Emma's+Lab+Notebook+Spring+2011

__Secondary Stress Trial__ Bivalves in large tank were fed at 8:15 am (see spreadsheet). The feeding jars for the pH trial were fed at 9:15, 20 mL into each jar.
 * September 29, 2011**

__Secondary Stress Trial__ Bivalves in large tank were fed at 9:15 am (see spreadsheet). In the morning, all jars were sampled for spec pH, salinity, temperature, and ammonia (for jars containing animals and 1 control). In general, pH dropped to about 6.9 in all the oyster jars regardless of starting pH, stayed consistent in the non-animal controls, stayed consistent in the clam jars with 5 clams, and dropped a little (~0.2 units) in the clam jars with 10 clams. After sampling for pH, all animals were fed 40 mL of algae. In the afternoon, 1 jar from each replicate was sampled for spec pH and total alkalinity. Duplicate alkalinity samples were taken for time series of beer bottles and for verification of the Forestry spec method. All samples were poisoned with ~70 µL HgCl2. After sampling, water was changed in all the jars. The ambient water was at pH 8 so we added 0.4% of high CO2 water to 4.75 L of ambient to get the low pH treatment. Dave will feed all the animals in the evening. Only 1 jar from each replicate will be fed for the next 24 hours to see if feeding causes changes in pH (jars 1 are the fed jars). The new treatment water is pH 8.03 for low CO2 and 7.77 for high CO2. For detailed pH, see [|water chemistry spreadsheet].
 * September 28, 2011**

__Secondary Stress Trial__ Bivalves were fed at 8:20 am (see spreadsheet). Took 2 cuvettes full of water from each trash can (low and high CO2) and added 70 µL of 0.1 N NaOH or HCl to low and high, respectively. The low CO2 Mukilteo water is 7.7 and the high is not calculable because it is so high. Mixed 50/50, 25/75, and 10/90 high/low CO2 water and took pH. The 10/90 mixture was at pH of ~4. Turned off pure CO2 bubbling into high CO2 trash can. Made mixes of 10, 20, and 50 mL of high CO2 water in ~2L of low CO2 water. From these pHs, we made a regression of pH on % volume high CO2 water. To get a low pH 0.2 units below the control, we need to add 0.18% volume high CO2 water, or about 8.8 mL to 5L. We have 2 pH treatments: high CO2 and low CO2 (pH of about 7.7 and 7.9). In each pH, there are 2 densities of oysters (4 or 8) or clams (5 or 10) per jar, in duplicate. There are also 2 control jars per pH without any animals. We took duplicate spec pH measurements of both treatments and 5 total alkalinity samples - 2 in PMEL bottles and 3 in beer bottles. The animals were put in their treatment jars around 13:30. Wet masses were taken of groups of bivalves as they were put into their jars. The jar labels are type of animal (Cg or clam) followed by # in jar-replicate.
 * September 27, 2011**

Low CO2: Cg 4-1 80g Cg 4-2 109.7 g Cg 8-1 156.2 g Cg 8-2 156.5 g clam 5-1 56.3 g clam 5-2 42.8 g clam 10-1 90.8 g clam 10-2 90.1 g

High CO2: Cg 4-1 84.5 g Cg 4-2 63.4 g Cg 8-1 148 g Cg 8-2 149.1 g clam 5-1 38.1 g clam 5-2 45.6 g clam 10-1 88.7 g clam 10-2 90.6 g

Dave fed all animals, 20 mL algae per jar (~60,000 cells/mL) in the evening.

__OA FHL 2011__ qPCR of gDNA from 9/26/11 qPCR (DH 12) and new gDNA (0627 gill tissue) using 18s, ATP synthase, citrate synthase, and new Prx6 primers. DH12 did not amplify at 18s, indicating that the results from yesterday were spurious. the new gDNA amplified at 18s, ATP synthase, and Prx6, but not at citrate synthase. media type="custom" key="10647688"

__Secondary Stress Trial__ Bivalves were fed at 9:30 am (see spreadsheet). Switched from aquarium water to Mukilteo water to try to remedy low pH. Bubbled one trash can with pure CO2 and the other with CO2-free air.
 * September 26, 2011**

__OA FHL 2011__ Reconstituted primers designed Sept 14, 2011 in TE buffer (pH 8.5). SR IDs are as follows: IMSP5 = 1387, 1388; ATP synthase = 1386, 1385; citrate synthase = 1384, 1383; Prx6 = 1382, 1381. Did qPCR using all primers on gDNA (DH 12 from Sam's -20C gDNA Box 1) and cDNA (C. gigas gill vibrio exposed). All primers amplified cDNA, only IMSP5 amplified gDNA, but is probably primer dimer because it also amplified in the negative control where no other primer pair did. Also did a gradient between 50 and 57°C and all primers amplify well at 55°C. Need to make sure that gDNA was actually amplifiable by checking with 18s primers. media type="custom" key="10647686"

__Secondary Stress Trial__ Dave had started bubbling 2000 ppm CO2 and CO2-free air into trash cans on Saturday. Took 2 cuvettes full of water from each trash can and added 10 µL of 0.1 N NaOH to one low CO2 cuvette and 10 µL 0.1 N HCl to the other. The low CO2 water was about pH 7.87 and the high CO2 only at 7.81.
 * September 25, 2011**

__Secondary Stress Trial__ Bivalves were fed at 10:20 am (see spreadsheet). Counted clams and oysters: 205 oysters, 211 clams. Water ammonia was at 0.25 ppm. Flushed out all excrement and did 50% water change.
 * September 23, 2011**

__Secondary Stress Trial__ Bivalves were fed at 10:50 am and 6:30 pm (see spreadsheet). During morning feeding, ammonia was at 0.5 ppm. Did 100% water change and rinsed out feces/pseudofeces.
 * September 18, 2011**

__Secondary Stress Trial__ Bivalves were fed at 9:45 am and 6:15 pm (see spreadsheet). One oyster was found dead at the morning feeding. During evening feeding ammonia was at 0.5 ppm. Did 80% water change.
 * September 17, 2011**

__Secondary Stress Trial__ Bivalves were fed at 9:45 am (see spreadsheet). Made F/2 media with 1 L of sterile seawater and 132 µL each of Procul A and B.
 * September 16, 2011**

__Secondary Stress Trial__ Bivalves were fed at 9:30 am (see spreadsheet). One oyster was dead.
 * September 15, 2011**

__Secondary Stress Trial__ Bivalves were fed at 9:15 am (see spreadsheet).
 * September 14, 2011**

__OA FHL 2011__ Two samples - both from treatment 105B (1500 µatm) taken on 8/1/11 - were extracted for demonstration purposes and discarded.

Primer design for qPCR - primers must span introns. Since I have not been able to get rid of all gDNA in the RNA samples, I'm going to design primers that will amplify only cDNA. I have found gDNA sequence in C. gigas or a related species for Prx6 (C. gigas sequence AM265552), ATP synthase (C. gigas sequence EE677774), citrate synthase (EE677716), and IMSP-5 (a protein from the oyster shell,HS208448). I was not able to find sequence for SOD. Dave supposedly has primers that might amplify only cDNA for Hsp70, but they have not yet been tested with gDNA. To find gDNA to align with the RNA off of which the primers were originally designed for these genes, I first did a megablast of the RNA sequence against Crassostrea sequences only. If that yielded gDNA, then I used that gene sequence and aligned it with the RNA in Spidey (indicating it was C. elegans, which probably has the most similar splice sites). If the original blast did not yield any results, then I blasted against S. purpuratus only, searching for somewhat similar sequences. If that still did not work, then I opened up the blast parameters to all organisms in genbank, searching for somewhat similar sequences. The following are the species for which I found gDNA corresponding to the RNA that I was blasting: Prx6 - primers were originally designed off of a DNA sequence with 6 exons (C. gigas,AM265552); exons occur on DNA 38-123, 918-1074, 2091-2234, 2952-3025, 3190-3262, 3352-3483 ATP synthase - S. purpuratus (NM_214578); exons occur on mRNA sequence 262-437 and 577-623 citrate synthase - Aspergillus niger (HQ407432); exons occur on mRNA sequence 96-157 and 638-708 IMSP-5 - Lotus japonicus (AP004959); exons occur on mRNA sequence 466-563 and 611-662

