Friday 7/31/15

Nine larvae/setting tanks to clean! Michael and I had an evening flight out east for his cousin’s wedding, so today was a pretty rushed day trying to get everything done and still make the 4:05p ferry.

Larvae tanks

  • NF_Tank2_160 (224) -> 31,200 total: 500 for DNA, 11,000 to cultch set B, 19,700 dumped out
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) -> swimmers only added back
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 17,281 total: 500 for DNA, 11,000 added to cultch set B, 5,781 added back to HC_Tank2_160
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> swimmers only
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 11,537 total, 537 to DNA, 11,000 to cultch set B
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> swimmers only
  • SS_Tank1_new (160) -> SS_Tank2_160

Cultch Setting System

Set up a 2nd silo replicate for all 3 populations. As SS only had ~11,000 224-sized larvae, I added 11,000 larvae to each of the silos. 5,781 extra HC larvae were added back to HC_Tank2_160. With NF, there were ~9,000 dead 224-sized larvae- most likely due to my adding the the ones I wasn’t using back in to Tank2_160. If a larva that is ready to set can’t find an appropriate surface, it will eventually die. I dumped out the 19,700 larvae that were not used in the cultch set (see previous post for my regret about this).

New larvae

Only saw some larvae in HC and SS. I ran out of time to take samples for counting, so just screened out the larvae, cleaned the broodstock buckets, and dumped the larvae back into the buckets.

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Thursday 7/30/15 (Lab work!)

I got to do some bona fide lab work today, which was a nice change of pace. I’ve been taking samples of larvae for DNA sequencing at various points throughout the experiment:

  • From all newly released larvae (either from each family or combined, depending on how I filtered them out)
  • From larvae in the “New” tanks that reach 160 microns
  • From larvae in the “160” tanks that reach 224 in size
  • Occasionally pooled larvae from a tank

These samples have mostly been stored at -20degC in .5-1 mL of RNALater, but duplicates of many were also stored in ethanol (1st in 75%, then in 95%). Earlier in the summer I wanted to do a test extraction to see if there was a particular storage method that worked best and figure out which extraction kit to use, but then the oysters needed maintenance 6 days a week and all of a sudden in was almost August. With the growth rate experiments and larval production essentially done, I finally had a day to do the test extraction.

Continue reading

Tuesday 7/28/15 and Wednesday 7/29/15

Tuesday 7/28/15

New Larvae

  • HC: 0
  • SS: some
  • NF: 0

Settlement

Set up an “overflow” setting system for larvae that do not go into the tile set-up. Silos (15 cm diameter, 20 cm height) with 180 micron screens are suspended in a tote with a draining outlet. 224 sized larvae are added into the silos along with 1-3 tablespoons of 450 micron cultch. An airstone is in the totes and water/algae is dripped into the silos. The water level is 13 cm deep, making the volume of water in the silo to be 2,296 cm^3 (equalling to 2,296 mL). I’ve been told that ~5 larvae/mL is good for this type of set-up, with no more than 10 larvae/mL. At 7 larvae/mL, the max to add to each silo would be 16,000 larvae.

Wednesday 7/29/15

Larvae tanks

  • NF_Tank2_160 (224) -> 56,700 total: 600 for DNA, 15,300 to cultch set, 41,400 to NF_Tank2_160
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) 0> swimmers only added back
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 15,937 total: 600 for DNA, 15,300 added to cultch set
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> swimmers only
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 9,675 total, 600 to DNA, 9,075 to cultch set
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> swimmers only (a lot on bottom)
  • SS_Tank1_new (160) -> SS_Tank2_160

New larvae

  • Some new larvae from all 3 populations

Setting Systems

To clean the tile setting systems, I first fill up a clean, empty 100 L larval tank with seawater and fill up a misting sprayer designated for setters with fresh water. I empty a tile setting tank over a 100 micron screen to catch the larvae that haven’t set yet. I put these in a tripour beaker with seawater and place the tiles in the 100 L tank full of water. I clean the tank with Vortex and fill it up as quickly as possible. Meanwhile, I spray the tiles and poultry wire gently with freshwater before returning the tiles and larvae into the cleaned tank. This took a little getting used to, especially figuring out how to minimize the time the larvae sat in the beaker as they would try to set on the bottom if they were in there too long.

