"This experiment was conducted at the Taylor Shellfish Hatchery in Quilcene, WA. The goal of this experiment was to identify causes of oyster seed mortality. Given that we know mortality occurs at 23C and not 29C- a comparison of protein expression between cohorts was carried out along with assessment of seed morphology, ciliate presence, and microbial diversity.
Notebook(s): GitHub.io
Sample collection date(s) and personnel: 7/8/16 to 7/25/16. Experiment conducted by @Ellior2
Background
In past years at the Taylor Shellfish Hatchery, we have experienced high mortality of Pacific oyster seed grown at 23C by Day 10-14 post-set. Oyster larvae grow best at 23C so it is not well understood why this is an intolerable temperature for young seed. We often see a bacterial infestation and invasive ciliates that embed themselves inside the mantle cavity during these mass mortality events shortly after settlement. This creates a substantial bottleneck in oyster seed production as it takes over a month and considerable resources to get the oysters to this post-settlement stage. Only recently in August 2015 have we seen exceptional growth and survival when we tried setting oysters at 29C. Preliminary research suggests the invasive ciliates that are present in the dying oysters cultured at 23C are not found in oysters cultured at 29C. However, we still do not know the underlying reason for the mass mortality of oysters grown at 23C. Do these invasive ciliates thrive best at 23C thereby making the newly set oyster seed susceptible during this energy-taxing metamorphosis? Or are these newly set oysters unable to survive at 23C due to other reasons, and the invasive ciliates are only opportunistic by feeding on already dying seed? Perhaps we can begin to answer these questions by using proteomics to understand what is physiologically happening during these critical life stages.
Protocol
Started with 8 million competent triploid oyster larvae (holding on a 250um screen).
Eight 18” diameter 180um silos - 80ml of microculch (graded between screen sizes 180um and 315um)
Each silo was placed in a 200L conical and fitted with a standpipe to keep the water approximately 6” above the level of the seed. One million larvae (20.0 g of 250’s) was set in each of the silos. Conicals 1-4 were supplied with 23C seawater and Conicals 7-10 with 29C throughout the length of the experiment. Unlike the 2015 experiment, the oysters stayed in their respective treatments for the length 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 dinoflagellates: 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 0.8gpm and were downwelling in each silo by a spray nozzle for the whole experiment. All seed were rinsed with freshwater daily.
Sampling Description
Sample Identification key: MMDDYY.Temperature.ConicalDuplicate For example: 063016.23C.1A
Day 1 (7/8/16): Before putting larvae into the system today we took larvae samples for proteomics, histology, and microscopy following protocols outlined below. We also took water samples from 23C and 29C before larvae were introduced into the system.
Day 3, 5, 7, 9, 11, 13, 15, 17:
- Collected water samples (3.5L) for each conical for microbiome studies by dropping the standpipe and collecting the effluent water.
- Removed silo from conical and rinsed gently with freshwater shower nozzle. Used a clean paper towel to dry the bottom of the silo. Once the oysters were dry, I used a metal spatula to scoop out oysters for proteomics, histology (bacteria / ciliates), and microscopy (larval development) sampling.
- Repeated steps 1&2 for the each of the other silos.
Day 6: We screened all of the seed to estimate settlement rates and discard any unset larvae and unused microculch. We estimated quantities of seed within given size ranges. After screening, we put back all seed holding on a 450 micron screen into each respective silo. Quantities of seed within each silo varied.
Day 18: We got a final count of oyster seed in each silo on the last day of the experiment. We collected samples from each size class within each silo to preserve and assess survival at a later date.
Water samples for Microbiomes To collect the water samples, we dropped the standpide to the conical and collected the effluent seawater in a carboy. It is important to note that we did this step before agitating the silo in anyway as to prevent suspending too much particulate matter. On the first day, we ended up filtering one 6L sample of 23C and one 6L sample of 29C (without larvae in the system). We used a 0.5um GFF filter to screen off bigger particulates and then used the 0.22um to retain bacteria. On Day 3, we realized this volume would take too much time to filter because we now needed to filter water samples from each of the eight conicals. On Day 3 we reduced the volume to 3.5L per sample for the rest of the experiment. We also changed the top filter to either a 0.7 or 0.8 um filter, depending on what was available at the time (documented on each sample). Once the water was filtered through the 0.22um, we rinsed 0.2 filter with 1ml of 25% cold TCA. We then folded the filter paper in half (microbes on the inside!) and stored in a small ziploc bag at -80. Samples were transferred to Steven's -80C freezer on 9/21/16 with dry ice.
Oyster samples for proteomics Scooped 250ul 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 on 9/21/16 with dry ice.
Oyster samples for histology Scooped 500ul 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 will be sent to Dr. Ralph Elston of Aquatechnics for histological analyses (ciliates, bacteria, and digestive gland quality).
Oyster samples for microscopy Removed ~100 larvae for fixation for each sampling event. Rinsed oysters using 1mL or less of seawater into a tube. Added 1 mL 7.5% MgCL2 in ultrapure water and let sit for 15 minutes so oysters relax. Oysters settled to bottom of tube. Pipetted off liquid and replaced with 1 mL of 4% paraformaldehyde in filtered seawater. Let sit in PFA overnight. After overnight incubation, rinsed 2 times with 1xPBS; let tubes stand 15 minutes between each wash. Then washed in increasing concentrations of ethanol: 30, 50, and 70%, again letting stand 15 minutes between each. Store larvae in 70% ethanol. These oysters will be used to determine developmental stages.
On the last day of the experiment, I also collected samples from every size class within each replicate to assess survival at a later date. I preserved the samples for microscopy using the methods described above.