Prx6 SRID: 1381, 1382 ATP synthase SRID: 1385, 1386 citrate synthase SRID: 1383, 1384 IMSP-5 SRID: 1387, 1388

__Secondary Stress Trial__ Bivalves were fed around 8 am (see spreadsheet).
 * September 13,2011**

__OA FHL 2011__ Did spec (Nanodrop) of Dnased RNA samples (n=6). The concentrations and estimated remaining volumes of the samples are below. media type="custom" key="10452986"

__Secondary Stress Trial__ Bivalves were fed around 10 am and details were entered in the spreadsheet.
 * September 12, 2011**

__OA FHL 2011__ Re-DNased samples that were DNased on August 22, 2011 (except for 105B/1500 ppm samples). Followed regular DNase protocol. Diluted DNAsed RNA 1:20 in nanopure water. Did qPCR using 18s primers using 1 and 2 µL of diluted RNA as template. The thermalcycler protocol is 2step amp+melt for EvaGreen.

__Secondary Stress Trial__ At 9:30 am, the oysters and clams were fed as described 9/9/11. The volume of algae was about 300 mL. The information was entered in the bivalve feeding [|spreadsheet]. Bivalves were fed again around 6 pm. Ammonia levels were checked in the water and were at 1 ppm (threshold = 0.1 ppm). Did a full water change.
 * September 10, 2011**

__Secondary Stress Trial__ We got ~200 pacific oysters and 200 manila clams from Joth's farm. We put the oysters and clams in a large fiberglass tank with water circulating through a chiller to keep it at 16°C and through biofilters. At 4pm we reduced the water in the tank to 40.5 L and fed the bivalves 60,000 cells of algae mix (from Taylor) per mL, about 575 mL. After about 1/2 hour, when the water had been cleared of algal cells, we then put the same water back in the tank. Plans for the trial and links to data on feeding the bivalves can be found [|here].
 * September 9, 2011**

__Notes from Richard Strathmann on larval rearing__ To make a water bath, use aquarium heater and circulating pump with a stopcock to regulate flow. Do water changes with a sieve and bowl. At 22°C with feeding, water changes need to be every day in a static system. If there is flow, need to monitor amount - does the outlet have material stuck in it? Scottie Henderson (dissertation in FHL library) used pulse food supply. IV bag could drip food in. Could shine a light on the bag to keep culture photosynthesizing. Could also slow siphon using valve and small stopcock. If pulse feeding, can do ad libitum, ~ >100,000 cells of isochrysis per mL (based on M. edulis), bigger cells would be less. This amount does not scale with larval age. Possible mix for larvae: Isochrysis galbana + Pavlova lutherii or nanochoropsis or chitopserois. Organic content is about equal to cell volume, but much larger cells have bigger vacuoles. When collecting hatched larvae, make sure they are not malformed. Spawning: take straight from lagoon and spawn immediately or feed and keep at a higher temperature. Can wedge open the shell and inject serotonin into the adductor (or anywhere) - may yield better eggs. could adjust larval density for a constant biomass Ciliates should wash out in a continuous flow. When strip spawn, rinse tissue well with filtered seawater. quality of food is important with sterilized seawater. Settlement: Would be cleaner with tiles, but shell may stimulate them better. Try adding K+ or cesium ions, check concentration to get maximum effect (Yool et al. 1986 Biol Bull). Yool's results: Greater synchrony, downstream of normal sensory input, normal metabolism and good survival. What do the lab lights do to larvae? old paper published with c. virginica.
 * September 2, 2011**

__OA FHL 2011__ Nanodrop concentrations of 105A and 105B cDNA. The concentrations are very similar to 103A3 so there should be enough cDNA for a qPCR since that sample amplified well. Redid qPCR of the 105 samples with the same primers. Also did qPCR of all samples using Hsp70 primers (SR ID 971 and 972). Protocol is 2Step Amp+Melt Eva Green.
 * August 25, 2011**

__OA FHL 2011__ Nanodropped all DNased RNA samples 3 times. Concentrations were higher (~8-16 ng/µL), but this may be because the background blank of pure water is no longer a true blank after DNAsing. Went ahead and reverse transcribed all samples. Since concentrations are so low, reverse transcribed the maximum volume (still less than 1 µg) of 17.75 µL. Added 0.5 µL Oligo dT primers to sample and incubated at 70C for 5 minutes, followed by a few minutes on ice. Then added 5 µL MMLV 5x buffer, 1.25 µL 10 DNTPs, and 0.5 µL of reverse transcriptase to each sample - mixed and incubated 42C for 1 hour + 3 minutes deactivation at 95C.
 * August 23, 2011**

Did qPCR of cDNA using 1 µL template a full concentration (equal volume pooled cDNA from all samples), diluted 2x, 4x, and 10x for a dilution test. Primers used were superoxide dismutase (SR ID 600 and 601) and EF1a. qPCR protocol used was 2Step Amp+Melt Eva Green (annealing = 55C). media type="custom" key="10263777" All NTCs were clean. The only samples that amplified were EF1a full concentration and 2x dilution.

Did qPCR using the same EF1a and SOD primers, glutathione peroxidase (Rachel's primers from 441), and peroxiredoxin 6 (SRID 634 and 635). Used 2 µL of full concentration cDNA template. Did positive controls of gDNA and cDNA. media type="custom" key="10263789" All NTCs were clean. The 105A and 105B samples did not amplify. It's not a problem with the primers because there was amplification of the 103A and 103B samples and positive controls.

__OA FHL 2011__ DNased all RNA samples (Ambion's Turbo Dnase kit, regular protocol). Due to low concentrations of RNA had to DNase entire volume of RNA. Diluted DNased RNA 1 µL in 19 µL water for qPCR test of gDNA contamination. Stored DNased RNA in -80•C. qPCR of DNased RNA using 18s primers. Did qPCR using 1 and 2 µL of RNA template (both diluted 1:20). media type="custom" key="10263745" gDNA contamination in all samples, but NTCs were clean.
 * August 22, 2011**

__OA FHL 2011__ qPCR of RNA extracted August 18 to check for genomic DNA carry-over. Diluted RNA 1:20 and 1:10 and amplified in 20 µL reactions using Sso Fast EvaGreen mix. The thermal cycler program used was 2 Step Amp+Melt EvaGreen. There was gDNA contamination in all the samples (NTCs were clean). All samples need to be DNased. media type="custom" key="10237519"
 * August 19, 2011**

__OA FHL 2011__ Extracted RNA from all samples collected July 26, 2011. Used Tri Reagent and followed manufacturer's protocol. Did not see any pellets in any of the samples. Resuspended the RNA in 0.1% DEPC, 50 µL and pipetted 3 times to mix. Nanodropped RNA 3 times (1 µL each) per sample to get concentration. All concentrations are very low and entire volume will need to be used for reverse transcription. RNA is stored in NOAA OA May-June 2011 box in -80C. media type="custom" key="10234689"
 * August 18, 2011**

__OA NOAA August 2011__ NOAA is doing another OA experiment with 6 different treatments: 400, 600, 800, 1000, 600 +/- 200, and 800 +/- 200 µatm. They are rearing C. gigas larvae in each of the treatments. I sampled 2 entire jars per treatment for RNA analysis. The entire jar volume was decanted onto a 20µm mesh sieve. The larvae were washed down into a small volume at the bottom of the sieve and transferred to a 2 mL tube. The tubes were spun down at 4,000 rpm for 1 minute. The water was drawn off and remaining material was flash frozen on dry ice. The samples were stored at -80C at NOAA.