I initially thought that the cultch set would be an experiment to see if the populations have differential success in the number of single oysters produced. Because of this, I cared a lot about having replicate silos, adding similar numbers of larvae to each, adding larvae on the same day, adding the same amount of cultch to each, and randomizing the order of silos. This led to me not using all of the 224s that I screened out, particularly from NF which somehow peaked in the number of 224s before the other 2 groups. I’ve since realized that to truly make it a viable experiment would take a lot of work (especially in the number of silos needed once I started screening them out by size) and that the main benefit of the cultch set is to grow up F2 oysters for future experiments. So I regret throwing out some of the 224s, but should still have enough for the project goals.

  • NF: 15,300 added to Cultch_SetA
  • SS: 9,075 added to Cultch_SetA
  • HC: 15,300 added to Cultch_SetB

Setting up for Setters (Monday 7/27/15)

On Monday 7/20/15 and Wednesday 7/22/15 I screened out the “160” tanks over 224 micron screens to see if any larvae were ready to settle. This is the same size class used by the hatchery to separate larvae out to put in their setting system. There were less than 5,000, so I figured that by the next Monday there would be enough to set up my setting system. As a reminder, I’m placing 15 4in x 4in white PVC tiles that have been roughed up on one side in a 100 L larvae lank with the hopes that enough larvae set on the tiles. After a few weeks, I will cull larvae to ~20-30 per tile to avoid overcrowding impacting growth. The benefit of using tiles over large shells or cultch (small ground up shell) is that they are easier to standardize for replicates among groups and provide a flat surface for measuring growth rate. Obviously they aren’t a great representation of nature, but will work well to explore the differences in growth rate among the populations.

Tiles are attached to plastic coated

Tiles are attached to plastic coated “poultry wire” with cable ties

Tile

The poultry wire is bent to fit inside a 100 L larvae tank

The poultry wire is bent to fit inside a 100 L larvae tank

Larval tank counts

  • NF_Tank2_160 (224) -> 30,000 to experiment; 41,175 to NF_Tank2_160
  • NF_Tank2_160 (100)
  • NF_Tank1_new (100)
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> all 30,000 to experiment
  • HC_Tank2_160 (100)
  • HC_Tank1_new (100)
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 30,000 to experiment; 6,000 to SS_Tank2_160
  • SS_Tank2_160 (100)
  • SS_Tank1_new (100)
  • SS_Tank1_new (160) -> SS_Tank2_160

I had at least 30,000 larvae at 224 microns in each group, so I decided to add 30,000 to each of my tile systems. Ryan Crim at the hatchery recommended I add at least four times as many larvae as I want to set. With 15 tiles per tank and at least 200 larvae per tile (to then be culled to 20), this meant I only needed ~12,000 larvae per tank so 30,000 should be plenty.

For the rest of the larvae from NF and SS that weren’t placed in the setting system, I added them back to their respective “160” tanks to deal with later in the week.

New larvae

There was between 32,000-57,000 larvae from all populations.

Friday 7-24-15 (S. Roberts)

On Wednesday night I left to spend a few days in Chicago to start setting up the Committee on Evolutionary Biology’s new high performance computer, so Steven Roberts and Sam White came to the hatchery to do the Friday cleaning and counting. I wrote up some instructions– also good exercise so we can reference them when writing up Materials and Methods. Check out their great write-up here (lots of pictures!).

Monday 7/20/15 -Wednesday 7/22/15

Monday 7-20-15

This was the day I finally felt like I had things under control (only took 6 weeks!). The system of screening out the 160 tanks, then screening out the New tanks and transferring the 160s over to the 160 tank seems to work well and my counting has gotten faster.

