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
Immersion heaters are pesky
Overall, today was pretty smooth. Olivia and Grace helped me screen my larvae. At first, I thought I would be able to screen on both an 80 micron and 60 micron screen. However, after screening the first two buckets, Bucket 11 and 6, I didn’t count any larvae from the 80 micron screen. I decided to continue just screening on a 60 micron screen, and try again with two screens on Wednesday. I did something different today where I added food to the bucket before restocking, instead of adding food to all buckets at the end. I think this method is better because the larvae get fresh algae after being stressed by the screening process, so I’ll continue to do this. While screening Bucket 12, Grace accidentally screened through a 48 micron screen first. She caught her mistake very quickly, but it’s possible some larvae were lost with this additional screen. All of my larvae are at about 2 larvae/mL, which is the stocking density I want at this point. You can see my counts and feeding log here.
Now here’s the annoying part. Totes 3, 4, 5, 7 and 9 all had their high alarms (27ºC) go off after the buckets were restocked. Tote 4 and 7 had their alarms go off multiple times! To reduce temperature, we used tripours to drain out some of the water in the tote, and then we refilled with ambient freshwater instead of using the water from the heating reservoir line. This quickly brought our temperatures down. I think refilling totes with ambient freshwater should be our standard now. Adding heated water to a tote that’s already at temperature may set it over the edge.
- Record tote positions
- Add new HOBO loggers buckets without them
- Feed oysters
- Finalize help schedule for this weekend
- Prepare task lists for those helping
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Anoxia is no bueno
My main goal today was to flush out any anoxic water from the heated line. When I got to the hatchery, I started draining the header tank and opened the heated line to flush any water out. I let this run while I prepared tripours for tomorrow’s screening and fed Joth’s scallops. After 30 minutes, I shut the heated line off and replaced the 1 micron filter. I noticed that the filter casing didn’t have the black rubber ring around it anymore, but water wasn’t leaking from it. I rinsed the casing and put a new filter in, then turned the heated line back on. I let the line run for 2 minutes while I took a freshwater hose and cleaned the inside of the header tank. After a few more minutes of letting the tank drain, I closed the bottom valve and started filling it.
While the tank was filling, I fed my larvae. I noticed that the buckes were relatively clear, and most of the algae had settled to the bottom.
Figure 1. Tank color upon arrival.
I figured I would increase the algal density to about 65,000 cells per mL and check the color of the buckets tomorrow morning before screening. I don’t want the larvae to go without food dispersed throughout the water for more than an hour, so maybe increasing the amount of food will help maintain the algae level I want. It’s also possible they’ve started to eat more as they’re growing.
After feeding my larvae, the header tank was about halfway filled. I plugged all three heaters back in, kept the heated line open so the tank could finish filling, and rushed out the door to catch the 11 a.m. ferry back. In my haste, there were a few small screening preparation tasks I forgot to do, so they’re at the top of my list tomorrow!
- Record which buckets are in which totes (I need this before I rerandomize my buckets!!)
- Turn off cold water input
- Label tubes for larval samples
- Measure out amount to feed ahead of screening, so larvae can be fed while being restocked
- Screen larvae on 80 and 60 micron screens
- Count larvae and drop stocking density to approximately 2 larvae/mL
- Based on my current count data, Buckets 3, 9, 10 and 11 are the four that are above this stocking density
- Drain heating header and unplug heaters
- Clean and/or replace filters
- Return Rick’s silver heater
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Smooth Saturday screening
Today I screened and counted larvae using a 60 micron screen. I’m getting much faster at it! Shoutout to Kaitlyn for coming and helping me screen.
Figure 1. Kaitlyn screening larvae
While counting, I took this picture of a dead larvae and alive larvae side-by-side to use as a reference if I was ever unsure if something was dead. Using the compound scope and Sedgewick Rafter cell, it’s much easier to focus on the larvae see if a shell is completley empty.
Figure 2. Dead larvae (left) and alive larvae (right).
