ºC lines
• Added filter cartridges to all tanks
• Added crabs to tanks!
• Number of crabs in each tank
  • T1: 13
  • T2: 13
  • T3: 13
  • T4: 13
  • T5: 13
  • T6: 13
  • T7: 12
  • T8: 13
  • T9: 13
  • T10: 13
  • T11: 12
  • T12: 13
• Checked cold room temperatures

6/23/23:

• Went through facility checklist with Lauren, McCaela, and Julia
• Fed 1/4 tsp food to each tank

6/24/23:

• Cleaned all tanks
• Added new crabs to tanks
• Number of crabs in each tank
  • T1: 17
  • T2: 17
  • T3: 17
  • T4: 18
  • T5: 17
  • T6: 18
  • T7: 18
  • T8: 18
  • T9: 17
  • T10: 18
  • T11: 17
  • T12: 17
• Fed 1/4 tsp food to each tank

6/25/23:

• T11 and T12 airstones floating on arrival. T12 all crabs huddled next to filter.
• Cleaned all tanks
• Checked ammonia in all tanks. All tanks read at 4.0 so I added 1/2 tsp conditioner to each tank
• Replaced 1/3 water in all tanks
• Fed 1/4 tsp food in each tank
• No mortalities!
• Checked cold room temperatures
• Labelled and re-launched HOBOs in all tanks
  • Added HOBOs with weights to all tanks except T10
  • Floating HOBOs in all tanks except T1 and T2
• Updated Google Sheet versions of survivorship and measurements

6/26/23:

• Replaced filter cartridges
• Labelled newly added crabs in T1-T6, T7 and T8
• T7 only had 16 crabs?! In the words of Steve Martin’s character in 30 Rock…I miscounted the men.
• No mortalities!
  • T1: 17
  • T2: 17
  • T3: 17
  • T4: 18
  • T5: 17
  • T6: 18
  • T7: 16
  • T8: 18
  • T9: 17
  • T10: 18
  • T11: 17
  • T12: 17
• Added T10B, T1A, and T2A HOBO loggers
• Updated Google Sheet versions of survivorship and measurements

The temperature saga…continued

Ed and Rick twinned the large water bath tanks together so I could have three tanks at the 5ºC temperature! Of course, I ran into some issues. Once the tanks were twinned, I wasn’t achieving the 5ºC temperature. I noticed the edge water baths were reaching ~6ºC, but the middle was at 12ºC.

Figures 1-2. Temperatures of edge water baths on 6/22/23

Figures 3-4. Temperatures of middle water bath and tank on 6/22/23

When I looked at the tanks, it was clear that water was not circulating through the middle tank. Dave gave me a few pumps to try and circulate the water. I also asked Ed if there was a way to pump water throughout. He and Rick modified the tank set up on Friday so that water was actively going from the right water bath to the chiller, feeding into the left water bath, dripping into the middle water bath, then once again leaving to the right water bath. Since they added new water to the baths, the chiller was not at the setpoint.

I checked the chiller on Saturday, and it read that water was 32ºF! I was suspicious…and rightfully so. The pump in the right water bath was almost running dry! Looking at the plumbing, it looked like the tube that fed from the chiller to the left water bath had yeeted itself out of the bath and onto the floor of the cold room. I placed the tube back in the left water bath and refilled water in all the water baths. I checked the tank temperatures on Sunday.

Figures 5-8. Temperatures in cold room on 6/25/23

Okay, we’re still hovering around 6-7ºC instead of 5ºC. That’s slightly better. However, there’s this weird periodicity in the measurements where the water heats to 10ºC. I outfitted the HOBO loggers in the cold room with some weights so they sank to the bottom of the tank and stayed there, instead of floating on top and getting sucked through the plumbing.

In the shower, I thought to check if the chiller got plugged into a timer outlet. WHICH WAS RIGHT. I walked into ESL first thing and moved that plug to a non-timer outlet. I’ll check temperatures tomorrow to see if I achieved my desired 5ºC set point.

Going forward

1. Finalize cold room temperatures
2. Add remaining HOBO loggers to tanks
3. Label and measure all crabs
4. Obtain leg joints for genotyping
5. Test formatting and analysis for one set of respirometry data
6. Respirometry analysis
7. Update methods
8. Update results
9. Cover respirometry chambers with black trash bags and duct tape, and add the probe holders with silicone glue