Larval tank counts

  • NF_Tank2_160 (224,100)
  • NF_Tank1_new (100)
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224,100)
  • HC_Tank1_new (100)
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224,100)
  • SS_Tank1_new (100)
  • SS_Tank1_new (160) -> SS_Tank2_160

New larvae

  • Some: SS2,SS3,HC5,HC3,HC1,SS1
  • Lots: NF2,NF5

Growth

Took the last samples from the 2nd growth experiment today and fixed them in 80% ethanol.

Tuesday 7/21/15

Salinity

Looking at the samples taken Friday 7/17/15, there was a lot of white precipitate in many of the wells and it was a little more difficult to distinguish between live and dead. Ryann and I decided that larvae with any color were alive and those that were completely clear were dead. We also took new samples from each of the silos to count that day and avoid a similar complication. I’m still waiting on some of the data for Ryann, but the link to the datasheet is below. It seems that they all pretty much died at 6 ppt, but above that mortality is variable. The goal was to get at least 100 larvae per sample when counting but that wasn’t met for most of them.

Salinity counts datasheet

New larvae

  • Some: SS, HC
  • Lots: NF

Wednesday 7-22-15

Larval tank counts

  • NF_Tank2_160 (100)
  • NF_Tank1_new (100)
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224,100)
  • HC_Tank1_new (100)
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224,100)
  • SS_Tank1_new (100)
  • SS_Tank1_new (160) -> SS_Tank2_160

Salinity

Ryann cleaned up the salinity experiment.

New larvae

Some: HC5

Screened out all of the buckets but got no larvae for NF or SS. With so few larvae, I decided to go ahead and collapse my 15 bucket system down to 3. All families in a population are now in the same bucket, but still in their separate bags. This will considerably reduce cleaning time, and although it will change the genetic makeup of the offspring there should be about a 2 week lag given the length of brooding time.

Larval counts datasheet

Tuesday 7/14/15 – Sunday 7/19/15

Tuesday 7-14-15

Today I primarily checked for new larvae and did a mortality count for the HC tanks (as these had some of the most culling of non-swimmers during the tank cleanings on Monday). I also drilled holes into my 10cmx10cm PVC tiles so that I can tie them on to a plastic coated wire mesh for the settlement stage.

Checking for larvae

  • Some: SS3,HC3,HC1,NF1,SS1,SS2,HC2(?)
  • Lots: NF3

Larval tank counts

  • HC_Tank2_160 (100)
  • HC_Tank1_new (100)
  • HC_Tank1_new (160) -> HC_Tank2_160

Wednesday 7-15-15

S. Roberts came in today to help out with cleaning, counting, and setting up the settlement system. He roughed up one side of the PVC tiles with sandpaper, so as to make that side more preferential to oyster settlement.

Larval tank counts

  • NF_Tank2_160
  • NF_Tank1_new (100)
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160
  • HC_Tank1_new
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160
  • SS_Tank1_new
  • SS_Tank1_new (160) -> SS_Tank2_160

New larvae

With the extra help today, I decided to do individual counts for each family. There were very few, with none for NF, making me think that the broodstock are winding down.

  • Some: SS3,HC2,HC3,HC4

Growth experiments

  • I took some more samples from the growth rate experiments today for size measurement, and will be taking down the 1st growth experiment on Friday.

Friday 7-17-15

The seawater was shut off for a couple hours today as they were doing some reorganization of the hatchery. I was able to clean and take samples for counting from the larvae tanks before the shut off and then finish the broodstock buckets afterwards. I made sure everyone had enough food during the break by splashing some extra algae in.

Larval tank counts

  • NF_Tank2_160
  • NF_Tank1_new (100)
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160
  • HC_Tank1_new
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160
  • SS_Tank1_new
  • SS_Tank1_new (160) -> SS_Tank2_160

New larvae

Well I was wrong by thinking that they were all winding down. There was 441,100 larvae from SS and 205,133 from HC, but barely any from NF.

