Yaamini’s Notebook: Manchester Conditioning Update

It’s been a slow burn

As I expected, there were problems getting the heater to warm the water in the header tank fast enough to meet the desired temperature schedule I set. Laura mentioned that the temperature fluxuated during the day, so she was wary of increasing the temperature.

Table 1. Actual water temperatures read from AVTECH. Temperature listed was the peak temperature that day.

Day Date Temperature
6 6/16/17 17.06 ºC
7 6/17/17 17.31 ºC
8 6/18/17 18.56 ºC
9 6/19/17 18.50 ºC

The temperature is holding around 18.5 ºC, so I increased the temperature setting on the AVTECH two degrees. My goal is to get the temperature up to 20ºC. This will only set back my conditioning timeline two days!

Table 2. Desired temperatures for the remainder of conditioning.

Day Date Temperature
10 6/20/17 20 ºC
11 6/21/17 21 ºC
12 6/22/17 22 ºC
13 6/23/17 23 ºC
14-26 6/24/17-7/6/17 23 ºC

I’ll be back on Day 13 to check on things and Laura will continue to raise the set point and check the AVTECH.

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Laura’s Notebook: Manchester, 6/19

Screening day and more! Things went very smoothly today. Grace, Olivia and I make a good team.

Arrival inspection:

  • Banjos very dirt, a few pseudo-clogged. Due to heavy use of Tet algae.
  • HL-10 Ambient, which spawned, banjo overflowing but luckily lots was caught in an empty trio pour that was sitting adjacent to the catchment bucket

Screened larvae (Olivia, Grace, and me).

  • Mortality ranged dramatically between groups. Very high mortality in the following groups: – SN-6 Low 180um – lots of ciliates present – SN-10 Low 224um, 180um – lots of ciliates present – NF-10 Low 224um, 180um

imageimage

Collected new larvae, counted, stocked, and sampled. Grace did the majority of this process today.

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Cleaned gigas (Olivia)

  • Drained all three tanks, cleaned with vortex- LOTS of stringy stuff in the top tank w/o animals. I now am certain that the mucus is a result of the Reed’s paste, and not coming from the animals: file_009
  • Rinsed gigas, checked for morts – there were none.
  • Refilled tanks, then turned off flow and allowed header temp to increase.
  • Grace adjusted flow for 1.2 L/Min on each side.
  • Increased set point temp to 21degC.
  • Fed with 490mL Reed’s paste
  • Installed new pump (had been using small PSRF pump).

Cleaned Broodstock (Olivia):

  • First, recorded broodstock location on manifold
  • [image here]
  • Dumped all broodstock & larval catchment buckets, rinsed oysters, cleaned buckets with vortex.

Larval experiment water change:

Katherine recommends having 5 replicates.

  • [insert Katherine’s post here]
  • I heeded her advice, and thus split my 3 reps into 5 via the following:
    • Math!:
      • Assuming 800 larvae in each tripour, total # larvae per treatment = 800*3 = 2,400
      • Larvae per rep for 5-reps = 2,400/5 = 480
      • larvae to remove from each tripour = 800-480 = 320. Split these up into 2 new silos = 160 larvae per new silo per triplicate
    • How this was done:
      • Set 2 new silos in FSW. Label with designated treatment group.
      • Working 1 silo at a time, transfer larvae to a tripour, fill to 200mL
        • mL needed per new silo = 800 larvae/200 mL = 4 larvae/mL, 160 larvae / l/mL = 40 mL
      • Plunge 20 times, pour 40mL into 2 falcon tubes. Add the contents into the two silos.
      • Repeat with other 2 triplicates.
      • Result:
        • 2 new silos will have 160*3 larvae = 480
        • Original silos will have 800-(2*160) = 480
    • New setup: file_000 4
    • Changed water.
      • Filled a 5-gal bucket with 18L.
      • Harvested 450mL Tiso, CGW & counted density:

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  • Mimicking Katherine’s experiment, I will sample & image larvae on Thursday 6/22 (day 7), then also on 6/29 (day 14)
  • Reminder: I’m working with the 4 treatment groups in the South Sound (SN) population.

current summary data:

imageimageimageimage

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Laura’s Notebook: Manchester, 6/18

Today’s vista: file_007file_008

Water change on larval experiment

  • Harvested 450 mL Tiso + 450 mL Chagra
  • Counted algae. Here’s what they look like under the scope. I count 5 out of the 9 large squares in the hemocytometer grid (each large square contains 16 small squares). This first photo shows one large square, scope is at 10x: file_000

And here’s algae at a higher lens; I believe this is 30x, but will need to confirm: file_002

Counts: a44fec13-6ad3-4789-a768-781e5224e140a44fec13-6ad3-4789-a768-781e5224e140

  • Filled 5gal bucket with 13L FSW, added 690 mL algae cocktail, plunged and distributed among 12 clean tripours
  • Transferred larval silos to new medium

Changed banjos in SN & NF groups (hadn’t done that yesterday)