Totes 2, 6 and 8 all had their immersion heater alarms go off because they reached 27ºC. The immersion heater is generally about 2ºC warmer than the bucket, so this means the larvae were at 25ºC. That temperature is the maximum they should be kept at in the larval period. I took out the buckets in those totes and we screened them immediately. We waited for the temperature alarm to go off before putting the buckets back in, which was about 15 minutes. I think the alarm goes off when there’s only one bucket in the big gray totes. Because the water level drops when we take the buckets out and there’s less water for the heater to heat, the temperature may rise quickly. For future screening days, I think it’s a good rule of thumb to ensure there are at least two filled buckets in each gray tote at at time.
After Kaitlyn helped me screen, I had her count the number of algae cells in a mix of C.iso, 609 and Chagra in a 2:1:1 ratio. We then fed each bucket 250 mL C.iso, 150 mL 609 and 150 mL Chagra. I fed the larvae a more 609 and Chagra than I measured because I didn’t want to use a tripour to measure out 125 mL, and to give the larvae a little extra food to compensate for the stress of screening. I then filled all buckets to 4 gallons and drained the header tank.
- Feed Joth’s scallops
- Feed larvae
- Change 1 micron filter
- Flush heated line
- Drain and fill header
- Heat new water
- Prepare tripours and screening table
Here’s a copepod I found while counting larvae, just for fun:
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Lots of maintenance today
Aside from daily feeding, I don’t have much urgent work on days I don’t screen and count. I used my off day to get my system fully set up and catch up on counting larvae from my first screening.
What I did today:
- Recorded which tote each tank was in
- Wireless monitoring
- I have four AVTECHs available for my larvae: Temp 1, Temp 4, WISH 9 Ext 1 and WISH 9 Ext 2. Temp 3 is for my heating header tank
- Changed names on OA Monitor Dashboard on GoToMyDevices and placed in totes
- Temp 1 = Gigas-Larvae-1 = Tote 5
- Temp 2 = Gigas-Larvae-2 = Tote 1
- WISH 9 Ext 1 = Gigas-Larvae-3 = Tote 7
- WISH 9 Ext 2 = Gigas-Larvae-4 = Tote 9
- Temp 3 = Gigas-Larvae-Header
- Labelled AVTECHs with tape so I knew which one was which
- Set up alerts if temperature gets above 27ºC or below 22ºC
- Decided against relabelling HOBOs because I didn’t want the Sharpie to leech into the water. This is something I need to think of an alternative for
- Set high (27ºC) and low (24ºC) temperature alarms on immersion heaters
- Flushed header and heated line
- Turned off all heaters
- Removed and cleaned Rick’s heater
- Added a third heater to replace Rick’s
- Drained header tank and flushed heated line
- Refilled header tank
- Heated water
- Fed Joth’s scallops
- Cleaned tripours for Saturday screening
- Replaced PSRF’s blue screens with green screens
- Counted Plate 2 and 3
- While I was counting Bucket 10 (Plate 2, Wells C1-C3) I found there dark blobs that were bigger than the larvae. I sent images to Rhonda and she didn’t know what it was. I couldn’t zoom in and make the image clear enough to take a photo, but here are the blurry images I took. I found 30 in Well C1, 38 in C2 and 18 in C3.
Figures 1-3. Big blobs found in Bucket 10
- Because of this weird contamination and the fact that I incorrectly counted Plate 1, I these data may end up being an outlier.
Fed larvae a mix of Chagra, C.iso and 609. Again, the algae was really dense!
- I fed 15 and 16 extra 100 mL of 609 and 100 mL Chagra by accident
- Feed Joth’s csallops
- Screen and count on 60 micron screen
- Feed larvae
- Drain header tank
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My larvae are 60 microns now!