__OA FHL 2011__ I spent the last week counting the larvae I fixed during the OA experiment, assessing levels of calcification and measuring the larvae. Polarization was used for calcification and it doesn't look like there is much of a difference between treatments. For measurements, shell hinge length and depth were measured at 10x using CF's Nikon. Using a two-sided t-test in R, there is a significant difference in hinge length between 400 and 1000 µatm on day 3 and a difference in shell depth between the control larvae and all treatments on day 3 (4 days post-fertilization). p-values are in pink on the images; on the shell depth graph, all p-values refer to a comparison between that treatment and 400 µatm. The colors correspond to the following treatments: blue = 400, green = 700, yellow = 1000, orange = 1500. media type="custom" key="10220123"media type="custom" key="10220129"
 * August 17, 2011**

__Ceramide__ [|Steven's stats] for gene expression data
 * August 9, 2011**

__Ceramide__ Assembled information for a table in the manuscript that includes gene accession number (sptlc1=JN315146, 3KDSR=JN315143, AC=44, GlcCer=45), sequence length, similar oyster ESTs and top blast hit in NCBI. The similar oyster ESTs were found by doing a blastn of the amplified nucleotide sequence, selecting "other EST" and constraining the search to C. gigas. The top blast hit was found by running a blastx of the nucleotide sequence within the swissprot database.
 * August 8, 2011**

__OA FHL 2011__ Started counting samples from 1st timepoint of experiment - 7/25 1 hour post fertilization. Counted total number of eggs in each tube, number of eggs that have evidence of a polar body or fertilization envelope (indicating successful fertilization), and cleavage stage if present. The fixation seemed to work well, but there are fewer eggs than I expected in each sample. Entire sample is emptied from the tube into a welled plate and then 0.5 mL of 70% EtOH is used to rinse out the tube into the well. When done counting, the entire volume from the well is pipetted back into the tube and the well is rinsed with 0.5 mL of 70% EtOH.
 * August 5, 2011**

__Ceramide__ qPCR of tissue distribution of the second set of primers for 3KDSR (3KDSR qPCR.2). media type="custom" key="10152469"

__OA FHL 2011__ Finished fixation of samples from 8/3.
 * August 4, 2011**

__OA FHL 2011__ End of experiment Did TA and spec pH on cooler water and 2 containers in 105B (because flow is still a problem in these containers). From each remaining container, took 2 separate aliquots of 2x 1000 µL each for live/dead counts. Filtered out remaining larvae and fixed in PFA (fixation finished 8/4).
 * August 3, 2011**

__OA FHL 2011__ Ran all remaining total alkalinity samples and DIC. Took and ran spec pH and TA for all 4 coolers. Fed larvae by putting in 30 mL of hatchery algae mix and turning off water for 2 hours.
 * August 2, 2011**

__OA FHL 2011__ Sampled 2 containers for RNA and counting from each treatment: 103A-5, 103A-6, 103B-1, 103B-2, 105A-4, 105A-5, 105B-2, 105B-3. Containers were chosen because they had the highest densities based on last week's counts for the 4 remaining larval containers in each treatment. Larvae were spun down in 2 mL of water and flash frozen in liquid N2. For counting, 4 aliquots of 500 µL were taken per container and for fixation, 2 aliquots of 500 µL was taken. Counts were done for live/dead and totals. Photos were taken for SEM: 9 photos for 103B, 10 for 103A, 5 for 105B, and 1 for 105A (there was only one larva counted in all the subsamples taken for the 105A treatment). Samples were taken from the cooler water for DIC, TA and spec pH. For each container sampled for RNA, water chemistry was done (pH and TA). Some samples from previous days were run for TA and DIC. The larvae were fed ~60,000 cells per mL of the hatchery algae mix by turning off the water flow for 2 hours.
 * August 1, 2011**

__OA FHL 2011__ Laura Newcomb fed the larvae with 50 mL of each Iso. and Dunaliella, leaving water shut off to system for 2 hours.
 * July 31, 2011**

__OA FHL 2011__ Finished total counts of larvae sampled 7/28. Also took SEM of the same larvae: 7 larvae photographed from treatment 103A, 10 from 103B, 5 from 105A, and 4 from 105B.
 * July 29, 2011**

Took salinity and temperature from all outside cooler water and took spec pH of same water.

Sampled containers for RNA (emptied all larvae onto mesh, poured into 2 mL vial, spun down for 1 min at 2,500 rpm and flash frozen): 103A-3, 103A-4, 103B-3, 103B-5, 105A-1, 105A-2, 105B-4, 105B-6.

Finished fixation of larvae from 7/26 and 7/28.

Fed larvae as described 7/27.

__OA FHL 2011__ Crisis at 6 am - cooler 106B pH was down to 6.5. Switched containers over to 105B, which had been pre-equilibrated at 7.52 starting yesterday. Took a spec sample to see how low pH had gotten within the container (sample was of 106B-3). The containers will stay permanently in 105B. Without temperature correction, the pH in 106B-3 was about 6.8.
 * July 28, 2011**

Sampled all containers for counting (3x 500 µL aliquots). Live/dead counts were done today, but total counts will be done tomorrow morning (as well as SEM!). Sampled containers 1, 3 and 4 for fixation with PFA (2x 500 µL aliquots). Took pH and TA samples from containers 3 and 4. Took pH, TA and DIC samples from outer cooler water.

Used what I thought was a 50 µm mesh sieve for the first container (103B-1), but I think it was actually much larger because no larvae were retained. Switched back tot he 35 µm for the remainder and larvae were present in every well.

Ran total alkalinity for cooler water samples collected 7/25 and 7/27 (8 samples).

Made new 4% PFA in seawater.

__OA FHL 2011__ Made new 50 µm mesh for outflows on 105A and 106B (see 7/26). Took samples for DIC, TA and pH and ran DIC from 7/25 and 7/27. Fed larvae.
 * July 27, 2011**

Continued fixation of larvae from yesterday. Only did 2 PBS washes, leaving larvae in second wash at 4C.

Around 11 am we ran out of CO2 and all the pHs went to ~8 in all the treatments. Moose and I installed another cylinder, and the systems re-adjusted, but in most cases the pH was overshot and went too low before it re-equilibrated. All seem OK now.

At 11:30 am took samples from outer cooler water for DIC and TA and measured, salinity, temperature, and took spec pH.

Counted algal cells for larval feeding. Counted 3 squares in hemacytometer, 25 of which equal one square millimeter. For Dunaliella, there were ~366 cells per square mm and for Isochrysis ~ 216 (diluted 1:1 and 2:1, algae : EtOH, respectively). This scales up to 1.8 million cells per mL for Dunaliella and 1.3 million for Isochrysis. To feed the larvae approximated 45 million cells oof each (30,000 cells per mL in a 3 L container), they need 24 mL of Dunaliella and 34 mL of Iso. At 4 pm, turned off water flow and fed larvae. Turned water flow back on at 6 pm. Tubing has not arrived yet, so had to feed larvae by adding food and turning off water flow for 2 hours.

At 6 pm, took spec samples from outer cooler water and salinity and temperature.

Ran DIC for all samples collected 7/25 and 7/27 standardizing against CRM 111.