Imaged all larvae screened on 6/15. These were not killed with lugols, and were kept in the fridge. Changed up my imaging protocol: – Used the dissecting scope. I think the photos are much nicer: file_006file_005

 - Imaged a ruler (mm) at 12x and 32x ![file_003](http://ift.tt/2rykJ0h) ![file_004](http://ift.tt/2sRC3kS) - Imaged all wells at at 32x. - New protocol for imaging will be: - Don’t add lugols - Store in fridge - image 1-3 days post collection - Use dissecting scope, image only at 32x, image as many larvae as possible. Larvae collect in the center of the well, in a cluster, so should be able to capture most in one image. Fed - Setting/K&HL broodstock table: - 1/2 609 - 1/4 Tet - 1/4 Chagra - Larval table/SN&NF broodstock table: - 1/2 Tet - 1/2 Chagra Gigas: - Increased set point temp to 21degC. - Since temp is not achieving set point, I tried switching to the heated line, but the flow on the heated line was not sufficient enough to achieve 2.4L/min. Swapped back to the ambient line, and adjusted flow to achieve 2.4L/Min. - **When swapping lines on gigas, the high pressure on the ambient line blew the tube off the labcock valve, which sent a ton of ambient, very dirty water through and into one of my 100um larval buckets - NF-6 Ambient. I likely lost some larvae, and the bucket was extremely dirty. I screened this bucket through 200 onto 100um, trying to remove some gunk. I did, but not all of it. I returned the remnants back to the larval bucket. Luckily, the flow had been pushing live larvae into the 180um bucket since yesterday afternoon, and there were very few 100um larvae in this bucket to start with, only ~2,700: - Screening counts: ![2f62fbc7-99c4-4969-9e55-59741ad20f3d](http://ift.tt/2ryoRxk) - And none stocked since 6/15:  

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Laura’s Notebook: Manchester, 6/16 & 6/17

6/16

  • Counted rest of larvae from 6/15 screening
  • Imaged all new larvae from 6/12, 6/14 & 6/15
  • Prepared FSW+algae for larval experiment water change:
    • Filled Harvested 400mL Tiso & 400mL Chagra. I want 100k cells algae/mL in my larval experiment tripours, which are filled to 800mL. To determine algae concentration, I plunged and sampled 20ul and injected into hemocytometer.
    • Counted 5 of the 1mm^2 squares. Here are my calcs from the past few days:

6/17

Arrived, collected new larvae, of which there were lots:

image

Noticed that the algae dosing pump @ the small table (setting tank/K & HL broodstock) was not turned on. They were without food since X, so for ~X hrs. I fed each setting silo 200mL of the algae cocktail in the algae header.

Prepared FSW+algae for larval experiment water change:

  • I used a new water pre system, based on Katherine’s protocol from last summer:
  • Filled a bucket with 13L FSW (taken from my small table manifold, with the algae pump turned off).
  • Harvested 400mL Tiso & 400mL Chagra. I want 100k cells algae/mL in my larval experiment tripours, which are filled to 800mL. To determine algae concentration, I plunged and sampled 20ul and injected into hemocytometer.
  • Counted 5 of the 1mm^2 squares. Here are my calcs from the past few days: image
  • Mixed 630mL algae cocktail into 13L FSW, plunged, and filled 12 tripours to 800mL.
  • Transferred larval silos into fresh water.

Larval bucket maintenance:

  • Set up 2-bucket challenge by connecting the 100um and 180um within the SN & NF groups, and added a second bucket for the HL & K groups. This ends up taking ~1-1.5hrs; should determine how I can streamline this task.

Cleaned outside upwelling tank: turned of inflow, drained, filled with fresh water & added 400mL Vortex. Left pump on. Will leave it overnight to clean.

Gigas maintenance:

  • Vacuumed as best I could. Noticed that there was lots of mucus in the east tank.
  • Moved temperature probe & Durafet 3 into the east tank.
  • Increased immersion heater set point to 20degC..
  • Filled algae header with 485mL Reeds + fresh water.

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Laura’s Notebook: Manchester, 6/15

Screening day. Some hiccups, but nothing devastating. Grace screening, Olivia working buckets/setup, me sampling/counting. Initial visual inspection of larval buckets look like larvae aren’t doing as well as they were last week. But, we’ll see!

Data: imageimage

Snafoo: the SN-10 Ambient group was mislabeled as SN-10 Low, and before realizing I stocked the 224um larvae (of which there were ~30k!) in the SN-10 Low setting tank. This error was caught moments later, and labeling/data entries were corrected. Unfortunately, the SN-10 Low setting tank, which had been stocked with ~4,300 larvae since 6/5 and which I know there were still swimmers, is now mixed with ~30k SN-10 Ambient larvae. I started a new SN-10 Low setting silo. I kept the mixed up batch, labeled as such. Most of the animals in this group are from SN-10 Ambient.

