Presenting at Evolution and Updating Github

For the past 3 weeks I’ve been hanging around the Pacific Northwest, first going out to Tatoosh Island with my adviser, followed with some hiking, and then finishing up at the Evolution meeting in Portland, OR.  I gave an oral presentation of the first chapter of my dissertation on the population structure of Olympia oysters that you can check out here, and a poster of a paper that’s going to be submitted soon looking at adaptive differences among 3 populations of oysters in Puget Sound, WA (here). I’ve also been cleaning up my Github so that scripts and notebooks are better organized by project and have more documentation to them. In particular, I cleaned up the notebooks detailing the figures I made in my talk at Evolution.

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Goals June 2017

This is my first summer in Chicago and first summer during my dissertation without any fieldwork! Instead I’ll be doing lots of lab work to process samples from previous field seasons and a bit of writing on the first and second chapters of my dissertation. Since our adviser and most of our lab is away for the summer, my lab mate and I are instigating monthly summer goals. To accountability!

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DDIG is DDONE!

Excuse the bad pun… finally finished my proposal for the NSF Doctoral Dissertation Improvement Grant! Definitely one of the most time-consuming and stressful things I’ve done, as there are so many little parts that go into it and just one formatting error can get you disqualified without review. Check it out on my github here if you want an idea of what I have planned for the rest of my dissertation (pending funding, of course).

Monday 8/24/15 – Thursday 8/27/15 (Last week of fieldwork!)

Monday 8/24/15

Had a long chat with my advisor, Cathy, about the status of my fieldwork- what data I had collected, what I was leaving behind, plans for a stressor experiment- as well as plans for funding next summer. Puget Sound Restoration Fund has approved me to conduct a common garden experiment next summer with olys from California, Puget Sound, and British Columbia. My hope is to use this rangewide common garden to contextualize the regional-scale one I did this summer.

Tile culling

  • SSA_7: 9
  • SSA_8: 83
  • HCB_1: 160
  • HCB_2: 310
  • HCB_3: 384
  • Took pictures of tiles that were culled last Thursday but didn’t have pictures for.

Growth Rate Experiment

Started taking pictures of larvae from the growth rate experiments conducted earlier in the summer. This was done using an adaptor to attach a phone to the eyepiece of a microscope. Labelling scheme (until I think of a better one) is Population+Replicate_Growth rate experiment (1 or 2)_date sample was taken. Also did live/dead counts of the entire sample.

  • SS3_G1_7/12: 25L/15D
  • SS1_G1_7/12: 57L/8D
  • SS3_G1_7/15: 38L/14D
  • SS2_G1_7/12: 64 L/9D

Tile Set B

  • Cleaned out the tanks from tile set B. Cut off the tiles and randomized them among the wire “cages” in the large tank with the other tiles from Set_A.

Tuesday 8/25/15

Cultch Set

  • SS>450A + SS>450B -> SS> 450B; SS>1000
  • SS_new -> SS>450B; dump
  • HC > 450 ->  HC > 1000
  • HC_new -> HC > 450; dump

Tile culling

  • HC_B_1: 42
  • HC_B_2: 103 from front, 164 from back
  • HC_B_3: 67 front, 42 back
  • HC_B_4: 4
  • HC_B_7: 4 front, 13 back
  • HC_B_8: 12 front, 3 back
  • HC_B_10: 9 back
  • HC_B_11: 1 back
  • HC_B_12: 2 back
  • HC_B_14: 86 front, 5 back
  • SS_B_1: 7 back (none on front)
  • SS_B_2: 4 back (none on front)
  • SS_B_3: 0/3
  • SS_B_7: 0/10
  • SS_B_8: 0/2
  • SS_B_9: 0/5
  • SS_B_10: 0/7
  • SS_B_11: 0/6
  • SS_B_13: 0/1
  • NF_B_1: 0/27
  • NF_B_2: 95/120
  • NF_B_3: 0/13
  • NF_B_4: 77/270
  • NF_B_5: 8/12
  • NF_B_6: 208/92
  • NF_B_7: 0/2
  • NF_B_8: 18/10
  • NF_B_9: 8/19
  • NF_B_10: 70/48
  • NF_B_11: 11/17
  • NF_B_12: 10/27
  • NF_B_13: 1/12
  • NF_B_14: 0/2

Wednesday 8/26/15

  • Dropped off samples and borrowed materials at the Roberts lab.

Thursday 8/27/15

The little oyster babies are leaving the nest for the big open ocean! I started out the day finishing up any culling that was needed. I also looked over previously culled tiles to make sure that at least 1 cm of space was around each oyster.

Culling

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Steven Roberts came to help with the deployment. While I finished up culling, he made labels for the trays but cutting small PVC pipe into 1 inch pieces and etching numbers onto them. We’re also putting waterproof paper in a tube with the tray number.

12 tiles (4 per population) were attached to each tray with zipties. The populations were ordered in the same way for each section of each tray (NF, HC, SS).

Tray with tiles attached

Tray with tiles attached

Pictures were taken of each tile next to a ruler to measure size at deployment of the oysters.

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7 trays were filled up with with tiles. To minimize the effects of location within a stack of trays, we put 4 trays in a stack with a 2′ spacer between the top 2 and bottom 2 trays. An additional tray was used as a cover to keep out predators. We made 2 of these stacks, with an empty tray in Stack 2.

Stack of trays with oyster tiles ready for deployment

Stack of trays with oyster tiles ready for deployment

Order of tiles and trays

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The trays were hung of the dock at Manchester by ~20 foot rope. One of the stacks seemed to float (which was odd), so we tied on clam bags with rocks to both stacks.

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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.

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

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)