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

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.

Setting up an Oyster Garden

Monday (June 8) was my first day out at the NOAA Manchester Research Station in Washington State. Specifically, I’m working in the Kenneth K. Chew Center for Shellfish Research and Restoration. This shellfish hatchery is the result of collaboration between many groups and funding agencies, in particular the Puget Sound Restoration Fund (PSRF).

http://www.nwfsc.noaa.gov/news/features/hatchery/

The hatchery (right) and algae greenhouse (left)

My project this summer is to raise oysters descended from three Puget Sound populations under common conditions in order to measure differences in fitness. This type of experimental design is commonly referred to as a “common garden”, and allows one to control environmental variables so that phenotypic disparities among individuals can be attributed to their genetic differences. My fitness metrics are reproductive output, survivorship at different life stages, and growth rate. I will also be taking DNA/RNA samples along the way to see if mortality is random in respect to genotype, or due to purifying selection. With the RNA, I plan to look at differences in gene expression to help detect cryptic differences in phenotype between these populations.

Three source populations for common garden experiment

Three source populations for common garden experiment

This project is a collaboration with Steven Robert’s lab at the University of Washington, who previously conducted a reciprocal transplant experiment with offspring of wild oysters from these same populations. For that experiment, they outplanted the young oysters from each group at four different sites and measured growth rate, mortality, and reproductive characteristics. They observed significant variation at these metrics among populations and sites (informative slides and manuscript preprint available here). My experiment will be following up on these results by testing if population-level differences are consistent in a second generation under controlled environmental conditions.

As I’ve never raised shellfish before, this week has had a bit of a learning curve. Fortunately for me, the staff at the hatchery have been super helpful in showing me the ropes and advising on how to set up my experiment. I’m starting with about 100 adult oysters for each group (see lab notebook entries for data). These are the first generation (F1) offspring of wild oysters, and have been living in common conditions their entire lives- mostly hanging off the docks near the hatchery. Their offspring will be 2nd generation (F2) from the original broodstock, and should have any influence from maternal effects erased.

The adults were brought in to the hatchery on May 28 and placed in three separate buckets to avoid cross fertilization. To maximize genetic diversity and minimize the chance that one male fertilizes all of the females, I split each group into 5 buckets of ~20 oysters. These “families” will be marked, so that I can genotype them later and follow their offspring’s success throughout the experiment. Their water temperature was switched to a balmy 20°C this week, which will encourage them to start spawning and producing larvae.

(sorry for the lack of pictures, I’ll take some and put them up soon!)