2015 Oyster Seed experiment 23C vs. 29C - RobertsLab/project-pacific.oyster-larvae GitHub Wiki
This experiment was conducted at the Taylor Shellfish Hatchery in Quilcene, WA. In previous studies, we have seen increased growth and survival of oyster seed grown at 29C during settlement and post-settlement stages as opposed to 23C. The goal of this experiment was to determine when we could transfer the seed back to 23C and still see the same high survival as oyster seed grown at 29C throughout the whole experiment. Growing oysters at 29C for any great length of time is not only energetically expensive but could ultimately become stressful as oysters continue to metabolize at an abnormally high rate. We collected samples to determine differences in the proteome between oysters under these different temperature regimes. Additionally we collected samples for Dr. Ralph Elston with Aquatechnics for histological analyses to complement this proteomic study.
Notebook(s): Github.io
Sample collection date(s) and personnel: 9/6/15 to 9/20/15. Experiment conducted by @Ellior2
On 9/6/15 we harvested competent diploid oyster larvae from the production department at Taylor Shellfish and distributed 1.1 million larvae per each of the 8-18" diameter silos, labeled 1-6,8, and 9. We used the following conversion to estimate the number of larvae: 18.0 grams=1 million larvae (larvae holding on a screen size of 236 um). Silos 1-6 and 8 were set in 29C conditions and Silo 9 was set in 23C. Both control silos 8 and 9 were sampled daily throughout the experiment starting at Day 5. Silos which were transferred (1-6) were sampled immediately before the transfer and every subsequent day after the transfer. Because this was more of an exploratory type of experiment we did not have replicates. Control silos 8 and 9 were placed in individual conicals for the length of the experiment, however the transferred silos shared a common vessel during all or part of the experiment. All seawater was pumped in from Dabob Bay, filtered through 25um, 10um, and 5um filter bags, and treated with sodium carbonate to reach a pH set point of 8.4. All seed received the same mixed high-density microalgae diet produced by our in-house algae bag system. Species include flagellates: Isochrysis spp., Pavlova spp., Nannochloropsis spp., Rhodomonas spp., and Tetraselmis spp. Effluent algal densities were targeted at 100K cells/ml. Incoming seawater flow rates were 1.0gpm and were downwelling in each silo by a spray nozzle for the whole experiment. All seed were rinsed with freshwater daily using a gentle shower nozzle. Microculch graded from 180-315um was used for the settlement stage (Day 0-5). On Day 5, we screened each silo separately to assess survival to settlement. Additionally we removed any leftover microculch and unset larvae (anything not catching on a 315um screen). Finally on the last day of the experiment (Day 14) we screened again to assess final growth and survival for each silo.
Before sampling, all seed was rinsed with freshwater to remove waste. I refilled the vessels with seawater. Then I removed the silos to scoop out the seed with a metal spatula when sampling-so oysters were covered in seawater prior to freezing.
Oyster samples for proteomics Scooped 500ul of dry larvae or seed into labeled cryotube (duplicate- labeled A and B). Flash froze in liquid nitrogen and stored in -80C freezer. Samples were transferred to Steven's -80C freezer with dry ice and Emma and Rhonda processed the samples in December of 2015.
Oyster samples for histology Scooped 1ml of dry larvae or seed into a 19ml falcon polystyrene round-bottom tube with 4.5ml of seawater and 0.5ml of formaldehyde solution (37%). Stored samples upright at room temperature. These samples were sent to Dr. Ralph Elston of Aquatechnics for histological analyses (ciliates, bacteria, and digestive gland quality). We have the report.
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Post-screening (Day 8) | Post-screening (Day 9) | Post-screening (Day 10) | Post-screening (Day 11) | |
|---|---|---|---|---|---|---|---|---|---|
| Silo # | Date | 9/6/15-9/10/15 | 9/11/2015 | 9/12/2015 | 9/13/2015 | 9/14/2015 | 9/15/2015 | 9/16/2015 | 9/17/2015 |
| 1 | Day 5 transfer | 29 | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
| 2 | Day 6 transfer | 29 | 29 | 23 | 23 | 23 | 23 | 23 | 23 |
| 3 | Day 7 transfer | 29 | 29 | 29 | 23 | 23 | 23 | 23 | 23 |
| 4 | Day 8 transfer | 29 | 29 | 29 | 29 | 23 | 23 | 23 | 23 |
| 5 | Day 9 transfer | 29 | 29 | 29 | 29 | 29 | 23 | 23 | 23 |
| 6 | Day 10 transfer | 29 | 29 | 29 | 29 | 29 | 29 | 23 | 23 |
| 8 | 29C control | 29 | 29 | 29 | 29 | 29 | 29 | 29 | 29 |
| 9 | 23C control | 16-23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
Samples: Here is entire list of samples collected. So far we have isolated the peptides and run MS/MS scan on 1,2,13,and 14.
