I took over for Yaamini at Manchester on Friday, August 11th and Sunday, August 13th for her gigas OA larvae screening and sampling.
On Friday, a volunteer named Stephanie helped with screening the larvae on 80 and 60 micron screens. Halfway through the day, Kelsey was able to help. Kelsey screened and Stephanie did the bucket and screen cleaning, replacing of air stones, and feeding.
I sampled larvae from each screen size from each of the 24 buckets. I counted the larvae from three samples of 250µl killed with googols and took a fourth sample and placed in a tube with ethanol for freezing in the -80. I then restocked the larvae.
Sunday was a similar day, and Kaitlyn came and helped. She was in charge of screening and cleaning and shuffling the buckets. I did the sampling and counting again.
On both days, I also drained and refilled the header tank to prevent anoxic conditions, and centrifuged and removed the ethanol from the larvae samples for the -80 freezer.
(1) Today I changed some things for the lab stickers – fonts, spacing, number of words, etc. Waiting on final decision of designs from lab members before purchasing 1×1 inch and 2×2 inch stickers.
(2) I also began looking at the methods of Rhonda’s paper: “Influence of temperature on larval Pacific Oysters (Crassostrea gigas) protein expression“ which can be viewed here: https://docs.google.com/document/d/11AjYtMvMImLUVDKnwy8aFNsGbKDVZWfSP8DNiUE1IOE/edit
The next steps from here are that I will be taught how to analyze proteomics data after I understand the methods in the paper.
Yesterday I assisted Yaamini at Manchester with her gigas larvae. It was a similar process to what was done with Laura’s, however Yaamini’s are not in a flow-through water system.
I screened larvae at 60 µm. 24 buckets.
Yaamini sampled, killed with googols, and counted larvae.
Olivia shuffled buckets, cleaned, and kept things organized.
I will be doing Yaamini’s counting job on Friday and Sunday, while she is out of town. I will have help from two of Joth’s helpers Friday, and Kaitlyn will come out with me Sunday.
Today is Day 2 of Lab HackWeek. Steven, Kaitlyn, and I cleaned up FTR 213.
My other tasks included:
- Creating a Lab Label that can be put on our equipment that we move to places like Manchester (made 1×1 and 2×2 inch: here)
- Researching options for searchable and public image sharing for lab images – Steven would prefer using something Google-related
Today I’ve been re-familiarizing myself with BLAST and Jupyter. I have done BLAST, sql-sharing, and grep-ing before using iPython (link to my old lab notebook with many iPython notebook links: here). However, it has been SO long, that I feel like I’m essentially learning everything for the first time. So perhaps “re-familiarizing” isn’t the best term.
To practice, I’m figuring out how to BLAST C. gigas against swiss prot.
Link to my practice Jupyter notebook: here.
Because it takes a lot of practice and repetition in order for me to remember and understand computer work, I had a hard time remembering how to change directories, make sure file names were correct, etc., since I haven’t really had the opportunity to do anything like this in nearly two years. I’m excited to continue to do more – there’s lots to learn! A lot.
Today, Laura was at SLU prepping for her SRM. So, Olivia, Kaitlyn, and I took over her Manchester oyster duties.
We began by doing the usual: screening the larvae.
Kaitlyn screened (224, 180, and 100 µm screens for live larvae, and 100 µm for mortality larvae).
Olivia brought Kaitlyn the buckets, cleaned them before they were re-stocked, rinsed the setting oysters outside, and sprayed the in-line filters with fresh water.
I sampled the larvae from the tri-pours, counted them, and restocked. If there were any 224 larvae, they were put in the setting tanks. The 180 and 100s were restocked into the larvae buckets. If within a sample group there were no 100 µm, then the 180 and 224 were stocked into the setting silos, and the large larvae buckets were taken down. That occurred for five bucket sets:
Below are images of the notebook I kept during the screening process:
Next, we fed her growth experiment. We grabbed 500 mL Chagra, and 500 mL Tiso. Kaitlyn counted the density and did the water exchange.
We increased the gigas’ set-point to 27 ˚.
Olivia vacuumed and checked for any mortalities. There were no mortalities.
Dana from PSRF fed the gigas.
(1). Extracting RNA from embedded Olys:
Looked through Laura’s histology slides and compared them to the extraction sites taken by Sam to determine whether gonad tissue was sampled or not. For the majority of the samples, I feel that I can confidently say that it was not gonad tissue. Samples were taken from the center of the tissues, while gonad tissue is located around the edges.
Below are the images of the histology blocks and the slides, with arrows indicating where gonad tissue can be found (determined through the use of a microscope) and the label indicates the oyster the sample was taken from.
The only samples that seem likely to have had gonad tissue sampled are NF-10-26 and SN-10-16.
(2) Laura’s geoduck tissue for SRM:
Added 50 ul “Final Solvent”
(1) Re-submitted the Heare et. al paper entitled, “Evidence of Ostrea lurida Carpenter, 1864 population structure in Puget Sound, WA” to Marine Ecology. After contacting the editor, we were able to get an extension on the resubmission deadline as we missed the original deadline of June 26th.
(2) Played around with Rhonda’s geoduck OA data and summarized the data and results:
Over the course of two weeks, the average sizes (microns) of geoduck larvae were recorded. The larvae were separated into six conicals. Three conicals were at pH 8.2, and three were at pH 7.1.
Figure 1. The larvae exposed to the lower pH (7.1) were smaller on average by about 90 microns at the end of the experiment on 5/28/17. (x-axis: date; y-axis: size in microns; key: conical numbers and the pH treatment value→ chart made by Rhonda – personally am unable to add chart features such as axes labels, title, etc.)
Figure 2. On average across all sampling dates, the geoduck exposed to pH 8.2 were larger by about 25 microns than those exposed to the lower pH (pH 8.2 average lengths – 230, 227, 228 microns; pH 7.1 average lengths – 198, 202, 201 microns).
Figure 3. The total mortality observed in each conical and treatment across all sample days. Total number of larvae sampled per treatment and conical = 350 larvae (50 larvae per day and 7 days of sampling).
Figure 4. Across the entire experiment, 350 larvae were sampled from each conical. To determine percent mortality: total number dead divided by 350 total larvae sampled times 100. pH 7.1 and 8.2 weren’t different for conicals 4,6,7,and 9. But conical 8 (pH 8.2) had more than double the mortality of conical 10 (pH 7.1)