Another week in review…6/29-7/2

So track record for daily posts aren’t great, but I’m working on it. This was a pretty eventful week in the hatchery- setting up a system for settlement, some unfortunate mortality, and getting locked out of the molecular lab (now I know to keep a stash of ethanol/RNALater/tips in the hatchery!).

Monday 6-29-15

Since I cleaned out the 100L larval tanks on Sunday, on Mon-Wed-Fri  I’m now only flushing the line with bleach, cleaning all the drippers, cleaning all the banjo filters, collecting any spawned larvae, and cleaning the broodstock/larvae catch buckets. On Tues-Thurs-Sat/Sun I’ll filter out the larvae from the 100L tanks and clean those, as well as the daily banjo filter and dripper cleaning. I’ll also check for newly spawned larvae on those days and filter those out to count and add to a 100L tank.

Checking for new larvae:

  • Some: NF1(?), HC3
  • Lots: SSS1, HC5, SS2, SS3, HC4, SS4, HC2
  • Got about 222,000 larvae from the SS buckets to add to SS_Tank2 and 230,000 from the HC buckets to add to HC_Tank2
  • None from NF

Had time to filter out what was spawned in the SS group bucket during cleaning but not the HC bucket, although there was no noticeable larvae in that one.

There’s been an issue with the algae cultures since Friday 6/26, where there hasn’t been any diatoms to feed the animals. The recommended diet is 50% diatoms and 50% flagellates, so the larvae might have a deficiency of necessary amino acids.

Tuesday 6-30-15

Checking for larvae:

  • Some/Maybe: HC3, NF1(?), NF2(?), SS1(?), NF5(?)
  • Lots: HC4
  • none in SS1 or NF families, just poop

Today I decided to try out doing “weight counts” as well as my usual counts for the 100 L larval tanks. With this method, you filter out a 100 L tank over a few different sized mesh screens. The larvae of each size class is put in a small (approx. 15cm diameter, 10 cm height) pvc silo with a 100 micron mesh screen at the bottom, where the weight of each silo when empty is known. You weigh the silo+larvae, subtract the weight of the silo, and use a conversion sheet to get the estimated number of larvae based on their size. I’ll add a picture of the conversion sheet later, but you can see my data from the counts on the 4th sheet of my Google doc.

Larval Counts Data Sheet

I screened each of my 5 tanks over 200, 160, and 100 screens. Since I’m not weighing every size class possible, I calculated a range for # of larvae- assuming that my 160 sample had larvae sized between 160 and 180 and my 100 sample had larvae between 100 and 150.

After counting, I threw out larvae from SS_Tank1 that was left on the 100 micron screen as these are mostly dead or sick.

When doing drop counts, I accidentally knocked over a tray with SS_Tank2 160, 100 and NF_Tank1 100 on it. I had already put the larvae back in their tanks, so except for weight counts I don’t have data for those.

Comparing the weight counts to drop counts, the weight counts usually overestimate compared to drop counts- particularly if there are less than 10,000 larvae.

Some Chagra (a diatom) is now available.

Wednesday 7-1-15

Steven Roberts came in to help today. I did all of the filtering of larval catch buckets, and he did all of the counts, helped with bucket cleaning, and helped me set up a growth rate experiment.

Checking for larvae:

  • Some: NF3, NF5, SS3, HC1,HC5
  • Lots: SS1,NF2,SS4
  • Filtered out all buckets
  • These were counted almost immediately after filtering out, but still saw a higher proportion of dead larvae in buckets than usual.

Growth rate experiment

As multiple families in each group spawned today, I started an experiment to look at differences in growth rate. Silos with 100 micron screens are put in beakers filled with seawater and algae. I have 3 replicate silo/beakers for each population, with ~900 larvae added inside of the silo.

Growth Rate Experiment

Cheesin’ hard next to the growth rate experiment

This is referred to as a “static system”, as water is not flowing through it. Every day I have to rinse off the silo and transfer it to a clean beaker filled with seawater/algae. I’m taking samples of the water that goes into the beakers and samples after the larvae have been in there for a day to get an idea of feeding rate. To calculate the algal cell density in these samples, I put 10 mL into a 15 mL centrifuge tube and spin them down. I then take out the seawater and add some back so that they are all at a volume of .5mL. With this concentrated sample, I can use a hemocytometer to count algal cells and scale up to estimate the density in my original samples.

Notes: algae is 50% Tiso, 50% Chagra

Hemocytometer

Hemocytometer

Thursday 7-2-15

On Wednesday night I bought a 24″ x 40″ sheet of PVC to make into 4″ x4″ tiles for oysters to settle on, as they were close to holding on a 224 micron screen on Tuesday (meaning they were almost large enough to be at the metamorphosis stage). However, when I got to the hatchery on Thursday there was no one around to show me how to cut it up. I noticed some larvae were piled up on the bottom of the SS_Tank1 and HC_Tank1. I thought this might be due to them being ready to settle. With the tiles not ready, I rigged a settlement system using large silos and cultch (ground up bits of shell around 450 microns in size). This system is static, like the growth rate experiment, so I have to change the water every other day and add in algae every day. I added 6,000 SS_Tank1 larvae to each of two silos.

I filtered out all of the 100L larval tanks over 224, 160, and 100 screens except for NF_Tank1 where I just did 160 and 100 (ran out of time). Based on the age of the HC_Tank1 and SS_Tank1 tanks, there shouldn’t be any screening below 160, but a significant number were in both cases. The larvae at all sizes did not move very much, even without ethanol added to the well. I took 20,000 of the SS_Tank1 larvae that were filtered on the 224 screen and added them to my settlement system. Not knowing what to do with the rest, I added them back into the SS_Tank1 tank. I threw out the 100 micron sized SS_Tank1 and HC_Tank1 larvae though.