| Number | Date | Contents |
|---|---|---|
| 1 | 9/11/2015 | 23C control seed from Silo 9 |
| 2 | 9/11/2015 | 29C control seed from Silo 8 |
| 3 | 9/11/2015 | 29C seed before the transfer to 23C (Silo 1) |
| 4 | 9/12/2015 | 23C control seed from Silo 9 |
| 5 | 9/12/2015 | 29C control seed from Silo 8 |
| 6 | 9/12/2015 | 29C seed before the transfer to 23C (Silo 2) |
| 7 | 9/12/2015 | 23C seed after the transfer (Silo 1) |
| 8 | 9/13/2015 | 23C control seed from Silo 9 |
| 9 | 9/13/2015 | 29C control seed from Silo 8 |
| 10 | 9/13/2015 | 29C seed before the transfer to 23C (Silo 3) |
| 11 | 9/13/2015 | 23C seed after the transfer (Silo 1) |
| 12 | 9/13/2015 | 23C seed after the transfer (Silo 2) |
| 13 | 9/14/2015 | 23C control seed from Silo 9 |
| 14 | 9/14/2015 | 29C control seed from Silo 8 |
| 15 | 9/14/2015 | 29C seed before the transfer to 23C (Silo 4) |
| 16 | 9/14/2015 | 23C seed after the transfer (Silo 1) |
| 17 | 9/14/2015 | 23C seed after the transfer (Silo 2) |
| 18 | 9/14/2015 | 23C seed after the transfer (Silo 3) |
| 19 | 9/15/2015 | 23C control seed from Silo 9 |
| 20 | 9/15/2015 | 29C control seed from Silo 8 |
| 21 | 9/15/2015 | 29C seed before the transfer to 23C (Silo 5) |
| 22 | 9/15/2015 | 23C seed after the transfer (Silo 1) |
| 23 | 9/15/2015 | 23C seed after the transfer (Silo 2) |
| 24 | 9/15/2015 | 23C seed after the transfer (Silo 3) |
| 25 | 9/15/2015 | 23C seed after the transfer (Silo 4) |
| 26 | 9/16/2015 | 23C control seed from Silo 9 |
| 27 | 9/16/2015 | 29C control seed from Silo 8 |
| 28 | 9/16/2015 | 29C seed before the transfer to 23C (Silo 6) |
| 29 | 9/16/2015 | 23C seed after the transfer (Silo 1) |
| 30 | 9/16/2015 | 23C seed after the transfer (Silo 2) |
| 31 | 9/16/2015 | 23C seed after the transfer (Silo 3) |
| 32 | 9/16/2015 | 23C seed after the transfer (Silo 4) |
| 33 | 9/16/2015 | 23C seed after the transfer (Silo 5) |
| 34 | 9/17/2015 | 23C control seed from Silo 9 |
| 35 | 9/17/2015 | 29C control seed from Silo 8 |
| 36 | 9/17/2015 | 23C seed after the transfer (Silo 1) |
| 37 | 9/17/2015 | 23C seed after the transfer (Silo 2) |
| 38 | 9/17/2015 | 23C seed after the transfer (Silo 3) |
| 39 | 9/17/2015 | 23C seed after the transfer (Silo 4) |
| 40 | 9/17/2015 | 23C seed after the transfer (Silo 5) |
| 41 | 9/17/2015 | 23C seed after the transfer (Silo 6) |
| 42 | 9/18/2015 | 23C control seed from Silo 9 |
| 43 | 9/18/2015 | 29C control seed from Silo 8 |
| 44 | 9/18/2015 | 23C seed after the transfer (Silo 1) |
| 45 | 9/18/2015 | 23C seed after the transfer (Silo 2) |
| 46 | 9/18/2015 | 23C seed after the transfer (Silo 3) |
| 47 | 9/18/2015 | 23C seed after the transfer (Silo 4) |
| 48 | 9/18/2015 | 23C seed after the transfer (Silo 5) |
| 49 | 9/18/2015 | 23C seed after the transfer (Silo 6) |
| 50 | 9/20/2015 | 23C control seed from Silo 9 |
| 51 | 9/20/2015 | 29C control seed from Silo 8 |
| 52 | 9/20/2015 | 23C seed after the transfer (Silo 1) |
| 53 | 9/20/2015 | 23C seed after the transfer (Silo 2) |
| 54 | 9/20/2015 | 23C seed after the transfer (Silo 3) |
| 55 | 9/20/2015 | 23C seed after the transfer (Silo 4) |
| 56 | 9/20/2015 | 23C seed after the transfer (Silo 5) |
| 57 | 9/20/2015 | 23C seed after the transfer (Silo 6) |