__OA FHL 2011__ Finished fixing samples from 7/25. Cleaned and replaced water in all tanks, started on flow-through. Took samples for counting live/dead and fixing for scope work later. Took chemistry samples (TA and pH) of all outer cooler water and inside 2 containers for each treatment.
 * July 26, 2011**

Details: Finished the fixation process from 7/25. Removed the PFA and washed 2x with 1x PBS, letting stand 15 minutes in between each wash. Then washed in increasing concentrations of EtOH: 30, 50, and 70%, again letting stand 15 minutes between each. Left larvae in the 70% EtOH and stored at 4C. Around 8:30 in the morning, took samples for spec pH from the outer cooler water of all treatments and from the following larval containers: 106B-2, 105A-2, 105A-3, 103B-1, 103A-2, 103A-4, 103B-3, 106B-3. Also took samples in Pyrex bottles (poisoned) for total alkalinity. In turn, emptied each larval container onto a 35 µm mesh sieve and then rinsed the larvae from the sieve into a 50 mL Flacon tube. Filled the tube to 50 mL with the appropriate pCO2 SW. Inverting 3x between each aliquot, aliquoted 450 µL (3x 150 µL)of larvae in seawater into a gridded welled plate. Aliquoted the same amount into a 2 mL microcentrifuge tube and added 7.5% MgCl2 for 15 minutes. Then followed same steps as 7/25 for PFA. For the larvae in the welled plate, counted live vs. dead on an inverted scope (went to 32x to verify mortalities) and total counts. Later in the afternoon, noticed that some of the containers in tanks 105 and 106 were overflowing. The problem seemed to be caused by not enough outflow from the containers. Tried to fix the problem for most of the evening and replaced most of the containers with new ones that had good flow around 1 am. By 2 am, all containers seemed to be holding a good volume and not overflowing. At 6:45 am on 7/27 came back and a couple containers looked like they were overflowing again. Replaced all the outflow mesh with 50 µm and the problem seems to be fixed.

__OA FHL 2011__ Took spec pH of the cooler water and of the water in one larval container for each treatment (6 am). Recalibrated the Durafet probes based on the spec pH for the cooler water. Turned off flow through on larval containers. At 7:30 am, collected 31 adult C. gigas from Argyle Creek. Strip spawned 10 females and 4 males, separating sperm and eggs. Fertilization (in the correct pCO2 water) began around 10:30 am. About 90,000 eggs were fertilized with about 1200 sperm. The fertilized eggs were incubated for 10-15 minutes to allow fertilization to occur and were then rinsed into the static larval containers (static for 24h). Fertilization of the falcon tubes used to monitor development and take samples for fixation within the first 24 hours begain at 11:25 am and proceeded as follows for each of the treatment coolers (2replicates fertilized per cooler): 106B - 11:25 am 105A - 11:40 am 103A - 11:55 am 103B - 12:10 pm The fertilized eggs were left to incubate for 10 minutes and then put into 40 mL of seawater at the correct pCO2. The tubes were placed in the coolers that contain the larval containers. At one hour post fertilization (hpf), the Falcon tubes were gently inverted 3x and 500 uL were aliquoted into a microcentrifuge tube (this was repeated for a total of 1 mL). The fertilized eggs were allowed to settle to the bottom of the tube for 5 minutes, then the seawater was drawn off and 1 mL of 4% paraformaldehyde (PFA) was added. The tubes were left to sit overnight. Starting at 5:25 pm (6 hpf) when there was evidence of the larvae hatching into the trocophore stage, the same steps were repeated except the larvae were all strained onto a 35 µm mesh screen and rinsed using 1 mL of SW into a tube. 1 mL of 7.5% MgCl2 was added to relax the larvae and they were left to sit for 15 minutes. The SW + MgCl2 was then removed and replaced with 1 mL of 4% PFA, left to sit overnight.
 * July 25, 2011**

At 2:30 pm, samples were taken in 500 mL Pyrex bottles for carbonate chemistry from each cooler. Samples were also taken for spec and temperature and salinity were measured with probes.

__OA FHL 2011__ Reset settings on tanks for new treatments. All are at 21C and the pCO2 are at 400, 700, 1000, and 1500 µatm.
 * July 24, 2011**

__OA FHL 2011__ Tank 103 is adjusted to the correct pH levels as is tank 106B. Tanks 105 and 106A are not appropriately equilibrating. Tank 106A was not flowing through and so was not allowing turnover of water - replumbed it and seems to be getting a little better. Turned off the CO2 to tanks 105 so that the pH could come up. Moose also fixed a problem with the vacuum compressor, which may help as well. Temperature is not getting high enough on any of the tanks (does not go above 22 C). On tanks 103, put a heater in the cooler that contains the cooling coil (set at 24C) to try and warm the incoming water. Each cooler containing the experimental replicates has 1 500W heater, it may be that they each need 2.
 * July 23, 2011**

__OA FHL 2011__ Made new m-cresol purple: dissolved 0.016 g of m-cresol purple (MW = 404.4 g/mol) in 20 mL of milliQ water to make 2mmol/L solution. Brought to pH ~7.9 using 0.1N HCl. Did a dye addition correction using 4 samples of seawater, 2 with added HCl for a pH range (10 µl and 20 µl). The slope (b) = -1.415 The intercept (a) = 0.996
 * July 22, 2011**

Durafet pH probe calibration: Each tank has a Durafet probe attached to the pump that measures the pH of the water in the tank. These need to be calibrated with the spectrophotometric pH. Turned off the water flow into the coolers around 9 am and let the water circulate through the pumps for ~1/2 hour. Took one cuvette of water from each tank to measure pH using m-cresol purple (MCP). Calibrated temperature for each probe using the Fluke thermometer. Tanks 103 A and B were at 10C, 105 A and B were 10.4C, and 106 A and B were 10.2 and 10.3 C, respectively. pH measured at 25C was adjusted for dye addition (see above) and adjusted for temperature using CO2calc and assuming that total alkalinity = 2060 µmol/kg. Probes were calibrated to the correct pH as follows: After calibration, tanks were set to the appropriate treatments outlined July 18, 2011.
 * TANK || MEASURED pH || ADJUSTED pH ||
 * 103A || 7.76 || 7.98 ||
 * 103B || 7.76 || 7.98 ||
 * 105A || 7.78 || 7.99 ||
 * 105B || 7.79 || 8 ||
 * 106A || 7.77 || 7.99 ||
 * 106B || 7.76 || 7.98 ||

__OA FHL 2011__ Turned on water in coolers and set flow through larval containers around 11:30 am.
 * July 21, 2011**

__OA FHL 2011__ Washed all larval containers and lids using vortex and fresh water. Made 35 and 50 µm large screens and 35 µm small screens.
 * July 20, 2011**

Made 7.5% MgCl2 in MilliQ water (0.7877 mol/L). Dissolved 7.49 g of MgCl2 (MW = 95.2115 g/mol) in 100 mL of water.

Made 1x PBS from 10x PBS (from Jaquan) by mixing 10 mL of 10x in 90 mL of DI water. Brought the pH down to 7.4 with 1N HCl. The 1N HCl was made from 10N HCl - 20 mL of 10 N HCl in 180 mL of DI water.

Made 4% paraformaldehyde in filtered seawater. Each ampoule of 16% PFA is 10 mL - mixed with 30 mL of FSW.

__OA FHL 2011__ Cleaned out all 6 coolers to be used for the experiment. Emptied all water in the coolers and wiped down with vortex. Let sit with the vortex for >5 minutes. Wiped down twice with fresh water.
 * July 19, 2011**

"Larvae-proofed" the 36 larval chambers for the experiment. All chambers have an outflow of ~1/2" diameter near the top of the container. I hot glued mesh of 35µm on the outflow so that larvae would not be able to escape.