  • Some: SS4,NF1,HC1,HC4
  • Lots: SS3,HC2,HC3,HC5,SS2

Salinity

Took samples of ~100 larvae from each of the 45 silos in the salinity experiment to do live/dead counts and save for taking pictures. Unfortunately Ryann, the PSRF intern helping me on the salinity project, and I ran out of time so I was just able to do a rough activity estimate (% swimming). I fixed the wells in 10% formalin and left covered in parafilm to look at next week.

Sunday 7/19/15

Came in for a couple hours on my way back from a camping trip in Olympic National Park. Cathy Pfister, my adviser from UChicago, stopped by for a tour of my setup and to catch up on how things are going. With Michael’s help, I also did the water changes for the 2nd growth experiment and the salinity experiment and changed out banjo filters.

Monday 7/13/15

Today I decided to change what I was doing with the 100L larvae tanks. Instead of each population having one tank for new larvae and one for the “sick” larvae that survived the previous mortality, I will now have a tank for new larvae and a tank for larvae that are >160 microns. For the larvae in the “sick” tanks, I screened them over 160, 140, and 100 micron screens and kept those on the 160 in the cleaned out “160” tanks, kept the swimmers on 140 in the “new” tanks, and dumped out the 100s as they were mostly dead or sick. On Monday, Wednesday, and Friday I will now screen the larvae in the “new” tank to separate out all those that have grown to be 160 microns and will move those to the second “160” tank. I’ve been taking DNA samples of all newly spawned larvae and will now also take samples of those that reach 160 microns to see if there is significant loss of certain alleles as the larvae grow up.
Larval tank counts
  • HC_Tank2sick (160) -> HC_Tank2160
  • HC_Tank2sick (140) -> swimmers to HC_Tank1new
  • HC_Tank2sick (100) -> culled
  • HC_Tank1new (160) -> HC_Tank2160
  • HC_Tank1new (100) -> HC_Tank1new
  • NF_Tank1sick (160) -> NF_Tank2160
  • NF_Tank1sick (100) -> culled
  • NF_Tank2new (160) -> NF_Tank2160
  • NF_Tank2new (120) -> NF_Tank1new
  • NF_Tank2new (100) -> swimmers to NF_Tank1new
  • SS_Tank1new (160) -> none
  • SS_Tank1new (120) -> SS_Tank1new
  • SS_Tank1new (100) -> swimmers to SS_Tank1new
New Larvae
  • Lots: HC2,SS3,HC4,NF1,HC5
  • Some: SS1

Larvae Count Datasheets

Friday 7/10/15 and Sunday 7/12/15

Friday 7/10/15

Salinity Experiment

Ryann changed the water for the 45 silos.

Checking for larvae

  • Lots: NF1,HC1
  • Some: NF4, SS4,SS1,SS2

Larval Tank Counts

  • NF_Tank2a (100,120,160) -> only swimmers of 100 size added back to NF_Tank2a with 120 and 160
  • HC_Tank1c (100,120,160) -> only swimmers of 100 size added back to HC_Tank1c with 120 and 160

Larvae counts datasheet

Sunday 7/12/15

Salinity Experiment

Using filtered/UV treated fresh water (not tap water) from a spigot at NOAA, I made up beakers for the salinity stress. So as to ensure all beakers got the same amount of algae (40,000 cells/mL), 160 mL of seawater in each beaker came from the same algae diet mix. The rest of the seawater required for a specific salinity came from the heated seawater line.

30 ppt
24 ppt
18 ppt
12 ppt
6 ppt
Algae diet
160
160
160
160
160
Seawater
640
480
320
160
0
Fresh water
0
160
320
480
640

The temperature was 19.5degC and salinity of each replicate was checked to make sure the mixing was correct.

