Grace’s Notebook: Thursday, December 14, 2017

(1) 2015 oyster seed data

I went through some more peptides on SkylineDaily with the 2015 oyster seed data. Protocol calls for checking ~100 peptides to ensure correct peak was selected (Step 5).

(2) Crab project

I met with Steven and Pam today to discuss the overall project and next steps.

Pam brought the most recent samples. There are three samples per crab, and 6 per warm-water treatment crabs (because only 3 survived). Total of 117 crabs.

I will save 50µl of hemolymph/RNAlater mixture for Pam from each crab (117 tubes total). Then I will spin down all the samples (114×3 + 3×6 = 360 samples total), remove and save the supernatant, and place supernatant and pellet tubes in -80.

In January, we will start processing the RNA and pooling the samples to be sent off for sequencing.

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Grace’s Notebook: December 12, 2017

Crab hemolymph samples:

Pink-capped were sampled 11/10/2017

I spun down the hemolymph samples (5000g for 10min) and saved the supernatant and pellets in separate tubes. The boxes were placed in the -80 (tray 14).

Grace’s Notebook: December 11, 2017

Crab Hemolymph Samples:

Green-capped were sampled 11/08/2017

I spun down the crab hemolymph samples and saved the supernatant and pellets in separate boxes. I spun them down at 5000g for 10 minutes. Then, I placed the boxes in the trays in the left -80. (Tray 8 and Tray 10)

I only got through the green-capped samples today.

Grace’s Notebook: Juneau, November 28, 2017

Today Pam and I cleaned and packed up the lab!

We sent the coolers, pumps, and other things to the Kodiak Lab, and sent some of her things back to Seattle.

We power washed the tanks and scrubbed them down.

Pam has to send samples with ethanol, and they can only be sent through UPS. In Juneau, UPS is only open from 7pm-8pm.

Tomorrow Pam is giving a talk around 11am about this project. We have a few more things to clean and pack up, and then we’ll be done!

Grace’s Notebook: Juneau, November 27, 2017

*I will double check some specifics of information (denoted by * or __) with Pam and edit post accordingly.

Effects of temperature change and Hematodinium sp. infection (Bitter Crab Disease) on Tanner crab (Chionoecetes bairdi)

Final sampling day of Tanner Crab (Chionoecetes bairdi) blood 

Pam Jensen and I arrived at the Ted Steven’s Marine Research Institute in Juneau at ~7:45am. We worked in the Annex (right image).

Experimental Set-up

There were three header tanks at different temperatures (4˚C – cold; 10˚C – warm; ~6˚C – ambient). The cold and ambient had coolers, and the warm had heaters. The water flows (2L/min) into the tanks.

There was a mass mortality in the warm tanks due to the temperature getting too high over a weekend. As a result, by today’s sampling day, there was only one crab in each of the three warm tanks.

whole-setup.jpg

Set-up of the lab

The header tanks with the coolers had brass floats. If the water went to high, it would close the valve with the water source (much like a toilet) to prevent the water from overflowing as well as the temperature becoming too cold. Additionally, both cooling tanks had pumps to circulate the water to homogenize the temperature.

The temperature was logged every 15minutes using HOBO Tidbits. One TidBit in each tank (9 total).

HOBOcrab

Tanner Crab phlebotomy 

The crabs were corralled into one end of the tank, and a screen was placed to hold them back. Additionally, bricks were laid at the bottom because the crabs could crawl under the gaps in the screen. In between samples, we kept foam over the top of the tank, to keep the temperature from fluctuating too drastically, since the room was likely quite a bit warmer (even though we did not have the heat on) than the water in the tanks.

crabsintank-screen

Crabs to be sampled.

Each crab has two tags of different numbers. There are two because sometimes the tags will come off, or their entire leg with the tag, will come off. All the crabs are male and young in order to keep from having too many variables.

holdingcrab-tags

After the tag numbers were recorded with a tube number, a Q-tip with rubbing alcohol was rubbed in order to clean the area of other dinoflagellates. Then, a syringe is used to sample the blood at the base of the front claw.

A drop of blood is placed on a labeled slide to create a blood smear, and then 0.2 ml is placed in three tubes containing RNAlater (6 for the warm water crabs, because we only had three total).

The blood smears will be stained by _____ at _____. She will use the slides to determine the life stage of the Hematodinium.

Clean up

The blood smear slides were let to dry and then placed in a slide box. The HOBO Tidbit temperature data were downloaded.

Pam and I began the process of taking down the lab. We had to sacrifice all the crabs even though they were collected from Stevens Passage in Juneau. Pam removed the tags and placed them in the dumpster.

We took apart all the plumbing and pumps. Rinsed everything with freshwater, drained the tanks.

Tomorrow we’ll clean, pack everything up, and ship things to the Kodiak Lab.

Other Info:

Pam said that while other types of infection can cause this mottled appearance in their legs, it is likely the result of Hematodinium infection because ~50% of Tanner Crabs are infected in the fall in  the Juneau area.

mottled

Grace’s Notebook: Tuesday, November 21, 2017

(1) I figured out a better way to image the larvae. I dump the ethanol in the tube into a 80ml beaker. Then, I dump it back into the tube. This dislodges all of the stuff at the bottom. Then I dump the ethanol back in the beaker, and it ends up containing the larvae, leaving behind the larger bits of junk. I wait for the larvae to settle, then pipette off the ethanol until I get to the larvae.

(2) Started the Analysis of the 2015 Oyster seed data. Emma performed the Pecan step, so I started in at Step 4, using the .blib file that she created. I am stuck at Step 4e . We need an isolation scheme file from Step 3b:

Screen Shot 2017-11-21 at 3.42.09 PM

We asked Emma to help us out. And here is what she provided: GitHub Issue

(3) Titrator: I worked through the SOP to calibrate the pH probe. It seemed to work well. Then Steven and I tried out the CSUN method from Hollie on pH buffers – just to try out the method. It seems a little odd to me because it did each sample one at a time. I thought that it would run like that pH calibration method where it went from sample to sample automatically.

Grace’s Notebook: Monday, November 20, 2017

(1) I made pie charts for the sex ratios of the Pre- and Post-OA C. gigas histology classification. I am still unsure about the sex for some of them, but here is what I have so far:

I’m searching for histology examples of different tissue types in C. gigas to try to determine if what I think is gonad, truly is.

(2) The Titrator Temperature Probe arrived today, so I connected that to the Titrator. I assigned it as a sensor using the touchscreen. I’m not sure if it’s reading, so I’ll wait to check that when we re-calibrate the pH probe.

(3) I have been taking images of oyster larvae. It’s not going so well. It’s quite difficult to get 100 larvae imaged when there is so much other stuff and ethanol in the tube with them. I’ve tried lots of different ways to get the excess ethanol off the top, as well as try to avoid the junk at the bottom. But the larvae are floating in the ethanol and I always end up taking too much ethanol up… and it just takes a really long time. I’m going to have to think of other ways to do this so that I don’t waste time.