Working on a detailed experimental protocol and materials list for the experiment. It can be found [|here]

__OA FHL 2011: Choosing pH for OA experiments__ The coolers representing different treatments need to be set at specific pH that represent the correct µatm treatments for CO2 in seawater. These values depend on total alkalinity (TA), salinity, and temperature. Students in the OA class have measured the TA and salinity of the incoming water into the tanks. TA = 2060 µmol/kg, salinity = 30.1 ppt. All measurements are taken at 25C. For each temperature treatment, the adjusted conditions are set to the correct T (24 or 28C). Constants used in CO2calc are Lueker et al. 2000 for CO2 constants, Dickson 1990b for KHSO4, Total pH scale, Wanninkhof 1992 for air-sea flux (Dickson, pers. comm.) pH at which the Honeywell controllers will be set are: 24C, 400 µatm: 8.04 24C, 700 µatm: 7.84 24C, 1000 µatm: 7.69 28C, 400 µatm: 7.98 28C, 700 µatm: 7.78 28C, 1000 µatm: 7.64
 * J****uly 18, 2011**

__How to measure total alkalinity__
 * June 17, 2011**
 * Find a clean, dry 200 mL beaker
 * Rinse beaker 3 times with a small amount of water from your sample (dispose of rinse water properly)
 * Fill beaker with sample to ~130 mL
 * Zero balance and weigh beaker with water and zero again. Remove beaker from balance, pour the sample into the insulated beaker on the stir plate and place empty beaker on the balance. The sample weight is the absolute value of the number on the balance display. Record the sample weight.
 * Stir contents of insulated beaker with a magnetic stir bar. Place TA and temperature electrodes in the sample. The sample should warm to 25 +/- 0.1 degree C.
 * Click “initialize instrument” in Controls tab.
 * When temperature is attained, enter the salinity and weight of the sample into the Controls tab in LabView. Click “begin titration”
 * An initial pulse of HCl will be added, then none for 2-5 minutes, then small pulses. The TA measured by the two different electrodes should be within 4 units of each other.
 * When TA measurement is done, rinse probes over sample with DI water and pat gently with kimwipes. Do NOT touch the electrodes on the bottom of the probes.
 * Use the peristaltic pump to suck out the water from the beaker into the SW + HCl waste. Rinse the insulated beaker with DI water a few times and turn off pump when done.
 * Without touching the inside of the insulated beaker, remove the stir bar with a kimwipe. Wipe out 3 times with kimwipes to dry

__Making m-cresol purple__ M-cresol purple needs to be kept at 4C once the powder is dissolved because the dye degrades in water over time. The concentration of the dye for a 10 mL cuvette should be 2 mmol/L, or 0.04044 g in 50 mL of nanopure/milliQ water. The dye needs to be brought to 7.9 +/- 0.1 pH, or the ratio of the absorbance peaks at 434 and 578 wavelengths should be ~1.6. This can be achieved by adding NaOH or HCl. The pH is determined by spec using a short path length (1 cm) cuvette at 25C.

__NOAA OA: C. gigas larvae__ The frozen larvae are stored in a freezer box in the -80C. They are on the second shelf from the bottom (currently on the righthand side). The tubes are labeled on the side with the jar, treatment, and date of collection. The three treatments are 280, 400, and 1000 ppm.
 * June 13, 2011**

__Ceramide: qPCR__ Designed new primers for 3KDSR qPCR (3KDSR_qPCR.2, 1341 and 1342 SRID). The primers were reconstituted with TE buffer (pH 8.5) to 100 uM. qPCR was done on 2 cDNA samples and 3 negative controls to test primers. The cDNA amplified and had a single melt curve; the NTCs were blank. qPCR was done on all control and vibrio-exposed samples (n=8 for each treatment) in duplicate. All samples amplified. One NTC had amplification that could be problematic. The difference in expression between control and vibrio-exposed is not significant (p=0.07). media type="custom" key="9761210"

__NOAA OA: C.gigas larvae__ Details about sampling Jars counted: 3A4, 3A5, 3B4, 1A4, 1A5, 1B4, 6A4, 1B5, 3B5, 6A5, 6B4 All jars sampled for RNA Spec done for jars: 1B4, 1B5, 3B4, 3B5, 6B5, 6A5 Counts: counts were done for live/dead, calcification, and size. Aliquots used for counting were 6x1000 uL divided between 2 wells. Calculations were done as described May 19. Calcification data were taken on June 7. As for June 1, there were barely any larvae left in the 1000 ppm treatment and densities were low in all jars. For the samples taken for RNA for 1A5 and 1B5 there were many shell bits that were too hard to remove without compromising the sample. 3A4 was the only jar that was first filtered with 180 um mesh to get rid of rotifers (it didn't seem to help much since the rotifers were still there, they were probably able to squeeze through the mesh). The algae used to feed the larvae had crashed but was still being delivered into the larval chambers. Copepods had proliferated in some of the jars and I saw many copepod eggs and some copepodites. The wells corresponding to 6B4 were completely covered by rotifers. When EtOH was added to these wells, the rotifers created a solid mat on the bottom.
 * June 6, 2011**

__NOAA OA: C.gigas larvae__ Details about sampling Jars counted: 1A4, 1A5, 1B4, 1B5, 3A4, 3A5, 3B4, 3B5, 6A4, 6A5, 6B5 No jars sampled for RNA Spec done for jars: 1A4, 1A5, 3A4, 3A5, 6A4, 6A5 Counts: counts were done for live/dead, calcification, and size. Aliquots used for counting were 3x 1000 uL. Calculations were done as described May 19. Sarah took size pictures for plate 2, wells A2, B2 and C2 on June 2. Trends included: a number of the jars at 1000 ppm had no larvae at all; there was no obvious trend in number of larvae swimming per treatment or in calcification or mortality. Since the anomalous results at previous time points for the 1000 ppm treatments drive the trends, it seems that the lack of data for this time point results in no obvious trends. The absence of larvae could be due to a mass mortality event between May 27 and June 1.
 * June 1, 2011**

Rotifers were in all jars counted and were very dense in some. This distracted from counting. The rotifers also "kick" the larvae, which seems to cause the larvae to close their valves and cease swimming. In 6B4, there was so much algae and rotifer bodies that it was difficult to see the larvae.

__NOAA OA: C.gigas larvae__ Details about sampling Jars counted: 1B3, 3B3, 6B3, 1B6, 3B6, 6B6, 1A4, 3A4, 6A4, 1A5, 3A5, 6A5, 1B4, 3B4, 6B4, 1B5, 3B5 Jars sampled for RNA: 1B3, 3B3, 6B3, 1B6, 3B6, 6B6* Spec done for jars: 1B3, 3B3, 6B3, 1B6, 3B4, 6B6 Counts: counts were done for live/dead, calcification, and size. Aliquots used for counting were 3x450 uL. Calculations were done as described May 19. Calcification for plate 4 was done on May 28 (in the morning around 7:30 am). Trends included: slightly greater number of larvae swimming in the 1000 ppm; fewer completely calcified larvae at 1000 ppm and greater partially calcified and uncalcified; overall least amount of total mortality at 1000 ppm but the single jar with the greatest total mortality was also at 1000 ppm; inconsistent instant mortality. Rotifers were observed in almost all the jars. There was a lot of algae in the jars from treatment 6, box B. Jar 6A5 was dropped and ~1/3 of the contents spilled. The jar was refilled with water and replaced in its box. Around 9 pm, ~60 g of adult C. gigas shell fragment were placed in all the remaining jars. In one box for each treatment, two jars were put in the system with only water and 2 jars with water and 60 g of shell fragment to monitor changes in total alkalinity.
 * May 27, 2011**
 * There are two tubes for 6B6 because there was so much algae in the sample that the volume couldn't be reduced enough on the sieve to fit in one tube.