TBD ….. more info…

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Laura’s Notebook: Manchester, 6/13 & 6/14

6/13

  • Temperature in system has fallen consistently over the past couple days- it was ~16degC. Noticed that the immersion heater I borrowed from Jon was reading 17degC, but was not heating. I swapped in the spare heater that we had purchased, and set it to 19degC. Will observe over the next couple days.
  • Checked on temp of upwelling tank outside: 17degC. This is sufficient- I will not install a 2nd heater.
  • Purchased pvc part and installed in a Calwell tank, which I will use tomorrow to feed the upwelling tank- hopefully I have some setters!
  • New larvae present in: SN-6 low B (some), HL-6 low (a tiny bit). Did not collect today, will do so tomorrow.
  • Connected the 100um and 180um buckets and adjusted flow/banjos for the 2-Bucket Challenge. Built new double-buckets for new larval groups.
    • Notes on bucket configuration:
    • I separate SN & NF groups into 2 buckets, 100um and 180um, after screening, but do not separate the K & HL groups (limited number of banjos). Mortality rate in the >180um group is considerably lower than in 100um; by separating the groups I minimize risk to the 180um, AND I maintain capacity in the 100um buckets to stock new larvae.
    • 2 days prior to screening I connect the 2 buckets in SN & NF groups, setting flow in the 1st (100um) bucket, where live 100um larvae will flow into the 180um bucket. For K & HL groups, I install a 2nd, bucket, pushing live larvae from old bucket to new bucket.
    • I screen on Mondays & Thursdays (usually); buckets are therefore connected in their 2-Bucket Challenge configuration on Tuesdays & Saturdays. The size groups are thus separated for 1 or 2 days.
  • Gigas:
    • Increased temp by 1degC – 17degC
    • Removed bags, sprayed with fresh water, inspected for morts – no morts observed
    • Tried vacuuming tanks, but instead drained completely with siphon, as water had become very dirty when removing bags.

6/14

  • Arrival inspection:
    • All larval banjos very dirty. A couple buckets nearly overflowing. Changed banjos immediately.
  • Screened and cleaned setting tanks and checked for set larvae:
    • Setters visibly inspected by first screening on to 450um, and viewing under microscope. Not very many, so I also sampled from the rest of the contents that did not hold on the 450um. Saw some newly set oysters, as well as lots of larvae that had yet to go through metamorphosis.
    • Cleaned setters/culch/larvae in freshwater bath
    • Oyster set observed in:
      • NF-6 Ambient – lots of swimmers too
      • SN-10 Low – lots of swimmers
      • NF-10 Low – very few swimmers file_000
      • NF-10 Ambient – very few swimmers, here’s a photo of an oyster set: file_000 1
      • HL-10 Ambient – a couple set
      • K-6 Low – no larvae visible in the sampled contents
      • K6 Ambient – just 1 setter, lots eyed larvae
      • K-10 Low – lots of morts, 1 larvae set visible
      • Here’s a video of the SN-10 Ambient group, in which I didn’t see any set oysters, but there were lots of swimmers: https://youtu.be/SomPKWeWvfc
    • Not enough setters to warrant using the outside upwelling tank. Returned all larvae/set to the 180um silos in the downweling tanks.
  • Collected new larvae: image
  • Changed filters
  • Rinsed larval catchment buckets
  • Increased Gigas temp to 18degC. Heater does not appear to be keeping up with the flow rate. Will continue to increase temp by 1 degC/day, as to not shock them.

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Laura’s Notebook: Manchester, 6/12

Screening day- Arrived at 8:30am, geared up to screen through the larvae. Grace worked the screening table, Olivia managed the buckets and whatnot, I counted and re-stocked larvae, and Katie took video and images. Finished at ~2:00pm. Things went very smoothly and we got a good routine down. Here are snap-shots of the data:

imageimage

After restocking, all buckets were fed 700mL algae cocktail of: 1/4 Tet, 1/4 609, 1/2 Chagra.

Images: using my phone & the new cell phone adapter, Katie imaged and observed activity. Images were taken using my phone. Here are her notes:

img_0565img_0566img_0567img_0568

I fixed all screened larvae with Lugols and stored in fridge for size imaging (under the scope with the scale) at a later date.

Collected new larvae, counted, sampled, and stocked:

  • HL-6 Ambient
  • HL-10 Low
  • K-6 Ambient
  • SN-6 Low B
  • NF-6 Low A

Other activities:

  • Arrival inspection:
    • All buckets very clear, likely have been without food for several hours.
    • It appears the dripper was left out of NF-10 Ambient B since Saturday. Air stone was still in there; no apparent mortalities.
    • Aeration was not sufficiently high on the west side of the larval table (mostly on K & HL groups).
    • Temperature in the whole system has dropped to ~16degC, possibly due to it being pretty cold outside…?
  • Rinsed all larval catchment buckets
  • Checked on the upwelling tank outside; it is holding at 17degC
  • Gigas:
    • Grace & Olivia cleaned, inspected for morts (none)
    • Grace measured and adjusted flow rate to set a 1.2L/min per side
    • I increased temp 1degC; temp was reading 15degC, so I set heater to 16degC
    • Installed aquarium pump in header tank, fed with 490mL Reeds Shellfish Diet, and set dosing pump to 57

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