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The following is a summary of the 2020 experiment in the works…
Experiment plan
8-month rearing trial starting from 30-day-old pediveliger (setter) stage well in to juvenile development. Experiment will contain three main Ωaragonite.sat treatments controled in triplicate
(1) ambient p_CO2 = _Ωaragonite.sat > 1; pHt.s. 7.8-7.9;
(2) moderate elevated p_CO2 = _Ωaragonite.sat 0.4; pHt.s. 7.4;
(3) severe elevated p_CO2 = _Ωaragonite.sat 0.1-0.2; pHt.s. 7.1;
There will be two reciprocal transitions to address both the duration and life-stage of hormetic-conditioning. The following is the timeline of trips to Jamestown Point Whitney Shellfish Hatchery and the corresponding treatments.
(1) Mid-march preparation (current and reported below): Clean and prepare 9 200L conicals with pump lines to a double heathstack of 9 trays with isolated flow - this gives us a strength of n = 3 treatment-1
(2) DAY 0 - mid-April: Start the experiment by adding 15k pediveliger post-larvae to each heath tray
| Trays total | Conical sumps total | Tray/sump treatment-1 |
|---|---|---|
| 9 | 9 | 3 |
| Age | pediveliger to 3 months |
|---|---|
| Treament 1 (control) | ambient Ω > 1 |
| Treament 2 | moderate Ω = 0.4 |
| Treament 3 | severe Ω = 0.1 |
(2) MONTH 2 - mid/late-June:
- Fix whole animals tray-1 in LN2 for DNA/RNA seq and cellular stress assays
- Scope for Growth measurements: P = [C - (R + U)]
- R respiration rate: measured with SDR dish at the hatchery; freeze all animals in -20C for AFDW as loss of ignition in Putnam Lab
- U ammonia excretion: freeze water samples from respiration rate measurements (~3-4 ml individual-1) for later analysis
- C ingestion rate and absportion efficiency: measured at the hatchery with submersible stir plate and live algae culture. Algae counts pre-post incubation (to calculate ingestion rate) will be measured at the hatchery with SD100 slides and the Nexcelom T4 Cellometer. Seston and fecal weights (to calculate absportion efficiency) will be aquired by loss of ignition of GF/F glass filters - filters will be stored at -20C for later analysis in Putnam Lab.
TREATMENT TRANSITION!!
All trays will be rinsed during this trip. Sixty animals will be allocated in 3 small tanks and 180 in 1 large tank each with rinsed sediment - the surface area of large to small is 3:1. One small tank and the large tank will remain in the original tray whereas the other two small tanks will be randomly positioned in the other two treatments! Thus, the treatments will begin as follows:
Total treatments, N = 9
| Age | pediveliger to 3 months | 3 months to 5 months |
|---|---|---|
| Treament 1 (control) | ambient Ω > 1 | ambient Ω > 1 |
| Treament 2 | ambient Ω > 1 | moderate Ω = 0.4 |
| Treament 3 | ambient Ω > 1 | severe Ω = 0.1 |
| Treament 4 | moderate Ω = 0.4 | ambient Ω > 1 |
| Treament 5 | moderate Ω = 0.4 | moderate Ω = 0.4 |
| Treament 6 | moderate Ω = 0.4 | severe Ω = 0.1 |
| Treament 7 | severe Ω = 0.1 | ambient Ω > 1 |
| Treament 8 | severe Ω = 0.1 | moderate Ω = 0.4 |
| Treament 9 | severe Ω = 0.1 | severe Ω = 0.1 |
| Tanks total | Small tanks tray-1 | Large tank tray-1 |
|---|---|---|
| 36 | 3 | 1 |
(3) MONTH 4 - mid/late-August:
Same sampling and measurments as month 2
TREATMENT TRANSITION!!
The Large tank (N = 180 juveniles) will be divided into the small tanks and randomly positioned each of the three pCO2 treatments! Thus, the treatments will begin as follows:
Total treatments, N = 15; replication, N = 3
| Age | pediveliger to 3 months | 3 months to 5 months | 5 months to 7 months |
|---|---|---|---|
| Small tanks will stay in current treatment | |||
| Treament 1 (control) | ambient Ω > 1 | ambient Ω > 1 | ambient Ω > 1 |
| Treament 2 | ambient Ω > 1 | moderate Ω = 0.4 | moderate Ω = 0.4 |
| Treament 3 | ambient Ω > 1 | severe Ω = 0.1 | severe Ω = 0.1 |
| Treament 4 | moderate Ω = 0.4 | ambient Ω > 1 | ambient Ω > 1 |
| Treament 5 | moderate Ω = 0.4 | moderate Ω = 0.4 | moderate Ω = 0.4 |
| Treament 6 | moderate Ω = 0.4 | severe Ω = 0.1 | severe Ω = 0.1 |
| Treament 7 | severe Ω = 0.1 | ambient Ω > 1 | ambient Ω > 1 |
| Treament 8 | severe Ω = 0.1 | moderate Ω = 0.4 | moderate Ω = 0.4 |
| Treament 9 | severe Ω = 0.1 | severe Ω = 0.1 | severe Ω = 0.1 |
| Large tanks divided in each treament | pediveliger to 5 months | 5 months to 7 months | |
| Treament 10 | ambient Ω > 1 | moderate Ω = 0.4 | |
| Treament 11 | ambient Ω > 1 | severe Ω = 0.1 | |
| Treament 12 | moderate Ω = 0.4 | ambient Ω > 1 | |
| Treament 13 | moderate Ω = 0.4 | severe Ω = 0.1 | |
| Treament 14 | severe Ω = 0.1 | ambient Ω > 1 | |
| Treament 15 | severe Ω = 0.1 | moderate Ω = 0.4 |
| Tanks total | Small tanks tray-1 | Large tank tray-1 |
|---|---|---|
| 45 | 5 | 0 |
(4) MONTH 6 - mid/late-October:
Same sampling and measurements as months 2 & 4
NO treatment transition!!
All trays will return to ambient conditions for a two-month recovery period! Thus, treatment total and replication remains the same
(5) MONTH 8 - mid/late-December or early January:
Same sampling and measurements as months 2, 4, & 6
Experiment take-down
Expected Results
-
Main effect
-
Moderate stress hormesis