__NOAA OA: C.gigas larvae__ Details about sampling Jars counted: 3A3, 3B3, 3A6, 3B5, 3B6, 3A6, 3A5, 6A3, 6A6, 6B3, 6A5, 1A6, 1A3, 1A5, 1B5, 1B3, 1B6 Jars sampled for RNA: 3A3, 3A6, 1A3, 1A6, 6A3, 6A6 Spec done for jars: 1A3, 3B3, 6A3, 1A6, 3A6, 6A6 Counts: counts were done for live/dead, calcification, and size. Aliquots used for counting were 3x333 uL (1 mL). Calculations were done as described May 19. Trends observed included: greater number of larvae swimming in the 1000 ppm; lower % calcified larvae at 1000 ppm; higher % partially and uncalcified larvae at 1000 ppm, lowest uncalcified at 280 ppm; greater total and instant mortalities at 1000 ppm. Rotifers had somehow gotten into the jars in treatment 6 (400 ppm). Ciliates were observed in some jars.
 * May 23, 2011**

__NOAA OA: C.gigas larvae__ Details about sampling Jars counted: 3B1, 3B2, 3B3, 3B4, 3A3, 3A4, 6B1, 6B2, 6B3, 6B4, 6A3, 6A4, 1B1, 1B2, 1B3, 1A3, 1A4, 1B4. Jars sampled for RNA: 3B1, 3B2, 6B1, 6B2, 1B1, 1B2 Spec done for jars: 1B1, 3B1, 6B1, 1B2, 3B2, 6B2 Counts: counts were done for live/dead and calcification. Photos were taken for size for plate 6. Sarah took photos for the other plates the next day (5/20). Aliquots used for counting were 7x100 uL (700 uL). Calculations and graphs were made to observe trends for % swimming larvae, % calcified, % partially calcified, % uncalcified, total mortality, and instant mortality. Since the number of empty shells were recorded, it is possible to separate out the larvae that have died recently (instant mortality) from those that have died over the course of the experiment but have just been counted (total mortality). % swimming is based on the total larvae, including the empty shells. All the calculations for % calcified, etc. are based on total larvae excluding the empty shells. Observed general trends include: more larvae are swimming at 1000 ppm; more larvae are totally calcified at 400 ppm > 280 ppm > 1000 ppm; more larvae are partially calcified and uncalcified at 1000 ppm; total and instant mortalities arehighest at 400 ppm and lowest at 1000 ppm. Ciliates were present in some jars.
 * May 19, 2011**

**May 18, 2011** __Ceramide sequencing__ Analysis/incorporation of new sequences 3KDSR to include stop codon: Assembled all previously sequenced reverse sequences. Based on alignment with the new reverse sequence (R2), deleted all Ns and the first 6 bp (GCCCTT) from the consensus (1626). Assembled with R2 and made consensus (1627). Assembled all previously sequenced forward sequences and made consensus (1628). Made reverse complement of the contig 1627 and assembled with the forward contig (1628) to make consensus full sequence 1630. Trimmed at bp 111 to start with ATG. Based on majority rule, base pairs were changed in the contig 1630 to have certainty in protein translation: bp 904 = G, bp 936 = G, bp 964 = C. Translated and trimmed amino acid sequence to include first stop codon at aa 329. Sptlc1 missing middle 26 bp: Sequenced purified gel band of Sptlc1 PCR (F and R) and resequenced plasmid prep from before (F and R) for 4 new sequences. Assembled all previous and new F and R sequences - F contig is 1631, R is 1632. They did not align. Aligned them with the original Sptlc1 EST and there are still 10 bp missing in the middle of the sequence.

__ NOAA OA: C.gigas larvae __ Shallin and Mike showed me how to take samples for PMEL to test total alkalinity (TA) and dissolved inorganic carbon (DIC). The jars (500 mL) are from PMEL and are made to be nonporous. 1. label the jars with a paint marker: "NWFSC/UW dd/mm/yy Tank #" 2. Grease the stopper and smooth out the grease by moving the stopper in the neck of the jar 3. Make sure the equilibrator pump is on and attach tubing to outlet from equilibrator. 4. Let water flow out the tube for at least 1 minute. 5. Rinse bottle well, upside down so water flows out. 6. Fill bottle completely with water from tank and let overflow for at least 5 seconds (about equal to half the volume of the bottle). 7. Pinch off tube tightly and quickly remove from jar. It is important to have adequate headspace so that the water does not overflow after the poison is added. Turn off water flow. 8. Poison jar with mercuric chloride (Shallin does this) and put in stopper.

__NOAA OA: C. gigas larvae__ The NOAA side of the experiment ended on Sunday (May 15) and our experiment now has access to 1 more box (=6 more jars) per treatment. The sampling schedule has been updated and is in the google doc of the sampling data. When I came in Monday morning, there was no water flowing to the jars in treatment 3 (280 ppm). Paul says that they had trouble with that tank on Sunday and that it probably stopped working Sunday night. After doing the pH spec, I verified that the pH in the jars had not been adversely affected by the static water. The jars were all moved to tank 4, which had been previously equilibrated to 280 ppm. The jars are still labeled as treatment 3 jars (i.e. 3A1 as opposed to 4A1) but will remain attached to tank 4 for the remainder of the experiment. Sampling for May 16: Total jars sampled for RNA - 1A1, 1A2, 3A1, 3A2, 6A1, 6A2 Jars counted (5 aliquots of 100 uL from the Falcon tube) - 1A1, 1A2, 3A1, 3A2, 6A1, 6A2, 1A3, 1B3, 6A3, 6B3, 1B1, 1B2, 3B1, 3B2, 6B1, 6B2
 * May 16, 2011**

Details about sampling RNA: All larvae were removed from jars using a 20 um mesh sieve. Larvae were transferred from the sieve into a 2 mL tube with 1-2 mL of seawater. The tube was spun down at 5,000 rpm for 1 min and the seawater was removed. The larvae were then flash frozen in liquid N2 and later transferred to the -80C freezer. Counting: Counts were done for live/dead, calcification, and size. In some of the wells (both wells for 1A1 and one well for 1A2) too much EtOH was added and it was difficult to find the partially/uncalcified larvae, so their representation may be missing from the final counts. For these wells, shells were fully calcified if they had they full D-hinge shape and were partially calcified if the shell was very small/was not the correct shape. No uncalcified larvae were found in these wells (which is unrealistic since it is the 1000 ppm treatment). All other wells were easy to score. For size, photos were taken of ~10 larvae per well on the dissecting scope (5x) of larvae that were positioned in the well so that a clear "D" shape was visible when looking down at them. Photos were taken 5.17.11. Measurements have not yet been taken.

Other notes All larvae were D-hinge or close to (kidney bean shaped) if calcification was apparent. Partially calcified larvae ranged from small dots of calcified material on the larval body to a full shell that did not show a well formed cross under the polarized light. Empty shells were included in counts of mortality, but not calcification. There were ciliates in some wells/jars (see data sheet). There seemed to be more algae in the jars from treatment 1 (1000 ppm) - maybe because the higher pCO2 is causing the algae to grow in the jars?

__NOAA OA: C. gigas larvae__ Sampled 2 jars from each treatment and took data on microscope. Began cleaning and sampling jars around 8:30 am. Cleaning was accomplished as described for May 10, 2011. Aliquots for counting were 3x 60 uL (180 uL). Calcification amount was also determined on the inverted microscope using double polarization. A few pictures were taken as examples of different levels of calcification: full, partial, uncalcified. Data can be found in link from May 10. Eggs were not included in total counts. A couple of ciliates were seen in one well from jar 3B2. Jar 6B5 was sampled in its entirety for RNA due to low densities of larvae in most of the jars from shelf 6. In general, densities are much lower than expected and the sampling schedule will have to be adjusted accordingly. Samples were taken for gene expression analysis from 6 jars: 1B1, 3B1, 1B2, 3B3, 6B2, 6B5. After preliminary density counts for each jar, the correct volume for the desired number of larvae (10,000 or 5,000) was taken from the 50 mL Falcon tube. This volume was filtered through a 20 um sieve and then pipetted into a 2 mL screw cap tube. The larvae were left to sit for ~1 min, tapped a few times on the counter to encourage larvae to go to the bottom, and then most of the seawater was pipetted off before the larvae were flash frozen in liquid nitrogen. The samples were later transferred to a -80C freezer, but it appears that the dewar had run out of liquid N2 and the samples may not have good quality RNA.
 * May 13, 2011**

Larval feeding began Thursday at ~4 pm, except for box 3B which was started Friday morning (and given a surplus of food at that time).