Sample(s): It is important to note our temperature control for 29C failed on 7/21 at 4PM and gradually became 16-18C overnight and remained at this temp for the rest of the experiment.
For every row we have collected the following:
- duplicate oyster seed samples flash frozen and stored at -80C in Roberts Lab
- oyster seed samples preserved in 70% etOH for morphology
- a 0.22um filter for microbiome study (Day 1 has 2 filters: 23C and 29C both before larvae were introduced)
- oyster seed samples preserved in formalin for Dr. Ralph Elston histological analyses (parasites, bacteria, etc.)
| Sample # | Day | Date | Silo # | Treatment |
|---|---|---|---|---|
| 1 | 1 | 7/8/2016 | Competent Larvae | N/A |
| 2 | 3 | 7/10/2016 | 1 | 23C |
| 3 | 3 | 7/10/2016 | 2 | 23C |
| 4 | 3 | 7/10/2016 | 3 | 23C |
| 5 | 3 | 7/10/2016 | 4 | 23C |
| 6 | 3 | 7/10/2016 | 7 | 29C |
| 7 | 3 | 7/10/2016 | 8 | 29C |
| 8 | 3 | 7/10/2016 | 9 | 29C |
| 9 | 3 | 7/10/2016 | 10 | 29C |
| 10 | 5 | 7/12/2016 | 1 | 23C |
| 11 | 5 | 7/12/2016 | 2 | 23C |
| 12 | 5 | 7/12/2016 | 3 | 23C |
| 13 | 5 | 7/12/2016 | 4 | 23C |
| 14 | 5 | 7/12/2016 | 7 | 29C |
| 15 | 5 | 7/12/2016 | 8 | 29C |
| 16 | 5 | 7/12/2016 | 9 | 29C |
| 17 | 5 | 7/12/2016 | 10 | 29C |
| 18 | 7 | 7/14/2016 | 1 | 23C |
| 19 | 7 | 7/14/2016 | 2 | 23C |
| 20 | 7 | 7/14/2016 | 3 | 23C |
| 21 | 7 | 7/14/2016 | 4 | 23C |
| 22 | 7 | 7/14/2016 | 7 | 29C |
| 23 | 7 | 7/14/2016 | 8 | 29C |
| 24 | 7 | 7/14/2016 | 9 | 29C |
| 25 | 7 | 7/14/2016 | 10 | 29C |
| 26 | 9 | 7/16/2016 | 1 | 23C |
| 27 | 9 | 7/16/2016 | 2 | 23C |
| 28 | 9 | 7/16/2016 | 3 | 23C |
| 29 | 9 | 7/16/2016 | 4 | 23C |
| 30 | 9 | 7/16/2016 | 7 | 29C |
| 31 | 9 | 7/16/2016 | 8 | 29C |
| 32 | 9 | 7/16/2016 | 9 | 29C |
| 33 | 9 | 7/16/2016 | 10 | 29C |
| 34 | 11 | 7/18/2016 | 1 | 23C |
| 35 | 11 | 7/18/2016 | 2 | 23C |
| 36 | 11 | 7/18/2016 | 3 | 23C |
| 37 | 11 | 7/18/2016 | 4 | 23C |
| 38 | 11 | 7/18/2016 | 7 | 29C |
| 39 | 11 | 7/18/2016 | 8 | 29C |
| 40 | 11 | 7/18/2016 | 9 | 29C |
| 41 | 11 | 7/18/2016 | 10 | 29C |
| 42 | 13 | 7/20/2016 | 1 | 23C |
| 43 | 13 | 7/20/2016 | 2 | 23C |
| 44 | 13 | 7/20/2016 | 3 | 23C |
| 45 | 13 | 7/20/2016 | 4 | 23C |
| 46 | 13 | 7/20/2016 | 7 | 29C |
| 47 | 13 | 7/20/2016 | 8 | 29C |
| 48 | 13 | 7/20/2016 | 9 | 29C |
| 49 | 13 | 7/20/2016 | 10 | 29C |
| 50 | 15 | 7/22/2016 | 1 | 23C |
| 51 | 15 | 7/22/2016 | 2 | 23C |
| 52 | 15 | 7/22/2016 | 3 | 23C |
| 53 | 15 | 7/22/2016 | 4 | 23C |
| 54 | 15 | 7/22/2016 | 7 | 29C |
| 55 | 15 | 7/22/2016 | 8 | 29C |
| 56 | 15 | 7/22/2016 | 9 | 29C |
| 57 | 15 | 7/22/2016 | 10 | 29C |
| 58 | 17 | 7/24/2016 | 1 | 23C |
| 59 | 17 | 7/24/2016 | 2 | 23C |
| 60 | 17 | 7/24/2016 | 3 | 23C |
| 61 | 17 | 7/24/2016 | 4 | 23C |
| 62 | 17 | 7/24/2016 | 7 | 29C |
| 63 | 17 | 7/24/2016 | 8 | 29C |
| 64 | 17 | 7/24/2016 | 9 | 29C |
| 65 | 17 | 7/24/2016 | 10 | 29C |
9/1//16
We have decided to a preliminary analysis on 3 separate silos- 2, 3, and 9 throughout the length of the experiment. Silos 2 and 3 were from the 23C treatment; Silo 2 had 2% survival and Silo 3 had 10% survival. Silo 9 was from the 29C treatment and had 10% survival. We are interested to see differences in protein expression between these three groups. Therefore we ran the following 22 samples:
1, 3, 4, 8, 11, 12, 16, 19, 20, 24, 27, 28, 32, 35, 36, 40, 43, 44, 48, 51, 52, 56
See #338"