 

 

Starting the Salinity Experiment (7/7-7/9)

Tuesday 7/7/15

With all of the populations spawning consistently and the mass mortality (hopefully) subsided, we started tanking about a “salinity challenge” experiment to see how larvae from the three populations respond to different salinities. A number of different techniques have been developed to measure salinity, and with these different units of measurement. One common way to express salinity is in parts per thousand (ppt)- harkening back to when titration techniques were used to measure the concentration of certain ions in seawater. Most seawater is considered to be around 30-32 ppt. In estuaries and bays with freshwater input (the favorite homes of oysters!) this can go much lower.

I found some information about salinity during the summer in Puget Sound, but not much (if you know of any good datasets, please let me know!). One review of Hood Canal studies in 1998-2000 included some figures that show surface salinities around 26 ppt in the summer.

Salinity data from transects of Hood Canal, from: Warner, M.J., Kawase, Mitsuhiro, and Newton, J.A., 2001, Recent studies of the overturning circulation in Hood Canal, in Proceedings of Puget Sound Research, February 12-14, 2001, Bellevue, Wash., Puget Sound Action Team, Olympia, WA, 9 p.

Salinity data from transects of Hood Canal, from:
Warner, M.J., Kawase, Mitsuhiro, and Newton, J.A., 2001, Recent studies of the overturning circulation in Hood Canal, in Proceedings of Puget Sound Research, February 12-14, 2001, Bellevue, Wash., Puget Sound Action Team, Olympia, WA, 9 p.

We decided to do a trial experiment on Wednesday where larvae from each population are exposed to either 30, 25, 20,15, 10, 5, or 0(freshwater) ppt for an hour. After which they are all put back into 30 ppt seawater and left for a day. Ryann, one of the PSRF interns, will be helping me plan and execute these salinity experiments.

Larval tank counts:

  • NF_Tank2a at 100 and 140

Did not filter out new larvae

Wednesday 7-8-15

Steven came out to help today and brought a handsaw to help with the PVC tiles. Ironically, we found a tool in the warehouse that worked even better than what he brought, but either way we finished up making the 10cm x 10 cm tiles.

There were very few larvae from Fidalgo Bay, so we set up our trial experiment with just Hood Canal and South Sound larvae. 7 salinities (30, 25,20,15,10,5,0), 700 larvae per silo, and 1 replicate per treatment. The experiment was set up at 2p and at 3pm we moved the silos into 30 ppt seawater with ~40,000 algae cells per mL. Water temp was 19degC.

Checking for larvae:

  • Lots: HC3, SS1, SS4
  • Some: HC1, SS5, NF1 (?)

Larval tank counts:

  • Steven counted
  • NF_Tank1sick (100,160): very few
  • HC_Tank2sick (100,160): half of the 100s were dead

Thursday 7-9-15

Checking for larvae:

  • Lots: HC3,SS3,NF5
  • Some: HC5,HC2,HC1(?),SS1,SS3
  • HC_All: 64,325
  • SS_All: 17,362
  • NF_All: 15,600

Salinity Trial:

Ryann and I took a few mL with larvae out of the salinity experiment silos for her to look at under the microscope. I’ll post her notes when I get them, but at 0 ppt all larvae looked at from both pops were dead. At 5 ppt there were some still alive, and she noted that the Hood Canal larvae looked hardier. After 15 ppt there was no noticeable mortality and larvae from both pops seemed active in general. We cleaned out those silos and then set up the full scale salinity experiment. 15 silos for each population were put in beakers with 30 ppt seawater and ~40,000 algae cells/mL of Reeds Algal Paste. Larvae from all families in a population were pooled together and then based on the drop counts ~700 larvae were added to each silo. Larvae were measured out with a graduated cylinder, with mixing in between and during the pours from beaker to cylinder. There is still likely to be some variation in the number of larvae per silo, but larvae will be counted at the end of the experiment when comparing live vs. dead.

  • 5 salinities: 30, 24,18,12,6 ppt
  • All 3 pops
  • 3 replicates per treatment with ~700 larvae per rep
    • HC: 7 mL per silo
    • SS: 20 mL per silo
    • NF: 22 mL per silo

Larval tank counts

  • SS_Tank1b (140,100)