__NOAA OA: C. gigas larvae__ Began cleaning and sampling jars around 8:30 am. Each jar was completely emptied into a 20 um mesh sieve and then rinsed out with the appropriate treatment water. A new, clean jar/larval chamber was filled with new water from the appropriate treatment and if that jar was not scheduled to be sampled, all larvae were rinsed into the new jar. If the jar was scheduled to be sampled (jars 5 and 6 for May 10), then a clean jar was prepped while larvae were washed into a 50 mL Falcon tube, which was then filled to 45 mL. The tube was gently mixed ~5x, 3 aliquots of a fixed volume were taken and put into a welled plate (1 well) and then these two steps were repeated so that for each jar sampled there were 2 wells of a fixed volume to count. For shelf 1, the volume taken was 45 uL x3, for shelves 3 and 6 the volume was 25 uL x3 (135 uL yielded too many larvae). After the aliquots were taken from the Falcon tube, it was poured into the new, clean jar and replaced in the appropriate box. At the end of sampling all the jars, they were plugged into the system so that the water would flow through. Counting of larvae began ~10 am. First, live/dead were determined on the inverted microscope at 40x. Then the larvae were dropped with 100% EtOH and total counts were taken per well on the inverted scope at 4x. Count data are [|here] Shallin did pH spec on the system and jars for demography. Jars were plugged into the system (flow-through) ~4 pm.
 * May 10, 2011**

Treatment 1 = 1000 ppm Treatment 3 = 280 ppm Treatment 6 = 400 ppm

__NOAA OA: C. gigas larvae__ Carolyn and Brent went to Taylor and fertilized eggs for the experiment. 21 females and 20 males were used in the fertilization. 80,000 eggs were aliquoted to each jar for transport (each jar would then be emptied into the larger larval chambers at NWFSC) and the eggs were fertilized in the jars with pooled sperm. We began putting the fertilized eggs into the chambers containing treatment water around 5 pm (treatment pCO2= 280 ppm, 400 ppm 1000 ppm). The system is being kept at 14C. Fertilized eggs in the larval chambers were left static overnight so that the larvae could hatch. 2 jars (~80,000) fertilized eggs were strained on 20 um mesh and stored at -20C for pop gen.
 * May 9, 2011**

__C. gigas larvae: OA & Vt__ extracted 2 samples for RNA: 1 control and 1 10^6 Vt. First spun down briefly at 5,000 xg and removed seawater supernatant. Then extracted with TRI reagent according to manufacturer's protocol. Resuspended in 50 uL 0.1% DEPC H2O and mixed by pipetting. Checked RNA concentrations on Nanodrop, doing each sample in triplicate. Average concentrations are reported below. control: 27.03 ng/uL 10^6 Vt: 30.68 ng/uL
 * May 3, 2011**

__S. glomerata: OA__ Extracted 2 samples for RNA: D9 B6, and D16 B6 from the sample set in the 2 mL tubes. Spun down at 5,000 xg for 2 minutes and removed larvae/sediment into a new tube. Saved supernatant in a 2 mL screw cap in -80C. Followed TRI reagent protocol. Resuspended in 50 uL 0.1% DEPC H2O and mixed by pipetting. Checked RNA concentrations on Nanodrop, doing each sample in triplicate. Average concentrations are reported below. D9 B6: 0.10 ng/uL D16 B6: 0.36 nguL

__NOAA OA: C. gigas larvae__ [|Raw data] from experiment April 11-18, 2011.
 * April 29,2011**

__Ceramide: sequencing__ Made 1% agarose plate (100 mL 1x modified TAE, 1 g agarose, 10 uL EtBr). Loaded 25 uL of PCR product into wells and used Bioline's Hyperladder. Ran for ~1 hour at 100V. Cut out gel bands from first 4 lanes. The contamination seen in the Sptlc1 is the same size as the desired product, so it should not adversely affect sequencing efforts. A new bottle of sterilized nanopure water was started yesterday - the new and old water in the different negative controls refers to this bottle ("new") and the bottle that had previously been in use ("old"). media type="custom" key="9219048"
 * April 27, 2011**

__Ceramide: qPCR vibrio exposure__ To confirm contamination of qPCR primer stocks for 3KDSR, did a cPCR following the qPCR thermal profile: 95C 10 min; 40x 95C 15s, 55C 15s, 72C 30s; 95C 10 min; 72C 10 min. The new bottle of sterilized water was used. The 10 uM working stocks for 3KDSR qPCR made 4.26 were used as well as the qPCR primers for acid ceramidase, which have never showed signs of contamination. Ran PCR products on a 1% agarose gel at 120V for 30 min. The primer stocks for 3KDSR are contaminated as evidenced by the amplification of the correct product size in the negative controls (which is not seen for acid ceramidase). Will order new primer stocks. media type="custom" key="9236946"

__C. gigas larvae: OA & Vt__ At 10 am (~24 hours post-inoculation) counted live vs. dead in the welled plates. There was differential mortality between the treatments. At ~2:30 pm, sampled all the jars in the experiment (vibrio exposure control and 10^2-10^6). Filtered entire 200 mL through a 30 um mesh screen and rinsed out jars with sterile seawater. Pipetted all larvae condensed in one corner of the screen into a screw cap tube. Flash froze in a mixture of dry ice and EtOH. Transferred tubes to the -80C box O.orca archived tissue from NWFSC.

__C. gigas larvae: OA & Vt__ Put ~2000 larvae in each jar and dosed with Vt (between 10:30 and 11:30 am). Also aliquoted 40 larvae into welled plates and added appropriate pCO2 seawater to bring volume to 4 mL. Larvae in plates were also dosed with Vt. Elene measured pH in each of the treatments using the spec. There wasn't much different between the treatments and the lowest pH was 8.09. Terminated the 2000 ppm and 840 ppm experiments, kept ambient to do LD50 with just Vt. Set up sterilized seawater bubbling with CO2 at 750 and 2000 ppm overnight to begin LD50s tomorrow.
 * April 26, 2011**

__Ceramide: qPCR vibrio exposure__ Repeated qPCR of 3KDSR from 4.22. Used new nanopure water for everything. Bleached bench and got new mat. Made new working primer stocks. Used a new plate and new immomix. NTCs still had contamination.

__Ceramide: sequencing__ Designed new primer to capture more of the 3' end of 3KDSR (Cg_3KDSRseq_R2). Also need to try to get missing middle 26 bp for Sptlc1. Made pooled cDNA for controls and 3-hour post exposure C. gigas gill samples. Did PCR on pooled samples with new 3KDSR reverse primer and original forward primer and with Sptlc1 primers (12.5 uL Apex, 8.5 uL water, 0.5 uL each primer, 3 uL cDNA; GENPCR in Emma's directory).

__Ceramide: qPCR vibrio exposure__ Box plot includes previous data for loci EF1a, Sptlc1, acid ceramidase, and acid sphingomyelinase. 3KDSR and ceramide glucosyltransferase are new based on April 22's qPCRs. The expression difference between control and exposed for 3KDSR is significant; the different is not significant for Cmglctrans (see zoomed in graph below). Significance is based on t-tests in R. media type="custom" key="9196438"media type="custom" key="9196442"
 * April 25, 2011**

__C. gigas larvae: OA & Vt__ Set up system in basement to do a LD50 with 3 levels of CO2 (ambient, 840 ppm, and 2000 ppm) and 6 concentrations of V. tubiashii (control, 10^2 CFU through 10^6 CFU). There are 2 replicate jars for each Vt concentration within each pCO2 for a total of 12 jars in each pCO2. Each jar holds 200 mL of sterilized filtered seawater. The jars were placed in airtight boxes with an inlet for the correct pCO2 and an outlet attached to tubing with the other end in water so that atmospheric air does not enter the box (see pictures below). At ~3:30 pm the boxes were sealed and the air was turned on so that the water in each jar will equilibrate to the correct pCO2. Also ~200 mL of water in a beaker was placed in each box that will serve as water in the plates to be used for counting. media type="custom" key="9196570"media type="custom" key="9196574"media type="custom" key="9196580"media type="custom" key="9196584" __Ceramide: qPCR vibrio exposure__ qPCR of 3KDSR and ceramide glucosyltransferase (CgT) of vibrio-exposed adult C.gigas gill tissue, 3 hours post-exposure. PCRs are of all 16 samples, 8 control and 8 exposed, in duplicate. Thermalcycler protocol was 3StepAmp+Melt_SJW. media type="custom" key="9192122" Results: 3KDSR was contaminated (high amplification of NTCs), CgT was not. Both primer pairs amplified only one product (one melt curve).
 * April 22, 2011**

Made new working stocks of 3KDSR (10 uM) and did a qPCR of all samples in duplicate. media type="custom" key="9192144" Results: Still slight amplification in the NTCs but it comes up much later than the other samples. Only one melt curve apparent at 75-75.5.

