Yaamini’s Notebook: Manchester Conditioning Update 7

The power is back!

Since Laura is out of town for a wedding, I took Kaitlyn out to Manchester today to help me take care of her oysters. My goal for today was to move my oysters to the bottom shelf since Laura no longer has any broodstock buckets down there.

The first thing we did was change out banjos in her larval buckets. Kaitlyn saw that the banjo in bucket HL6 AMB 180, so it was brimming when we got there. Steven then helped us switch all of the water lines back to the heated ones, as the power was back. The filters didn’t look too dirty and since replacing the one micron filter wasn’t a high priority, I left the filters untouched. While he was doing that, I bleached the algal lines and drained and cleaned the oyster and heating tanks. I found one dead oyster in Tank 4A.

Table 1. Revised oyster counts in each tank.

Tag Label A B Total
1 7 9 16
2 8 8 16
3 6 6 12
4 6 7 13
5 9 8 17
6 7 8 15
Heat Shock 5 6 11
Full Amb 7 8 15
Spare 2 3 5
Total 57 63 126

I had Kailtyn rinse the downwelled silos outside while Steven and I reconfigured the C. gigas set-up. We moved the heating tank to the bottom shelf and attached a longer PVC pipe to one end of the T-junction. Water was then able to flow into two different kiddie pools. Because the pipes are two different lengths and the water is flowing by gravity, adjusting the flow will be a bit more difficult than normal. I increased the setpoints on both heaters to get water up to temperature as well.

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Figures 1-2. Heater setpoints.

Since the bottom shelf was plumbed with Laura’s 19 ºC water, we also added in a tube that flowed 19 ºC water into my heating tank. This would help maintain water temperature. However, this means flow from the heated 14 ºC line needed to be decreased, which decreased the amount of food the oysters received.To compensate for the lower feeding from low flow, I increased the dosing rate to 50%. I then fed the oysters with 100 L of Tetraselmis and 250 mL of Reed’s paste. This isn’t a lot of food for them, so I need to ensure they get fed more when I get out there. I added two tripours of food directly to the heating tank and a tripour into each kiddie pool since the oysters didn’t have food while being cleaned and moved.

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Figures 3-4. Kiddie pool arrangement.

I then took care of Laura’s oysters. Kaitlyn and I filled her inside two algae headers and outside algae header with a 50/50 mix of C.iso and CGW. I used these same algae strains when feeding and doing a water change with her growth experiment.

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Yaamini’s Notebook: Manchester Conditioning Update 6

Power outage edition

But first, let me walk you through what I did before the power went out.

On Monday, Laura vacuumed my oysters and increased the temperature. She also fed them 1.5 L of Reed’s paste. On Wednesday, she saw that the left heater was on, but wasn’t heating. She unplugged it and plugged it back in. She also increased the setpoint to bring the water back to the temperature it was at, around 21 ºC. Temperature spiked around 22 ºC, then leveled back down to 21 ºC. Because we were running out of Reed’s paste, Laura fed 225 L of Tetraselmis and 1 L of Reed’s paste. She also moved Durafet 1 into Tank A, supplementing the Durafet already in Tank B.

Today, I drained and rinsed the oysters and oyster tanks. I found 3 mortalities: 1 from Tank 2A and 2 from Tank 4A. It’s a bit weird that most of my mortalities have been from Tank A…

Table 1. Revised oyster counts in each tank.

Tag Label A B Total
1 7 9 16
2 8 8 16
3 6 6 12
4 7 7 14
5 9 8 17
6 7 8 15
Heat Shock 5 6 11
Full Amb 7 8 15
Spare 2 3 5
Total 58 63 126

When cleaning the heating tank, I noticed that the Reed’s paste was much clumpier and globbier that it normally is. I think mixing it with Tetraselmis is changing the clumping pattern. When I fed the oysters today, I grabbed about 600 mL of Reed’s paste from the little we had left and mixed it thoroughly with about five gallons of water. I did this to dilute the Reed’s a bit more before mixing it with Tetraselmis. I then added in 100 L of Tetraselmis and topped off the algae header tank with freshwater.

The setpoints I left the heaters at got the water temperature to consistently fluctuate between 20ºC and 21 ºC.

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Figures 1-2. Heater setpoints upon arrival.

I increased the heater setpoints, as well as increased airflow to all tanks.

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Figures 3-4. Changed heater setpoints.

When I looked at the Durafet monitor, I noticed that the two probes were reading different pH values. Both tanks receieve the same water, so unless one tank is really respiring a lot more than another, this doesn’t make sense. I placed both probes in the same tank and saw that they were registering different pHs in the same tank.

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Figure 5. Durafet probe readings for water in Tank A.

I measured the flow rate at about 100 mL in 15 seconds, or 400 mL/min for each tank, which is pretty low. Before I had any time to think about it, the power went out! Apparently, a pipe burst and flooded the main power conduit. This meant that our power, even backup power, to the hatchery was gone. Our heated line was out and there was no water flowing to our tanks. We had to quickly ensure we could get water to our animals.

The first thing we did was switch our water over to the ambient lines. We temporarily unplugged our water heaters to plug in the mixing valve and allow the cold water to flow through. We also removed the one micron filter because it would get clogged too fast. Using an extension cord, we plugged in all of the heaters and probes so we can have heated water for the C. gigas and remote monitoring. We plugged in our dosing pumps and Laura’s water heaters into a power box as well.