Today, Laura helped me screen my larvae through 60 micron and 48 micron screens. You can see the data for yourself here, but the majority of my larvae held on a 60 micron screen! Since there was really nothing in the 48 micron screens, I tossed out those samples. While I probably threw some slower-growing larvae down the drain, I also got rid of dead larvae that could possibly disrupt the healthier ones. Laura screened while I counted right away. A few things I learned:
- It’s much easier to count my larvae on a Sedgewick Rafter cell than in a well-plate
- Yesterday, I was classifying larvae as dead if they didn’t show any signs of movement (both through the water column and their innards). This is incorrect! Larvae area dead when the shells are empty. I don’t remember seeing any larvae like that yesterday, but I think it’s safe to say that those data points are not high-quality. I shouldn’t use those counts in any analysis.
- What I thought was a shell deformation was actually the larvae’s…butt?? It’s just a normal D-hinge larvae from a different angle! I realized this when I saw one of those weirdly-angled larvae do a few spings and flips, then start swimming in a way that was recognizable as a D-hinge
I also preserved larvae in the freezer and fed a mix of C.iso and Chagra since those were the most dense. It’s likely that I undercounted algal cells on the hemocytometer since there were so many! I was a little short on time so I also only counted three hemocytometer cells.
Another thing we noticecd when filling buckets is that the water in the heating reservoir had been sitting so long that the 1 micron filter was showing signs of being anoxic. We flushed the line and replenished the header a bit. Laura talked to Stuart, and they suggest draining the header tank tomorrow and flushing fresh seawater through that line so that it sits overnight. I would also have to make sure my water is heating overnight.
- Figure out which AVTECH is which
- Add to larval tanks and totes strategically
- Relabel on Dashboard/OA Monitor
- Relabel HOBO probe tags
- Finish counting and imaging Plates 2 and 3
- Flush header and lines
- Replenish with new seawater
- Start heating process
- Feed Joth’s scallops
- Feed oyster larvae
- Prepare tripours for Saturday screening
- Clean up Sealab
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Counting larvae takes a while
Before I get into that, a quick rundown of other things I did today.
- Recorded probe numbers in each larval bucket
- 1: SN6 Amb pH A
- 2: SN6 Low pH B
- 3: SN6 Amb B
- 5: NF6 Amb B
- 6: NF10 Low pH A
- 9: SN10 Low pH A
- 10: SN10 Amb A
- 11: SN6 Low pH A
- 12: NF6 Amb A
- 12: NF10 Amb B
- 15: NF6 Low pH B
- 17: NF10 Low pH B
- 19: SN10 Low B
- 21: NF6 Low pH A
- 23: NF10 Amb A
- Recorded which bucket was in which tote
Figure 1. Tote layout. Totes go from 1 to 9, left to right.
- Did not record morning temperatures
- HOBOs already in tanks, no need
- Measured morning food presence
- Very dense! I think this is because I fed in the afternoon on Tuesday, so the larvae didn’t have a full 24 hours to really feed down
Figure 2. Color of larval tanks in the morning, before daily feeding.
- Steven rigged up the AVTECH in the SeaLab, so I now have 5 probes to work with
- He also started messing around with the OA system set-up
- Moved immersion heater probes to inside larval bucket, instead of in tote
- Counted heaters
- 8 new from Roberts Lab, 1 not being used right now
- 2 old heaters (one I ordered, one I took from the lab with the Farenheight controller)
- Gray heater borrowed from rick
- Fed larvae
Now to the main event: imaging and counting! I counted all of the larvae in Plate 1 collected yesterday. It took me much longer than I expected, mainly because I was trying to figure out how to work the dissecting scope while also taking pictures for future use. My count data can be found here.
There were lots of larvae that exhibited a normal D-hinge shape, but there were also some that looked like peanuts! I recorded how many of these deformed ones I saw as well.
Figures 3-4. Normal D-hinge larvae shape at 40x (top) and 20x (bottom) magnification.
Figure 5. Weird larvae shape.
I’m storing all of my larvae photos and videos from today here. I’m cataloging each day in a different folder, and labelling which bucket the larvae are from in the image name.
- Screen with 60 and 48 micron screens
- Sample and count
- Save samples in -80ºC freezer
- Finish imaging Plate 2 and 3
Since you made it through this lab post, here are some easy links to larvae videos. Both are from bucket 8:
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