Growth Experiment

Notes:

  • salinity: 29 ppt
  • temp: 20C
  • Food: Tiso, 609

Larval Count Sheet

Monday 7-6-15

Today I talked to Ryan, the hatchery manager, a little bit about the possible causes of the mortality I saw over the weekend. In his experience, mortality events are not uncommon when rearing larvae in the summer. He thinks it may have to do with water quality issues (which I can’t really do anything about). He recommended I be more vigilant about culling dead or sick larvae from the tanks.

As I emptied out HC_Tank1 and SS_Tank1 over the weekend to be cleaned for new larvae, I adopted a new labelling scheme on my data sheet. The first attempts in a tank have the letter “a” (i.e. HC_Tank1a, SS_Tank1b) and after totally culling or emptying out a tank of larvae I change the letter in the tank name. I’ll see how this scheme works when it comes to actual data analysis, but for now it’s helping with data entry and keeping track of how many are in a tank at a time.

SS_Tank2a smelled disgusting and had sheets of larvae on the bottom so was also emptied out entirely. For my NF and HC larvae, I made a “sick” tank and a “new” tank. In the sick tanks (NF_Tank1b and HC_Tank2b), I added back larvae that were swimming in the beaker after being filtered out from the tanks. I may have to throw out these too, but until I have too many larvae for the new tanks I figured I would try to save them.

Filtering out larvae tanks:

  • HC_Tank2a (160,120) -> swimmers into HC_Tank2sick
  • HC_Tank1 (100) -> swimmers into HC_Tank2sick
  • NF_Tank2a (160,120,100) -> swimmers into NF_Tank2a
  • NF_Tank1 (100) -> swimmers into NF_Tank1sick

Checking for larvae:

  • Lots: NF1,HC1,HC2,HC4,HC5,SS4,SS5
  • Some: HC3

Set up growth rate experiment #2:

Since I had lots of larvae from every family, I decided to start another growth rate experiment. I combined the larvae from all families in a population, took samples for DNA, and added 1,100 larvae to 3 replicate silos.

Friday 7/3/15 and Sunday 7/5/15

Friday 7/3/15

My fiancé Michael Alcorn had a vacation day, so was able to come out to the hatchery and finally find out what I do all day. He did some of the counts, but quickly found those to a bit too mind numbing for him so mostly helped out with cleaning. We also tried to cut up the PVC sheet into tiles, but weren’t able to do so with the tools in the hatchery workshop.

Became really concerned about the mortality going on in the 100L larval tanks, as all of them had a lot of larvae sitting on the bottom. With it being the holiday weekend, most of the hatchery staff were out of town so I didn’t have much advice on how to proceed. One of the hatchery technicians, Alice, said they were having mass mortality with their larvae as well making me think it was a hatchery-wide water quality issue. She gave me some advice to minimize time the larvae are spent in small containers or out of the water which I started implementing. I decided for today to empty out the HC1 larval tank, do counts for the different size classes, save some for a DNA sample, and then put all the newly spawned HC larvae in that tank.

Piles of dead oyster larvae on the bottom of the tanka

Piles of dead oyster larvae on the bottom of the SS2 larval tank

Checking for larvae

  • Lots: HC5, NF2, HC3
  • Some: HC2, HC1

Larval counts

Notes on growth experiment

  • CGW/Tiso/Chagra
  • 19degC
  • 30 ppt salinity

Sunday 7/5/15

I filtered out SS_Tank1, NF_Tank1, and HC_Tank2 over various sizes to look at how far they grew and the estimated mortality. I did not put SS_Tank1 larvae back in the tank, and instead cleaned it out for the newly spawned larvae. I also changed the water on my growth experiment, filtered out new larvae, and changed my banjo filters.

Checking for larvae

  • Some: SS4,NF5,HC5
  • Lots: HC2,HC3,HC4,SS5

Notes on growth experiment

  • CGW/Chagra
  • 19degC
  • 29 ppt salinity

Wednesday 6-24-15

Steven Roberts came to help out today, which made the Wednesday cleaning/collecting larvae only take about 2.5 hours. He did all of the larval counts, filtered out the SS larval catches, and cleaned many of the buckets. We decided in the interest of time to just qualitatively note individual family output of larvae and then combine the larval output from each family in a population for counting.

Families that were filtered for larvae:

  • very little/some larvae: HC2, SS5, SS2, SS4, SS3
  • lots: NF5, HC3
    • for both of these the larvae seemed to be clustering near the bottom (dead??)
SS5 seemed to release some sperm when put in the bucket with all the other SS families.
I’ve organized my datasheet for larval counts a little differently.
  • The 1st sheet, “Day 1”, is the raw data from counting the larval output from the broodstock.
  • The 2nd sheet, “Larval tank counts”, is the raw data from counting the number of live/dead larvae filtered out of the 100 L larval tanks. It also lists the estimated ages of the cohorts in the tanks.
  • The 3rd sheet- “Larval tanks running total”- is formatted like a bank account transaction list, with larval tanks for each population the “accounts”. This incorporates data from the first 2 sheets to let me know approximately how many larvae I have in the 100 L tanks at a time. The differences between counts is also my estimated mortality (although some of this is due to leakage during cleaning).

Update 6-25-15

  • On Tuesday 6-23-15, the seawater was shut off for about 3 hours. Also, there was an issue with the airline so that tanks may not have been bubbling for a while. When I went in on Wednesday, all tanks seemed to be bubbling fine.