**April 19, 2011** __NOAA OA: C. gigas larvae__ Took calcification data from the samples taken April 18. Some notes of concern: 1. a good number of mortalities were empty shells, meaning that the larval body had dissipated or been eaten - if uncalcified larvae suffered a similar fate they would be underrepresented in the counts. 2. the ethanol added to make the larvae drop can make cells lyse, which makes distinguishing unshelled larval bodies from debris difficult.

**April 18, 2011** __NOAA OA: C. gigas larvae__ Sampled all jars of larvae and changed water as outlined on April 12, 2011. Step 3 was again changed and 3 aliquots of 333 uL were taken for counting. The experiment was ended and all remaining larvae were strained from the water and preserved in RNAlater for analysis. Calcification data were not taken today but samples were saved to do tomorrow.

**April 15, 2011** __NOAA OA: C. gigas larvae__ Sampled all jars of larvae and changed water as outlined on April 12, 2011. Changes to the protocol include: 3 aliquots of 50 uL were taken for counting (step 3); a greater volume of larvae were removed for RNA isolation and were preserved in RNAlater (step 4); calcification data were taken.

__Ceramide sequencing: alignment and phylogenies__ Serine palmitoyltransferase Trimmed protein sequence to end with stop codon (468 amino acids). Aligned with protein sequences of C. elegans, D. melanogaster, D. rerio, H. sapiens, and M. musculus in ClustalX. Imported alignment into Geneious and made tree in PHYML. media type="custom" key="9093862"media type="custom" key="9093874"
 * April 14, 2011**

Updated all protein alignments and trees to include homologous genes from C. elegans, D. rerio, Xenopus spp., M. musculus, and H. sapiens. Not all sequences were available for all genes.

__Ceramide sequencing: alignment and phylogenies__ Serine palmitoyltransferase As mentioned below (April 7, 2011), the forward and reverse sequences for Sptlc1 do not overlap. There is a 26 bp gap between the two sequences. I generated consensus sequences from the contigs for the forward and reverse fragments (n=3 for each) and then assembled them to the original EST (which is made up of FQ665912 and 454 ESTs). Reverse complemented the consensus sequence and translated starting from the first sequence ATGTAT ( TAT = amino acid Y) based on how the translation of the original EST aligned with homologous sequences.
 * April 13, 2011**

__NOAA OA: C. gigas larvae__ Yesterday, Carolyn picked up diploid C. gigas larvae that were fertilized on 4/11 at about noon. They were brought to NOAA and put in the system at 6 pm. There are 3 treatment pCO2 (280, 400, 1000 ppm) and 3 replicates per treatment. They are being held at 14C. Starting around 9 am today, we began sampling each container (n=9) and Shallin took samples to determine the pH on the spec. Sampling involves the following: 1. Pour entire container through a 20 micron mesh sieve that is in a water bath (of the water you are pouring through). Rinse out the container with the correct treatment seawater 3x. 2. Rinse the larvae on the mesh into a 50 mL falcon tube and fill to 45 mL. 3. Invert the falcon tube gently 5x and take 3 aliquots of 30 uL (could be greater volume if larvae are less dense) and put in a welled plate. Which containers go in which wells is randomized. 5. Take 750 uL of water + larvae from the falcon tube and strain through mesh (should be >500 larvae). Remove larvae from mesh with a pipette and put in 2 mL screw cap vial and flash freeze. 4. Blind counts are done for number larvae dead, number alive but on bottom of well, total larvae (and then by subtraction, the number swimming), and calcification level (uncalcified, partially, or fully). Total larvae and calcification data are taken after the larvae have been dropped by EtOH. Calcification is determined using a polarized lens on the inverted scope. Mortality is verified at 40x.
 * April 12, 2011**

No calcification data were taken today because the larvae were still too young to be calcified. The larvae are so small that there are probably not enough frozen for qPCR. Tomorrow, feeding will begin for all the larvae.

__Ceramide sequencing: alignment and phylogenies__ Acid ceramidase Generated consensus sequence from alignment in Geneious. Most of the beginning and end of the sequence are Ns, the start codon (ATG) is at bp 89 and the stop codon (TAA) ends at bp 1258. Translated consensus nucleotide sequence into a protein sequence and exported FASTAs of C. gigas protein with protein sequences from GenBank for C. elegans, D. rerio, H. sapiens, and R. norvegicus. Aligned all sequences in Clustal and imported the protein alignment back into Geneious. Using the PHYML plug-in, generated a phylogenetic tree using the JTT algorithm, 100 bootstraps, 0 invariable sites, and 1 substitution rate category. media type="custom" key="9021524" media type="custom" key="9021586"
 * April 8, 2011**

Ceramide glucosyltransferase Same as for ACMase. The reverse complement of the sequence was used to generate the protein sequence. On the reverse complement, the ATG was 38bp after then Ns. The alignment was performed with sequences of CgT for H. sapiens, X. tropicalis, and C. elegans. media type="custom" key="9022226"media type="custom" key="9022246"

3-ketodihydrosphingosine reductase Same as above. The start codon was at bp 112 in the consensus sequence. Alignment was done with sequences from H. sapiens and D. melanogaster. media type="custom" key="9022314"media type="custom" key="9022334"

__Ceramide sequencing: alignment and verification__ Serine palmitoyltransferase (Sptlc1) Got back good quality sequence for 3 forward and 3 reverse. There are a few potential SNPs in the sequences. The fragments sequenced with the forward primer assemble together, as do the fragments sequenced with the reverse primer. When these 2 contigs are aligned with the original contig based on ESTs, there are ~25 bp missing between the two sequenced fragments.
 * April 7, 2011**

Ceramide glucosyltransferase Got back good quality sequence for 2 forward and 3 reverse fragments. All fragments assembled together to form a full sequence that assembles with the original (reversed) EST.

Acid ceramidase (ACMase) Got back good quality sequence for 4 forward and 4 reverse fragments. All fragments assembled together and aligned with the original EST used to design the primers.

3-ketodihydrosphingosine reductase (3KDSR) Got back good quality sequence for 8 forward and 7 reverse fragments. All fragments assembled together and aligned with the original contig used to design the primers.

__C.gigas Larvae Vibrio exposure__ Almost all larvae were dead in all treatments (including control), probably due to ciliates which had proliferated overnight.
 * March 31, 2011**

__C.gigas Larvae Vibrio exposure__ Counted larvae to determine mortality. Only counted larvae on the bottom of the well. On the last day of the experiment we will count total larvae in each well by dropping them with EtOH and then retroactively determine the number of swimming larvae for previous days. Dead vs. live larvae were determined at 40x magnification.
 * March 30, 2011**

__C. gigas Larvae Vibrio exposure__ Elene got 2 week-old larvae from the hatchery yesterday. This morning, we aliquoted ~40 larvae into wells of tissue culture plates and made sure the volumes in each well were at 4 mL. Different plates were inoculated with 10^3, 10^4, 10^5, 10^6, 10^7, or no V. tubiashii (control) that Elene had grown over the weekend. Plates were left on the benchtop to incubate over night.
 * March 29, 2011**