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Figure 6. Temporary set-up for dosing pump power.

Because the ambient line is colder than the 14 ºC heated line, I ramped up my heater setpoints, hoping to maintain the water temperature anywhere between 20 ºC and 22 ºC.

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Figures 7-8. Heating setpoints after power outage.

Before leaving, I checked the Durafet monitor to see how the pH and temperature were holding. The temperature was reading at 22 ºC, so I think we’re going to be okay!

img_7956

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Yaamini’s Notebook: SRM Assay Day 9

My samples are DONE.

The samples I needed to rerun to get data for (O01, O12, O22, O113, O118, O26 and O90) just finished running and all of them have data. Tomorrow, I will go to UWPR to prepare the oyster sample dilutions needed to create a dilution curve. The purpose of this is to verify that the assay is detecting peptides based on their presence in the autosampler vial. The oyster peptide detection should decrease as the concentration in the autosampler vial decreases, and vice versa.

Emma suggested pooling oyster samples to create my dilutions. I want to randomly pick oyster samples that I didn’t have to remake and rerun. I also don’t want to include my procedural blanks. Here are the samples I can choose from:

  • O17
  • O145
  • O128
  • O56
  • O71
  • O103
  • O04
  • O106
  • O49
  • O52
  • O14
  • O102
  • O10
  • O06
  • O122
  • O99
  • O08
  • O51
  • O21
  • O39
  • O137
  • O46
  • O43
  • O31
  • O66
  • O30
  • O100
  • O78
  • O35
  • O101
  • O40
  • O64
  • O91
  • O140
  • O124
  • O32
  • O147
  • O121
  • O24
  • O96
  • O131
  • O60

We want ten dilutions total. I first need to calculate how much of the pooled oyster protein sample I’ll need in each vial, as well as PRTC.

Table 1. Volumes of solutions needed to create peptide dilutions.

Vial Oyster Sample Volume (µL) Volume PRTC (µL) Remaining Volume (µL)
1 7.5 1.89 5.61
2 6.7 1.89 6.41
3 5.9 1.89 7.21
4 5.1 1.89 8.01
5 4.3 1.89 8.81
6 3.5 1.89 9.61
7 2.7 1.89 10.41
8 1.9 1.89 11.21
9 1.1 1.89 12.01
10 0 1.89 13.11

This means I need 38.7 µL of oyster peptide sample to start with. If I used five samples, I would need 8 µL of peptide from each sample.

Randomly choosing five samples:

  • O124
  • O39
  • O96
  • O14
  • O137

I will prepare my samples accordingly tomorrow!

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Yaamini’s Notebook: SRM Transition Protein Analysis

If you don’t have a visualization, did you really analyze data?

Now that I have some downtime before I can analyze my SRM data, I want to visualize my SRM transitions. I figured the easiest way to do this would be to focus on the proteins I chose. The past few times I made NMDS plots, heatmaps and REVIGO plots, so I thought I would do the same this time. I used code in this R script.

NMDS plot

NMDS

I ended up with all but one site and eelgrass condition nested on top of eachother. Not entire sure waht this means. I’ll need to consult Emma to ensure that I did this correctly.

Heatmap

heatmap

My heatmap is a much cleaner way to visualize the proteins I selected for my final transition list, and how the expression differs between sites.

REVIGO

After I exported a list of proteins, GOterms and p-values, I sorted through the list and picked the lowest p-value for each protein (all 20 replicates were listed) and only kept the first GOterm listed in the original “goterm” column. The resultant file can be found here.

When I put the GOterms and p-values into REVIGO, I only had 2 biological process GOterms:

biological process

The rest were for molecular functions:

molecular functions

While interesting, I’d rather have all of my GOterms be relevant to a biological process. I need to find an efficient way to sort through my list of GOterms and see if they are for biological processes. I posted this Github issue to figure it out.

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Grace’s Notebook: July 17, 2017 – Manchester

Today, Laura was at SLU prepping for her SRM. So, Olivia, Kaitlyn, and I took over her Manchester oyster duties.

For Laura:

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:

NF6 Low

SN10 Low

HL6 Amb

SN6 Amb

K10 Amb

Below are images of the notebook I kept during the screening process:
IMG_3892

IMG_3893

Next, we fed her growth experiment. We grabbed 500 mL Chagra, and 500 mL Tiso. Kaitlyn counted the density and did the water exchange.

For Yaamini:

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.

 

 

Yaamini’s Notebook: SRM Assay Day 8

Last day of my samples!

Before I went to Taylor Shellfish in Quilcene today, I checked the mass spectrometer progress around 5 a.m. I noticed that bivalve files 109 (O90) and 114 (O26) had no data, and QC25 still looked weird (similar to QC23 and QC24). I noted this down so I could prepare new samples when I got back from the hatchery. My suspicion is that I changed the vial position for QC25 to Plate 1, A3 after I queued the sample, so the change wasn’t registered.

When I got back from the hatchery, I checked the samples that ran. QC26 and QC27 looked normal, so I know that my QC is running well. I prepared more O26 and O90 and added it to the sample vials. I then added these samples to the bottom of the queue. I added 30 µL of the final acetonitrile solution to a new QC tube, then put that in the vial in Plate 1, A3. This will give us enough QC until QC42.

I stayed at UWPR to give Emma and Laura that mass spectrometer key, and to check that no other sample needed to be reprepared. There were none! My samples will finish early Tuesday morning.

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