Olympia oyster experiment is running well out at Manchester. Quick recap:
- Question: how do temperature & food availability during winter (or during “pre-conditioning”) impact Olympia oyster gonad quality, and subsequent larval survival through metamorphosis?
- Approach: precondition oysters in 4 groups, high/low temperature (~7C & ~10C) + high/low food availability (need from PSRF!) for 6 weeks and 12 weeks. Monitor gonad for evidence of resorption, maturation, via histology throughout (every 2-3 weeks); also collect and samle oysters from Mud Bay on same schedule to compare gonads in experiment to wild. Condition oysters up to 18C, monitoring gonad development via histology, and freeze 1/2 of gonad for lipid/glycogen/protein content. Spawn, collect & count larvae. Rear larvae in small static silos separated by day (~family). Measure survival to juvenile stage. Preserve subsample of larvae immediately upon collection from broodstock for lipid content (need to know how many/mass is necessary).
- Side experiment: larval trials! Subject larvae to various stressors, measure mortality rate: salinity, heat, pH – Erin Horken (PSRF) may help with this project.
- 1,700 Olympia oysters were collected from Mud Bay in Dyes Inlet on November, 6th 2017. The next day they underwent standard intake procedure (scrubbing, rinsing with fresh water, 1-hour freshwater+bleach soak to kill epibionts), then were acclimated to hatchery conditions in flow-through tanks (ask PSRF: feeding rate during this acclimation period?)
- On November 30th, subsample were sacrificed for histology (n=20) and DNA samples (n=100, including 20 used for histology) (DNA collected for PSRF’s purposes)
- 1,600 oysters were randomly sorted into 32 bags of 50, volume displacement of each bag was measured.
- The 32 bags were randomly sorted into 8 50L tanks (4/tank), for the following 4 treatments:
- A1 & A2: Low food, low temp
- B1 & B2: Low food, high temp
- C1 & C2: High food, low temp
- D1 & D2: High food, high temp
- Used recirculating chiller/heater to regulate temperature in 50L reservoirs, which then distributed SW to culture tanks.
- Temperature was gradually (1C/day) adjusted acheive ~7C & ~10C starting on December 1st.
- Animals were cleaned 3x per week, and checked for morts
- Half of the animals were removed at week 6 for spawning – PSRF managed this phase. They have been collecting and counting all larvae, but not rearing/monitoring survival.
- I will manage the 12-week treatment groups.
- See my Feb. 27th post for more detail on terminating the treatments and moving groups to conditioning/spawning buckets.
- Below are temperature plots from the Avtech system. I also have HOBO data loggers recording temperature on half of the buckets (one logger per treatment replicate), AND out at Mud Bay.
Temperatures from November 30th -> March 15th, when they reached 18C, the temperature used to induce spawning and rear larvae. The spikes correspond to cleaning events where the probes recorded temperature while they were out of the water
Here’s a closer look at temperatures during the conditioning phase
from LabNotebook http://ift.tt/2HzRjXy
I updated an excel spreadsheet so it has multiple stats that I thought might be useful to see any patterns in expression. There are multiple sheets on the file: combined data with few tags followed by silo 2, 3 and 9 with all tags. The tags and why they may be helpful in seeing protein expression patterns are listed below.
- Average- is this protein typically highly or lowly expressed?
- Standard Deviation- how much does each day deviate from one another on average?
- Coefficient of Variance – normalized variance; how dispersed the protein expression is
- Variance- less useful than (3) however another representation of the dispersion of protein expression
- Median- valuable if compared to the average protein abundance to understand if protein expression is consistent
- Slope- liner regression to understand overall trend of protein expression (decreasing vs. increasing)
- Kurtosis- understand if the protein has a sharp peak in protein expression
- Skewness- informs us if the protein is being expressed more in a certain hald of the experiment
- Max- is the protein expressed a lot at any point in the experiment?
- Min- is there a time when the protein is not expressed?
- Range- the overall change in protein expression (does not inform us whether it is increasing or decreasing)
- 1st quartile- What is the cutoff for 25% expression over the course of the experiment?
- 4th quartile- What is the cutoff for 75% expression over the course of the experiment?
- Sum- determines if the protein was highly abundant over the course of the experiment (relative to the sums of other proteins)
- Day0:Day15- a ratio of the day before treatment to the final day of the experiment; informs us if the protein significantly changed after treatment
- Day3:Day15- a ratio of the first day of measured day of treatment over the final day of treatment
- Average for Days 0-7- valuable when compared to the second average to see if there was a change in protein expression half way through the larvas’ lives
- Average for Days 9-15- a compliment to the above tag
- Range for Days 0-7- valuable when compared to the range for days 9-15; further elucidates changes in expression between the first half of the experiment and the second half
- Sum:Total Proteins Identified- what percentage of the total proteins in the experiment are caused by expression of this protein?
I’m not sure how else I should proceed with this data. I could potentially look at gene enrichment, but I believe that a significant portion of proteins should be eliminated before hand. Knowing which proteins to eliminate can be difficult because each ‘tag’ can highlight a new trait of that protein. Therefore, eliminating proteins will mostly depend on future interests for this data set.
Today Sam walked me through the process of using Agilent 2100 Bioanalyzer kit to assess DNA integrity (bp length) via fluorescence signal. Unique aspect of using this kit/analyzer is that it only requires 1ul of sample, at DNA concentrations between 0.5-50 ng/ul.
- Sam removed kit reagents from fridge to sit at room temperature for 30 minutes
- Prepared new gel-dye mix as per kit instructions. Prepared mix can be held at 4C for up to 4 weeks. All dye vials should be protected from light.
- Combined my sample reps into -A vial, e.g. transferred DNA from 1-B and 1-C to vial 1-A.
- Walked to Seeb lab in the 1st floor of the Marine Science building. Materials I needed: DNA samples, new DNA chip, prepared gel-dye mix, DNA marker & ladder from kit, DI water (for chip wells without my samples), 10ul pipette, 10ul pipette tips.
- Followed kit instructions – loaded gel, marker, ladder, samples, and DI water onto chip. Samples 1-8 were loaded into the wells of the same number. Wels 9-12 were loaded with DI water.
- Turned computer on, started DNA 1200 series software. NOTE: computer has login password – make sure I have that for future uses.
- Inserted chip into analyzer, closed lid carefully.
- For Assay Selection file, selected dsDNA -> DNA 1200 series 11.xsy
- For Destination file location, navigated to Roberts Lab folder
- Selected Start
The software displays data in real-time with fluoresence on y-axis, and time on the x-axis. Smaller DNA fragments “elute” faster, and likewise longer fragments take longer. Each sample well also has small (50bp) and large (17,000) standards/markers. We were hoping my DNA fragments would be long/as intact as possible. Here are some screen-shots of each fluoresence/time plot, along with the software’s calculated bp/concentrations:
Screenshot of all plots; my samples are labeled Sample 1 through Sample 8. Samples 9-12 are just DI water. Samples 1-7 look somewhat consistent, but Sample 8 looks very weird. Note different y-axis range.
Screenshots of the bp/concentration break-down: